WO2025087287A1 - Use of cholesin in modulating cholesterol homeostasis - Google Patents

Abstract

The present disclosure provides use of cholesin in modulating cholesterol homeostasis and a composition comprising cholesin, a nucleic acid encoding cholesin, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of endogenous cholesin protein in a subject. Specifically, the present disclosure relates to a method for treating or preventing a disease associated with an elevated plasma cholesterol level in a subject. The method comprises: administering to the subject an effective amount of cholesin, a nucleic acid encoding cholesin, or an expression vector comprising the nucleic acid, or enhancing the expression or activity of endogenous cholesin protein in the subject.

Description

Use of Cholesin in regulating cholesterol homeostasis

This international patent application claims priority to Chinese patent application No. 202311385254.5 filed on October 24, 2023, the entire contents of which are incorporated herein by reference for all purposes.

Technical Field

The present invention belongs to the field of biomedicine, and particularly relates to the use of the intestinal secretory hormone Cholesin in regulating cholesterol homeostasis, and the use of the intestinal secretory hormone Cholesin in regulating cholesterol homeostasis after being used in combination with other cholesterol-lowering drugs.

Background Art

Cholesterol homeostasis is coordinated by various tissues to maintain a balance between dietary cholesterol absorption, new synthesis, and bile clearance and excretion. Elevated blood cholesterol levels, especially low-density lipoprotein cholesterol (LDL-C), contribute to the development of atherosclerosis and are considered an important risk factor for cardiovascular disease (CVD). CVD is a pressing public health problem, accounting for more than 30% of deaths worldwide. Both dietary cholesterol and bile cholesterol are taken up by Niemann-Pick type C1 like 1 (NPC1L1) proteins on the apical surface of enterocytes in the intestine. After absorption, dietary cholesterol is released in the form of chylomicrons, which are then taken up by the liver, the major site of cholesterol synthesis. Cholesterol molecules from new synthesis and intestinal absorption are transported to cells for use via the circulation. To maintain cholesterol homeostasis, excess cholesterol is excreted from the body via bile secretion into the feces.

New cholesterol synthesis is an energy-consuming process in which cholesterol is generated from acetyl CoA under the control of a series of enzymes. Among them, HMGCR (3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase) is the rate-limiting enzyme, and its activity is controlled at the transcriptional and post-transcriptional levels. SREBP2 (sterol regulatory element binding protein 2) acts as a master transcription factor, governing the expression of HMGCR and other genes involved in cholesterol synthesis and uptake. Cholesterol biosynthesis and uptake are tightly regulated by a negative feedback mechanism that senses the cholesterol level of the cell. When cells are cholesterol-deficient, SREBP2, together with its escort protein SCAP (SREBP cleavage-activating protein), is transported from the endoplasmic reticulum (ER) to the Golgi apparatus in COPII vesicles. In the Golgi apparatus, SREBP2 is cleaved by site 1 and site 2 proteases in sequence. The N-terminal structure of SREBP2 released by this cleavage travels to the nucleus, where it acts as a transcription factor, enhancing the expression of genes involved in cholesterol synthesis and uptake. Conversely, when cellular cholesterol levels rise, cholesterol molecules bind to SCAP, triggering its interaction with INSIG (insulin-induced gene). This interaction retains SREBP in the ER and prevents subsequent activation of SREBP and expression of genes involved in cholesterol metabolism. This mechanism helps maintain cholesterol homeostasis. Both endogenously synthesized and exogenously acquired cholesterol are secreted into the blood in the form of very low-density lipoproteins (VLDL). After processing in the blood, VLDL produces circulating LDL, They can be taken up by cells via LDL receptor (LDLR)-mediated endocytosis. While the regulation of cholesterol homeostasis in response to cholesterol levels is well understood, the understanding of how signals other than cholesterol influence cholesterol synthesis remains limited.

The reciprocal relationship between cholesterol synthesis and absorption is known to regulate the response of circulating cholesterol to dietary or cholesterol interventions. However, the specific factors responsible for coordinating the balance between cholesterol absorption and synthesis, and how these factors precisely regulate this balance, remain poorly understood.

There are three major classes of cholesterol-lowering drugs commonly used to prevent and treat hypercholesterolemia. The first class of drugs, ezetimibe, inhibits NPC1L1 endocytosis, thereby blocking cholesterol uptake. The second class of drugs, statins, reduce cholesterol synthesis by inhibiting HMGCR and increase LDL uptake by upregulating LDLR. The third class of drugs, PCSK9 inhibitors, stabilize LDLR and promote liver absorption of LDL. Although ezetimibe can reduce intestinal cholesterol absorption, its compensatory effect of enhancing cholesterol synthesis limits its effectiveness in lowering plasma cholesterol levels. The cholesterol-lowering effects of statins and PCSK9 inhibitors are both LDLR-dependent, which poses a challenge for patients with defective or insufficient LDLR. In addition, compensatory increases in HMGCR levels worsen the outcomes of some patients who stop taking statins after long-term treatment. And statins can greatly increase the expression of Hmgcr and other cholesterol-causing genes, and their efficacy has been questioned.

Therefore, the search for new cholesterol-lowering drugs is of great biological significance for the treatment of diseases associated with elevated plasma cholesterol levels, including hypercholesterolemia and atherosclerosis.

Summary of the invention

The present invention aims to provide a method for treating diseases associated with elevated plasma cholesterol levels, including hypercholesterolemia and atherosclerosis. The inventors unexpectedly discovered that Cholesin is a hormone secreted by the intestine in response to cholesterol absorption, which has not been reported before, and can inhibit the synthesis of cholesterol and the secretion of VLDL in the liver, thereby reducing the level of plasma cholesterol to prevent diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Mechanistic studies have shown that Cholesin inhibits cholesterol synthesis controlled by PKA signaling and SREBP2 by binding to its receptor GPR146 (Figure 30). Therefore, the Cholesin-GPR146 axis is a molecular link between intestinal absorption of cholesterol and inhibition of cholesterol synthesis in the liver. Therefore, exogenous Cholesin or methods or agents that promote the expression or activity of endogenous Cholesin protein in a subject can reduce the level of plasma cholesterol and/or triglycerides, thereby preventing or treating diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Compared with the three existing cholesterol-lowering drugs, Cholesin has an LDLR-independent downregulation effect on the expression of genes involved in cholesterol synthesis and reduces plasma cholesterol levels, which means that Cholesin treatment is an effective strategy to combat diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. In addition, because Cholesin can reduce Combining Cholesin with existing cholesterol-lowering drugs such as statins is also a promising strategy for treating diseases associated with elevated plasma cholesterol levels, including hyperlipidemia and atherosclerosis.

Accordingly, in one aspect, the present invention provides a method for lowering plasma cholesterol level and/or triglyceride level in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In some embodiments, the plasma cholesterol is selected from plasma total cholesterol and plasma LDL-cholesterol.

In another aspect, the present invention provides a method for inhibiting hepatic cholesterol synthesis in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In some embodiments, the method inhibits the expression of a gene associated with cholesterol synthesis, and the gene is selected from Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk and Sqle. In some embodiments, the method inhibits the expression of a gene associated with cholesterol uptake, and the gene is Ldlr.

In some embodiments, the method inhibits the expression of a transcription factor that promotes the expression of genes involved in cholesterol synthesis or uptake, and the transcription factor is SREBP2.

In yet another aspect, the present invention provides a method for inhibiting hepatic VLDL-cholesterol secretion in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In another aspect, the present invention provides a method for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In an embodiment of the method of the present invention, the Cholesin protein is a human Cholesin protein.

In some embodiments, the nucleic acid is DNA or RNA.

In some embodiments, the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones.

In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia.

In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

In some embodiments, the method further comprises the step of administering a second therapeutic agent, preferably, the second therapeutic agent is selected from a protein or peptide, a nucleic acid, and a small molecule drug.

In some embodiments, the second therapeutic agent is a cholesterol-lowering drug selected from the group consisting of an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

In some embodiments, the HMGCR inhibitor is a statin.

In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin.

In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe.

In some embodiments, the second therapeutic agent is administered before, after, or simultaneously with the Cholesin protein, the nucleic acid, or the expression vector.

In some embodiments, the subject is a human.

In some embodiments, the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, for example, the subject has an LDL receptor (LDLR) defect.

In another aspect, the present invention provides a composition comprising a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of human endogenous Cholesin protein, and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the composition is used for one or more of the following:

1) reducing plasma cholesterol levels in a subject;

2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject;

3) reducing plasma triglyceride levels in a subject;

4) inhibiting hepatic cholesterol synthesis in a subject;

5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and

6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

In some embodiments, the Cholesin protein is a human Cholesin protein.

In some embodiments, the composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from protein Proteins or peptides, nucleic acids and small molecule drugs.

In some embodiments, the second therapeutic agent is selected from a cholesterol-lowering drug.

In some embodiments, the cholesterol-lowering drug is selected from the group consisting of an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor.

In some embodiments, the HMGCR inhibitor is a statin.

In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin.

In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe.

In some embodiments, the composition is formulated as an injection preparation or an oral preparation, such as a tablet, a capsule, a granule, a suspension.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Silver staining and immunoblotting results of serum proteins from mice fed a normal diet (RD) and a Western diet (WD).

Figure 2A: Localization of human Cholesin (hCholesin) and mouse Cholesin (mCholesin) in cells detected by immunofluorescence experiments.

Figure 2B: Secretion of human and mouse Cholesin in the culture medium detected by immunoblotting.

FIG. 3A : Plasma Cholesin levels of fasting, RD, and WD mice measured by ELISA.

FIG. 3B : Changes in plasma Cholesin levels in human clinical samples after fasting and 1 h of feeding.

Figure 4: Cholesin protein levels in different mouse tissues. Cholesin whole body knockout (KO) mice were used as controls.

Fig. 5A: Plasma Cholesin levels and small intestinal total cholesterol levels in fasted and WD-fed WT and IKO mice.

FIG. 5B : Plasma total cholesterol levels and plasma triglyceride levels in fasted and WD-fed WT and IKO mice.

FIG. 5C : Plasma Cholesin levels and small intestinal total cholesterol levels in WT mice and IKO mice gavaged with corn oil (chol-) and cholesterol (chol+).

FIG. 5D : Plasma total cholesterol levels and plasma triglyceride levels in WT mice and IKO mice gavaged with corn oil (chol-) and cholesterol (chol+).

Fig. 6: Cellular localization of Cholesin observed by immunofluorescence experiments.

Figure 7: Cholesin levels secreted in the culture medium and total cholesterol levels in cells after cholesterol stimulation of HCT116 cells overexpressing Cholesin.

Fig. 8A: Cholesin secretion from Cholesin-overexpressing HCT116 cells with NPC1L1 knockdown (NPC1L1 KD) detected by immunoblotting. Cholesin-overexpressing HCT116 cells without NPC1L1 knockdown (KD) served as a control.

Fig. 8B: The secreted Cholesin level in the culture medium and the total cholesterol level in the cells after co-culture with cholesterol in NPC1L1 knockdown (NPC1L1 KD) Cholesin-overexpressing HCT116 cells. NPC1L1 non-knockdown (NT) Cholesin-overexpressing HCT116 cells served as a control.

Figure 9A: Cholesin levels in plasma and total cholesterol levels in the small intestine of mice treated with ezetimibe (Ezetimlbe+) after oral administration of cholesterol (chol+). Mice not treated with ezetimibe (Ezetimlbe-) served as controls.

Figure 9B: Total cholesterol levels in plasma of mice treated with ezetimibe after oral administration of cholesterol. Mice not treated with ezetimibe served as controls.

Fig. 9C: Cholesin secreted by Npc1l1 knockout (Npc1l1 ^-/- ) mice detected by immunoblotting. Wild-type (Npc1l1 ^+/+ ) mice without Npc1l1 knockout served as controls.

Fig. 9D shows the level of Cholesin in plasma and the level of total cholesterol in small intestine of Npc1l1 knockout (Npc1l1 ^-/- ) mice after oral administration of cholesterol. Wild-type (Npc1l1 ^+/+ ) mice without Npc1l1 knockout were used as controls.

Fig. 9E shows the total cholesterol level in plasma of Npc1l1 knockout (Npc1l1 ^-/- ) mice after oral administration of cholesterol. Wild-type (Npc1l1 ^+/+ ) mice without Npc1l1 knockout were used as controls.

FIG. 10A : Correlation between plasma Cholesin level and plasma total cholesterol (TC) level in humans.

FIG. 10B : Correlation between plasma Cholesin level and plasma low-density lipoprotein cholesterol (LDL-C) level.

FIG. 11A : Plasma total cholesterol and triglyceride levels in wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD.

FIG. 11B : Levels of total cholesterol and triglycerides in the liver of wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice fed RD and WD.

FIG. 11C : Plasma total cholesterol levels and levels of HDL, IDL/LDL, and VLDL in wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD as measured by fast protein liquid chromatography.

FIG. 12A : Body weight of wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD.

Figure 12B: Wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice fed RD and WD After body fat percentage.

FIG. 12C : Food intake of wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice fed RD and WD.

FIG. 12D : Water intake of wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice fed RD and WD.

FIG. 12E : Small intestinal total cholesterol and triglyceride levels in wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD.

FIG. 12F : Chylomicron breakdown rates in the small intestine of wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD, as assessed by lipid tolerance assay.

FIG. 13A : Gallbladder volume, bile total cholesterol concentration, and bile acid concentration in wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD.

FIG. 13B : Fecal total cholesterol and triglyceride levels in wild-type (WT) and intestinal Cholesin-specific knockout (IKO) mice after feeding RD and WD.

FIG. 14A : mRNA levels of cholesterol synthesis-related genes in the liver of WT and IKO mice fed RD and WD as determined by qPCR.

FIG. 14B : Protein expression levels of SREBP2, HMGCR, HMGCS1, and LDLR in the livers of WT and IKO mice fed RD and WD as determined by immunoblotting.

FIG. 15 : Poloxamer 407-injected plasma VLDL secretion rate in WT and IKO mice fed RD and WD.

FIG. 16A : Immunofluorescence staining of Cholesin binding to various tissues of wild-type (Gpr146 ^+/+ ) and Gpr146 knockout (Gpr146 ^−/− ) mice.

FIG. 16B : Immunofluorescence staining of Cholesin binding in primary hepatocytes from wild-type (Gpr146 ^+/+ ) and Gpr146 knockout (Gpr146 ^−/− ) mice.

FIG. 16C : Saturation curves of Cholesin binding to wild-type (Gpr146 ^+/+ ) and Gpr146 knockout (Gpr146 ^−/− ) mouse primary hepatocytes.

FIG. 17 : Binding affinity curves of Cholesin to wild-type (WT) and mutant (Mut) GPR146.

FIG. 18 : Interaction of GPR146 with GNAI1 detected by co-immunoprecipitation in the presence and absence of Cholesin.

FIG. 19 : cAMP levels in primary hepatocytes of wild-type (Gpr146 ^+/+ ) and Gpr146 knockout (Gpr146 ^−/− ) mice after Cholesin treatment.

FIG. 20 : Hmgcr mRNA levels in mouse liver after intraperitoneal injection of Cholesin.

FIG. 21A : Experimental scheme for intraperitoneal injection of Cholesin into mice.

Figure 21B: Hmgcr mRNA levels in the liver of mice injected intraperitoneally with Cholesin.

FIG. 21C : Total cholesterol levels in plasma of mice injected intraperitoneally with Cholesin.

FIG. 21D : Total cholesterol levels in the liver of mice injected intraperitoneally with Cholesin.

FIG. 22A : Experimental scheme for injection of adenovirus and Cholesin into mice.

Figure 22B: mRNA levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, and Pmvk in the liver of wild-type (Gpr146 ^fl/fl ) mice and Gpr146 knockout (Gpr146 LKO) mice injected with adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut), with or without Cholesin injection.

FIG22C : Protein levels of SREBP2, HMGCR, HMGCS1, LDLR, and pKA Sub in the liver of wild-type (Gpr146 ^fl/fl ) mice and Gpr146 knockout (Gpr146 LKO) mice injected with adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut) with or without Cholesin injection. The expression level of HSP90 was used as an internal control.

FIG. 22D : Total cholesterol levels in the liver of wild-type (Gpr146 ^fl/fl ) mice and Gpr146 knockout (Gpr146 LKO) mice injected with adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut), with or without Cholesin injection.

22E : Total cholesterol levels in plasma of wild-type (Gpr146 ^fl/fl ) mice and Gpr146 knockout (Gpr146 LKO) mice injected with adenovirus control (Vec), adenovirus expressing wild-type GPR146 (WT), or adenovirus expressing mutant GPR146 (Mut), with or without Cholesin injection.

FIG. 23 : Schematic diagram of the dosing regimen for Ldlr knockout (Ldlr ^−/− ) mice.

FIG. 24A : Changes in plasma total cholesterol levels in Ldlr ^−/− mice under different dosing regimens and statistics of the area under the curve (AUC).

FIG. 24B : Total cholesterol levels in the liver of Ldlr ^−/− mice after 8 weeks of drug administration under different dosing regimens.

FIG. 24C : Changes in plasma triglyceride levels in Ldlr ^−/− mice under different dosing regimens and statistics of the area under the curve (AUC).

FIG. 24D : Hepatic triglyceride levels in Ldlr ^−/− mice after 8 weeks of drug administration under different dosing regimens.

FIG. 25A : mRNA levels of genes related to cholesterol synthesis in the liver of Ldlr ^−/− mice after 8 weeks of drug administration under different drug administration regimens.

FIG. 25B : Expression levels of liver cholesterol synthesis-related genes Hmgcs1, Hmgcr and transcription factor SREBP2 in Ldlr ^-/- mice after 8 weeks of drug administration under different dosing regimens.

Figure 26: Oil red staining of the whole aorta of Ldlr ^-/- mice after 8 weeks of drug administration under different drug administration regimens and their statistical results.

FIG. 27A : Body weight changes of Ldlr ^−/− mice under different dosing regimens.

FIG. 27B : Changes in food intake of Ldlr ^−/− mice under different dosing regimens.

Figure 28: HE staining of liver tissue of Ldlr ^-/- mice after 8 weeks of drug administration under different dosing regimens.

FIG. 29A : Plasma alanine aminotransferase (ALT) levels in Ldlr ^−/− mice after 8 weeks of drug administration under different dosing regimens.

FIG. 29B : Plasma aspartate aminotransferase (AST) levels in Ldlr ^−/− mice after 8 weeks of administration under different dosing regimens.

Figure 30: Schematic diagram of the mechanism by which Cholesin regulates cholesterol metabolism through its receptor GPR146.

DETAILED DESCRIPTION

The above-mentioned features and advantages of the present invention and their additional features and advantages will be more clearly understood below by combining the accompanying drawings and the detailed description of the following embodiments. The embodiments described herein with reference to the accompanying drawings are explanatory, illustrative, and are used for a general understanding of the present invention. The embodiments should not be interpreted as limiting the scope of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For example, terms used herein such as "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Definitions as described in H. E., ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, the terms "a," "an," "one or more," and "at least one" may be used interchangeably. Similarly, the terms "comprising," "including," and "having" may be used interchangeably.

When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. For the purpose of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or containing at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists of only these embodiments.

As used herein, the term "and/or" is considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in phrases such as "A and/or B" is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The ranges listed in this article should be understood to include all values within the range (including the listed endpoint values). The range of 1 to 100 is understood to include any value, combination of values, or sub-range therebetween.

As used herein, the term "Cholesin" or "Cholesin protein" is a homolog of the unknown human protein C7orf50. The human Cholesin gene is located in the p22 region of human chromosome 7. The gene encoding human Cholesin has a gene ID of 84310 in NCBI, but its specific function has not yet been reported. The inventors discovered its inhibitory effect on cholesterol synthesis through characterization of the protein, so it was named "Cholesin". The amino acid sequence of Cholesin protein is highly conserved in various species. The amino acid sequences of Cholesin proteins in different species are as follows:

Homo sapiens (Gene ID 84310)

Mouse (Mus musculus) (Gene ID 73212)

Rhesus monkey (Macaca mulatta) (Gene ID 696867)

Domestic cattle (Bos taurus) (Gene ID 522840)

Brown rat (Rattus norvegicus) (Gene ID 498154)

Junglefowl (Gallus gallus) (Gene ID 416455)

In this article, Cholesin protein should be understood in its broadest sense, including wild-type, isolated or purified, or recombinant Cholesin protein, its variants or derivatives, as long as the desired biological activity (e.g., activity of reducing plasma cholesterol level and/or triglyceride level in a subject) is retained. Variants or derivatives of Cholesin protein encompass, for example, variants or derivatives of Cholesin protein described herein that retain the ability to reduce plasma cholesterol level and/or triglyceride level in a subject to a degree similar to that of wild-type Cholesin protein, to the same degree as that of wild-type Cholesin protein, or to a degree higher than that of wild-type Cholesin protein. The variant or derivative of the Cholesin protein retains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% of the biological activity of the wild-type Cholesin protein, or has a higher biological activity than the wild-type Cholesin protein, particularly the activity of reducing the plasma cholesterol level and/or triglyceride level in a subject.

Variants of Cholesin protein include polypeptides that have significant sequence identity to wild-type Cholesin protein and retain the biological activity of Cholesin protein (e.g., activity that reduces plasma cholesterol levels and/or triglyceride levels in a subject). Variants may have one or more amino acid mutations compared to wild-type Cholesin protein. Mutations may include, for example, amino acid insertions, deletions, or substitutions. The amino acid sequence of the variant may, for example, have at least about 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 98.2%, 98.4%, 98.6%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher identity to the amino acid sequence of wild-type Cholesin protein.

Derivatives of cholesin proteins generally refer to those that have been chemically modified, especially by one or more substituents. Covalently linked compounds prepared from wild-type Cholesin protein or its variants. Therefore, the derivatives of Cholesin protein refer to chemically modified Cholesin protein or its variants. Typical derivatives can be, for example, amidated, glycosylated, alkylated, acylated, esterified, pegylated Cholesin protein or its variants.

Herein, "a step of enhancing the expression or activity of an endogenous Cholesin protein in a subject", "a method of enhancing the expression or activity of an endogenous Cholesin protein in a subject" and "an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject" refer to any method or substance that can enhance the stability or expression of a nucleic acid encoding a Cholesin protein, enhance the transcription and translation of Cholesin, increase the level of Cholesin protein, enhance the activity of Cholesin protein, enhance the stability of Cholesin protein, and increase the effective action time of Cholesin protein. For example, an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject can be a small molecule, a protein, a polypeptide, a nucleic acid, etc.

The term "GPR146" as used herein is a G protein-coupled receptor (GPCR) that is involved in the regulation of cholesterol metabolism and inhibits cholesterol synthesis controlled by PKA signaling and SREBP2. Previous studies have found that GPR146 regulates the secretion of very low-density lipoprotein (VLDL) in the liver by activating the extracellular signal-regulated kinase (ERK) signaling pathway and promoting the activity of liver sterol regulatory element binding protein 2 (SREBP2), thereby regulating the levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in the circulation. The GPR146/ERK axis has a regulatory role in systemic cholesterol metabolism, and inhibiting GPR146 may be an effective strategy to reduce plasma cholesterol levels and atherosclerosis.

The human GPR146 gene ID is 115330, and the amino acid sequence is: MWSCSWFNGTGLVEELPACQDLQLGLSLLSLLGLVVGVPVGLCYNALLVLANLHSKASMTMPDVYFVNMAVAGLVLSALAPVHLLGPPSSRWALWSVGGEVHVALQIPFNVSSLVAMYSTALLSLDHYIERALPRTYMASVYNTRHVCGFVWGGALLTS FSSLLFYICSHVSTRALECAKMQNAEAADATLVFIGYVVPALATLYALVLLSRVRREDTPLDRDTGRLEPSAHRLLVATVCTQFGLWTPHYLILLGHTVIISRGKPVDAHYLGLLHFVKDFSKLLAFSSSFVTPLLYRYMNQSFPSKLQRLMKKLPCGDRHCSPDHMGVQQVLA(SEQ ID NO:7)

The mouse GPR146 gene ID is 80290, and the amino acid sequence is: MWSCGPLNSTAWAEEPLCRNLRLGLWVLSLLYLGAGVPVSLGYNALLVLANLASKNTMTMPDVYFVNMAVAGLVLTALAPAYLLGPAHSRWALWSLSSEAHVTLLILFNVASLVTMYSTALLSLDYYIERALPRTYMASVYNTRHVCGFVWGGAVLTSFS SLLFYICSHVSSRIAECARMQNTEAADAILVLIGYVVPGLAVLYALALISRIGKEDTPLDQDTSRLDPSVHRLLVATVCTQFGLWTPYYLSLGHTVLTSRGRTVEGHYLGILQVAKDLAKFLAFSSSSVTPLLYRYINKAFPGKLRRLMKKMHCGRRHCSPDPSGIQQVMAQA(SEQ ID NO: 8).

Cholesterol is the main component of cell membranes and is also the main component of the synthesis of adrenal cortex hormones, sex hormones, bile acids and vitamins. The concentration of cholesterol (including total cholesterol and/or low-density lipoprotein cholesterol) in human blood can be used as an indicator of lipid metabolism to assess the risk of cardiovascular and cerebrovascular diseases, hyperlipidemia, atherosclerosis and other diseases.

Cholesterol often exists in the form of lipoproteins in the blood. Lipoproteins are divided into five types according to particle size and density, namely chylomicrons, very low-density lipoproteins, intermediate-density lipoproteins, low-density lipoproteins and high-density lipoproteins. The very low-density lipoproteins synthesized by the liver transport triglycerides into the blood, where they are broken down by lipase to produce very low-density lipoprotein remnants, also called intermediate-density lipoproteins. Part of the intermediate-density lipoproteins is metabolized by the liver; the other part is further converted into low-density lipoproteins by lipase and hepatic lipase in the blood. Low-density lipoproteins act on the whole body and are used by binding to low-density lipoprotein receptors of cells and tissues.

Low-density lipoprotein (LDL) in plasma is the main carrier for transporting endogenous cholesterol, which is degraded and converted by binding to the low-density lipoprotein receptor (LDLR) on its cell membrane. LDLR functional defects will reduce the clearance ability of plasma LDL, ultimately leading to the formation of atherosclerotic plaques in the arterial intima. Low-density lipoprotein cholesterol (LDL-C) is the main lipoprotein in fasting plasma, accounting for about 2/3 of plasma lipoproteins, and is the main carrier for transporting cholesterol to extrahepatic tissues. Therefore, the content of LDL-C is related to the incidence and degree of cardiovascular disease, hyperlipidemia, atherosclerosis and other diseases. It is considered to be the main pathogenic factor of atherosclerosis. Its concentration has a significant positive correlation with the incidence of coronary heart disease and is also an important indicator for evaluating the risk factors for the occurrence of coronary heart disease in individuals. In this article, "LDL" and "LDL-C" can be used interchangeably.

Very low-density lipoprotein (VLDL) mainly contains triglycerides, cholesterol and lipoproteins. VLDL is synthesized in the liver and transports triglycerides and cholesterol to tissues through the blood. Since this type of lipoprotein carries a relatively small amount of cholesterol and their particles are relatively large, it is not easy to penetrate the vascular endothelium. Therefore, normal very low-density lipoprotein generally does not cause atherosclerosis. However, since triglycerides account for 50%-70% and cholesterol accounts for 8%-12% in very low-density lipoprotein, once the level of very low-density lipoprotein increases significantly, in addition to the increase in triglycerides in the plasma, the cholesterol level also increases, which has the effect of causing atherosclerosis. In this article, "VLDL" and "VLDL-C" can be used interchangeably.

Intermediate density lipoprotein (IDL) is mainly an intermediate metabolite of very low density lipoprotein (VLDL), so it is also called residual VLDL. IDL can also be directly secreted by the liver, but its amount is very small. It is the last lipoprotein to be decomposed by lipoprotein lipase (LPL) among lipoproteins containing apolipoprotein B.

High-density lipoprotein (HDL) is a group of plasma lipoproteins with the smallest diameter but the highest density. In normal plasma, most HDL particles are spherical and consist of a hydrophobic core surrounded by phospholipids, unacylated cholesterol, and apolipoproteins. High-density lipoprotein cholesterol (HDL-C) can transport cholesterol from extrahepatic tissues to the liver for metabolism and excretion from the body by bile. In this article, "HDL" and "HDL-C" can be used interchangeably.

Chylomicrons (CM) are the largest lipoprotein particles in plasma and are the main lipoproteins for transporting exogenous triglycerides. Cholesterol and triglycerides contained in food are absorbed in the small intestine, through the lymphatic vessels, and absorbed through chylomicrons. After entering the blood, chylomicrons will become mature chylomicrons and, under the action of lipase, will be converted into free fatty acids. Free fatty acids are converted into energy by the human body on the one hand, and are also further synthesized and stored in fat.

Cholesterol synthesis occurs mainly in the liver. Cholesterol is generated from acetyl CoA under the control of a series of enzymes. Among them, HMGCR (3-hydroxy-3-methylglutaryl CoA reductase) is the rate-limiting enzyme, and its activity is controlled at the transcriptional and post-transcriptional levels. SREBP2 (sterol regulatory element binding protein 2) acts as a master transcription factor, dominating the expression of HMGCR and other genes involved in cholesterol synthesis and uptake. Cholesterol biosynthesis and uptake are strictly regulated by a negative feedback mechanism that senses the cholesterol level of the cell. When cells are cholesterol-deficient, SREBP2, together with its escort protein SCAP (SREBP cleavage-activating protein), is transported from the endoplasmic reticulum (ER) to the Golgi apparatus in COPII vesicles. In the Golgi apparatus, SREBP2 is cleaved by site 1 and site 2 proteases in sequence. The N-terminal structure of SREBP2 released by this cleavage travels to the nucleus, where it acts as a transcription factor, enhancing the expression of genes involved in cholesterol synthesis (e.g., Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, and Sqle). Conversely, when cellular cholesterol levels rise, cholesterol molecules bind to SCAP, triggering its interaction with INSIG (insulin-induced gene). This interaction retains SREBP in the ER and prevents subsequent activation of SREBP and expression of genes involved in cholesterol metabolism. This mechanism helps maintain cholesterol homeostasis.

Both endogenously synthesized and exogenously acquired cholesterol are secreted into the blood in the form of very low-density lipoprotein (VLDL). After processing in the blood, VLDL generates circulating LDL, which can be taken up by cells via LDL receptor (LDLR)-mediated endocytosis.

As used herein, the term "expression vector" is a nucleic acid molecule used as a medium for transferring (exogenous) genetic material into a host cell, where the nucleic acid molecule as a vector can, for example, be replicated and/or expressed. The term "expression vector" encompasses, but is not limited to, plasmids, viral vectors (including, for example, retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including, for example, BACs and YACs).

As used herein, the term "treatment" includes therapeutic or prophylactic treatment in a subject in need thereof. "Therapeutic or prophylactic treatment" includes prophylactic treatment aimed at completely preventing clinical and/or pathological manifestations or therapeutic treatment aimed at improving or alleviating clinical and/or pathological manifestations. Therefore, the term "treatment" also includes ameliorating or preventing a disease.

As used herein, the term "effective amount" means an amount of a therapeutic agent that, when administered to a subject for the treatment or prevention of a disease, is sufficient to achieve such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for prophylactic treatment.

As used herein, the terms "subject" or "individual" or "animal" or "patient" are used interchangeably herein and refer to any subject in need of treatment, particularly a mammalian subject. Generally speaking, mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

As used herein, the term "disease associated with elevated plasma cholesterol levels" refers to a disease, condition or disorder caused by elevated plasma cholesterol levels, which includes but is not limited to diseases caused by dyslipidemia and lipodystrophy-related diseases.

Dyslipidemia is an abnormal metabolism of lipoproteins in the human body, mainly including elevated total cholesterol, low-density lipoprotein cholesterol, triglycerides and/or reduced high-density lipoprotein cholesterol, etc. Dyslipidemia can lead to hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease, angina pectoris, malignant arrhythmia), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, liver failure, nephrotic syndrome, gallstones, pancreatitis and other diseases or symptoms.

Diseases related to fat metabolism disorder are a general term for related diseases caused by fat metabolism disorders, including, for example: hyperlipidemia, hypertension, atherosclerosis, obesity, fatty liver, cirrhosis, coronary heart disease, angina pectoris, myocardial infarction, inflammatory bowel disease, indigestion and gastrointestinal ulcers.

Hyperlipidemia usually refers to elevated levels of plasma cholesterol and/or triglycerides. Hyperlipidemia is a manifestation of abnormal fat metabolism in the human body and is mainly divided into three categories: hypercholesterolemia, hypertriglyceridemia, and mixed hyperlipidemia. Among them, hypercholesterolemia refers to elevated levels of total plasma cholesterol or low-density lipoprotein cholesterol. Hypercholesterolemia can lead to various life-threatening cardiovascular diseases, atherosclerosis and other complications.

Atherosclerosis is the most common and important type of vascular disease of arteriosclerosis. Lipid metabolism disorder is the pathological basis of atherosclerosis, and its characteristic is that the lesions of the affected arteries start from the intima. Generally, there is accumulation of lipids and complex carbohydrates, bleeding and thrombosis, fibrosis and calcification, and gradual degeneration and calcification of the middle layer of the artery. The lesions often involve elastic and large and medium muscular arteries. Once they develop to the point of blocking the arterial lumen, the tissues or organs supplied by the artery will be ischemic or necrotic. Because the lipids accumulated in the intima of the artery appear yellow and porridge-like, it is called atherosclerosis. Atherosclerosis is often accompanied by diseases such as hypertension, diabetes, and obesity.

As used herein, the term "HMGCR inhibitor" is also referred to as an HMG-CoA reductase inhibitor, and refers to a compound capable of inhibiting HMG-CoA reductase. It inhibits the biosynthesis of isoprenoids by inhibiting HMG-CoA reductase, blocks the isoprenylation of proteins, and thereby reduces plasma cholesterol levels.

As used herein, the term "PCSK9 inhibitor" refers to a class of compounds that inhibit PCSK9 (the ninth member of the Kexin-like proconvertase subtilisin family). PCSK9 is a liver-derived secretory protein that can reduce the number of LDLRs on hepatocytes, affect LDL internalization, and prevent LDL from being cleared from the blood, thereby leading to hypercholesterolemia. Therefore, inhibiting PCSK9 can stabilize LDLR and promote the liver's absorption of LDL, thereby reducing the level of LDL in the blood.

As used herein, the term "cholesterol absorption inhibitor" refers to a compound that is capable of inhibiting the absorption of cholesterol, for example, by inhibiting NPC1L1-mediated endocytosis, thereby blocking the uptake of cholesterol.

The term "composition" refers in particular to a composition suitable for administration to humans. However, the term also generally encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e., active agent and optional carrier or excipient) are preferably pharmaceutically acceptable, i.e., capable of inducing the desired therapeutic effect in the recipient without causing any undesirable local or systemic effects.

The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially toxic to the recipient thereof, and/or such carrier or excipient is approved or available for inclusion in the composition.

The inventors unexpectedly discovered that Cholesin is a hormone secreted by the intestine in response to cholesterol absorption, which can inhibit cholesterol synthesis and VLDL secretion in the liver, thereby reducing the levels of plasma cholesterol and/or triglycerides to prevent diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Mechanistic studies have shown that Cholesin inhibits cholesterol synthesis controlled by PKA signaling and SREBP2 by binding to its receptor GPR146. Cholesin has an LDLR-independent downregulation effect on the expression of genes involved in cholesterol synthesis and reduces plasma cholesterol levels, which means that Cholesin treatment is an effective strategy to combat diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. Therefore, exogenous Cholesin or methods or agents that promote the expression or activity of endogenous Cholesin proteins in subjects can reduce the levels of plasma cholesterol and/or triglycerides, thereby preventing or treating diseases associated with elevated plasma cholesterol levels, such as hypercholesterolemia and atherosclerosis. In addition, combining exogenous Cholesin or methods or agents that promote the expression or activity of endogenous Cholesin protein in a subject with existing cholesterol-lowering drugs such as statins is also a promising strategy for treating diseases associated with elevated plasma cholesterol levels, including hyperlipidemia and atherosclerosis.

Accordingly, in one aspect, the present invention provides a method for lowering plasma cholesterol level and/or triglyceride level in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In some embodiments, plasma cholesterol is selected from plasma total cholesterol and plasma LDL-cholesterol. In some embodiments, plasma cholesterol is plasma total cholesterol. In some embodiments, plasma cholesterol is plasma LDL-cholesterol. In some embodiments, plasma cholesterol is plasma total cholesterol and plasma LDL-cholesterol.

In another aspect, the present invention provides a method for inhibiting liver cholesterol synthesis in a subject, comprising administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or a nucleic acid comprising the Cholesin protein. The step of providing an expression vector for the subject, or the step of enhancing the expression or activity of the endogenous Cholesin protein of the subject.

In some embodiments, the method inhibits the expression of genes related to cholesterol synthesis. In some embodiments, the genes related to cholesterol synthesis are selected from one or more of Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk and Sqle. In some embodiments, the gene related to cholesterol synthesis is Hmgcr. In some embodiments, the gene related to cholesterol synthesis is Hmgcs1. In some embodiments, the gene related to cholesterol synthesis is Mvd. In some embodiments, the gene related to cholesterol synthesis is Mvk. In some embodiments, the gene related to cholesterol synthesis is Pmvk. In some embodiments, the gene related to cholesterol synthesis is Sqle. In some embodiments, the gene related to cholesterol synthesis is Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk and Sqle.

In some embodiments, the method inhibits the expression of a gene associated with cholesterol uptake. In some embodiments, the gene associated with cholesterol synthesis and uptake is Ldlr.

In some embodiments, the method inhibits the expression of genes associated with cholesterol synthesis and uptake. In some embodiments, the genes associated with cholesterol synthesis are Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk and Sqle; and the gene associated with cholesterol synthesis and uptake is Ldlr.

In some embodiments, the method inhibits the expression of transcription factors that promote the expression of cholesterol synthesis-related genes. In some embodiments, the method inhibits the expression of transcription factors that promote the expression of cholesterol uptake-related genes. In some embodiments, the transcription factor that promotes the expression of cholesterol synthesis and/or uptake-related genes is SREBP2.

In yet another aspect, the present invention provides a method for inhibiting hepatic VLDL-cholesterol secretion in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In another aspect, the present invention provides a method for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein.

In an embodiment of the method of the present invention, the Cholesin protein is isolated or purified. In some embodiments, the Cholesin protein is a mammalian Cholesin protein. Examples of mammalian Cholesin proteins include but are not limited to Cholesin proteins of humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows. In some embodiments, the Cholesin protein is a non-human primate Cholesin protein. In some embodiments, the Cholesin protein is a human Cholesin protein. In some embodiments, the human Cholesin protein comprises an amino acid sequence as shown in SEQ ID NO:1.

In other embodiments, the Cholesin protein is a mouse Cholesin protein. In some embodiments, the mouse Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:2.

In other embodiments, the Cholesin protein is a macaque Cholesin protein. In some embodiments, the macaque Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:3.

In other embodiments, the Cholesin protein is a bovine Cholesin protein. In some embodiments, the bovine Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:4.

In other embodiments, the Cholesin protein is a Rattus norvegicus Cholesin protein. In some embodiments, the Rattus norvegicus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:5.

In other embodiments, the Cholesin protein is a Gallus gallus Cholesin protein. In some embodiments, the Gallus gallus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid encodes human Cholesin protein.

In some embodiments, the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

The expression vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgene) and a larger sequence as a vector "backbone". Engineered vectors usually include an origin of autonomous replication in a host cell (if stable expression of the polynucleotide is desired), a selection marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally include a promoter, a genetic marker, a reporter gene, a targeting sequence, and/or a protein purification tag. As known to those skilled in the art, a large number of suitable expression vectors are known to those skilled in the art, and many are commercially available.

The Cholesin protein disclosed herein, the nucleic acid encoding the Cholesin protein, or the expression vector comprising the nucleic acid, or the agent for enhancing the expression or activity of the endogenous Cholesin protein of a subject can be used for treating and/or preventing diseases associated with elevated plasma cholesterol levels, i.e., diseases, conditions or disorders induced by elevated plasma cholesterol levels, for example, for treating and/or preventing:

1. Dyslipidemia and its sequelae, such as atherosclerosis, cardiovascular and cerebrovascular diseases, etc., especially those diseases characterized by one or more of the following factors (but not limited to):

- high plasma total cholesterol concentration,

- High plasma triglyceride concentrations, high postprandial plasma triglyceride concentrations,

- low HDL cholesterol concentration,

- low ApoA lipoprotein concentration,

- high LDL cholesterol concentration,

- low density LDL cholesterol particles,

- High ApoB lipoprotein concentration;

2. A variety of other conditions that may be associated with metabolic syndrome, such as:

- Obesity (overweight), including central obesity,

-Thrombosis, hypercoagulability and prothrombotic states (arterial and venous),

-hypertension,

- Heart failure, such as (but not limited to) heart failure following myocardial infarction, hypertensive heart disease or cardiomyopathy;

3. Lipodystrophy

- Hyperlipidemia,

- Atherosclerosis,

-hypertension,

- Obesity,

- Fatty liver, cirrhosis, liver failure,

4. Diabetes, especially type 2 diabetes, including its related sequelae

Specifically, it involves:

- Diabetes and diabetic complications, such as diabetic vascular disease, such as damage to large and small blood vessels leading to lesions of the heart, brain, kidneys, peripheral nerves, eyes, feet and other tissues and organs, including diabetic eye disease, diabetic heart disease, diabetic nephropathy, diabetic neuropathy and distal limb necrosis of the lower limbs, etc.

- Diseases that are common in diabetes, occur with diabetes, or are aggravated by diabetes, such as atherosclerosis, hypertension, coronary heart disease, myocardial infarction, cerebral thrombosis, cerebral hemorrhage, cerebral embolism, osteoporosis, etc.,

- Fat metabolism disorders and related diseases that are common in diabetes, occur with diabetes, or are aggravated by diabetes, including hyperlipidemia, hypertension, atherosclerosis, obesity, fatty liver, and cirrhosis;

5. Diseases or conditions involving inflammatory responses:

- atherosclerosis, such as (but not limited to) coronary artery atherosclerosis, including angina pectoris or myocardial infarction, stroke,

- Restenosis or reocclusion of the blood vessel,

- Chronic inflammatory bowel disease, such as Crohn's disease and ulcerative colitis,

-asthma,

- Other inflammatory conditions;

6. Other obstacles:

- Nephrotic syndrome,

-pancreatitis,

- Gallstones,

- vasculitis,

-Ischemia/reperfusion syndrome.

In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones.

In a preferred embodiment, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In other preferred embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

In some embodiments, the method further comprises the step of administering a second therapeutic agent. In a preferred embodiment, the second therapeutic agent is selected from a protein or peptide, a nucleic acid, and a small molecule drug.

In some embodiments, the second therapeutic agent is a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is selected from: HMGCR inhibitors, PCSK9 inhibitors, and cholesterol absorption inhibitors.

In some embodiments, the cholesterol-lowering drug is an HMGCR inhibitor. In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin. In a preferred embodiment, the second therapeutic agent is rosuvastatin.

In some embodiments, the cholesterol-lowering drug is a PCSK9 inhibitor. In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab. In some embodiments, the second therapeutic agent is Evolocumab. In some embodiments, the second therapeutic agent is Alirocumab. In some embodiments, the second therapeutic agent is Inclisiran. In some embodiments, the second therapeutic agent is Tafolecimab.

Evolocumab (Evolocumab, trade name ) is a fully human IgG2 monoclonal antibody. Evolocumab can bind to PCSK9 and inhibit the binding of circulating PCSK9 to the low-density lipoprotein receptor (LDLR), thereby preventing PCSK9-mediated low-density lipoprotein receptor degradation. Its approved indications are hypercholesterolemia and mixed dyslipidemia.

Alirocumab (Alirocumab, trade name ) is a fully human IgG1 monoclonal antibody that binds to PCSK9 and inhibits the binding of circulating PCSK9 to the low-density lipoprotein receptor (LDLR), thereby preventing PCSK9-mediated degradation of the low-density lipoprotein receptor. The drug is used to treat adults with heterozygous familial hypercholesterolemia and clinical atherosclerotic cardiovascular disease (e.g., heart disease or stroke requiring low-density cholesterol reduction).

Inclisiran (Inclisiran, trade name ) is a small interfering nucleic acid (siRNA) drug targeting PCSK9 for lowering low-density lipoprotein cholesterol. It has been approved for the treatment of adult patients with primary hypercholesterolemia (heterozygous familial and non-familial) or mixed dyslipidemia.

Tafolecimab (Tafolecimab, trade name ) is a fully human IgG2 monoclonal antibody. It can specifically bind to the PCSK9 molecule, increase the low-density lipoprotein receptor (LDLR) level by reducing PCSK9-mediated endocytosis of the LDLR, and then increase the clearance of low-density lipoprotein cholesterol (LDL-C) and reduce the LDL-C level. It is used for adult patients with primary hypercholesterolemia (including heterozygous familial and non-familial hypercholesterolemia) and mixed dyslipidemia who are unable to achieve the LDL-C target after receiving moderate or higher doses of statins, to reduce LDL-C, total cholesterol (TC) and apolipoprotein B (ApoB) levels.

In some embodiments, the cholesterol-lowering drug is a cholesterol absorption inhibitor. In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor. NPC1L1 inhibitors target the cholesterol transporter NPC1L1, and inhibit the intracellular transport of cholesterol by acting on the key transporter NPC1L1 for cholesterol uptake and absorption on the brush border of the small intestinal villi, thereby reducing the absorption of cholesterol in food and bile in the small intestine, hindering the transport pathway of cholesterol from the small intestine to the liver, reducing the cholesterol storage in the liver, and accelerating the clearance of cholesterol.

In some embodiments, the NPC1L1 inhibitor is selected from Ezetimibe and Hybutimibe. In some embodiments, the second therapeutic agent is Ezetimibe. In some embodiments, the second therapeutic agent is Hybutimibe.

In some embodiments, the second therapeutic agent is administered before, after, or simultaneously with the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein. In some embodiments, the second therapeutic agent is administered before the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein. In some embodiments, the second therapeutic agent is administered after the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein. In some embodiments, the second therapeutic agent is administered simultaneously with the Cholesin protein, the nucleic acid, or the expression vector, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein.

It should be recognized that treatment may require a single administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of an active agent of the invention. For example, depending on the type, half-life, and clearance rate of the active agent, the active agent may be administered one to three times a day, once every two days, once every 3 to 4 days, once a week, or once every two weeks, or once in a month.

The active agents disclosed herein (Cholesin protein, nucleic acid encoding Cholesin protein, or expression vector comprising the nucleic acid, or agent that enhances the expression or activity of endogenous Cholesin protein of a subject) can be applied to administration by various routes. Typically, administration is completed parenterally. Parenteral delivery methods include topical, intravenous, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is a human. In some embodiments, the subject is resistant to cholesterol absorption inhibitors, HMGCR inhibitors, and PCSK9 inhibitors. The subject may be unresponsive or intolerant to one or more of the agents, for example, the subject has an LDL receptor (LDLR) defect.

The effective amount of the Cholesin protein disclosed herein, the nucleic acid encoding the Cholesin protein, or the expression vector comprising the nucleic acid, or the agent that enhances the expression or activity of the endogenous Cholesin protein of the subject can be determined by those skilled in the art using known techniques. The appropriate dosage provides a sufficient amount of the active agent of the present invention, and is preferably therapeutically effective, that is, sufficient to cause, for example, a therapeutic or preventive response in the subject or animal within a reasonable time frame. For example, the dosage of the Cholesin protein of the present invention, the nucleic acid encoding the Cholesin protein, or the expression vector comprising the nucleic acid, or the agent that enhances the expression or activity of the endogenous Cholesin protein of the subject should be sufficient to produce a therapeutic or preventive response within a period of about 20 minutes, 30 minutes, 1 hour, 2 hours or longer, such as 12 hours to 24 hours or longer (e.g., 1 month, 2 months, 3 months, 6 months, 12 months, 24 months, etc.) from the time of administration. In certain embodiments, the time period can be even longer. As is known in the art, adjustments for the route, timing and frequency of administration, timing and frequency of administration of formulations, age, weight, general health, sex, diet, severity of the disease state, drug combinations, reaction sensitivities and tolerance/response to treatment may be necessary for therapeutic purposes.

Many assays for determining the dosage to be administered are known in the art. Typically, the attending physician determines the dosage of the disclosed active agent to be administered to each individual patient, taking into account a variety of factors such as age, weight, general health, diet, sex, the active agent to be administered, the route of administration, and the severity of the condition being treated.

In another aspect, the present invention provides a composition comprising a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of human endogenous Cholesin protein, and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the composition is used for one or more of the following:

1) reducing plasma cholesterol levels in a subject;

2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject;

3) reducing plasma triglyceride levels in a subject;

4) inhibiting hepatic cholesterol synthesis in a subject;

5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and

6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

In embodiments of the compositions of the present invention, the Cholesin protein is isolated or purified. In some embodiments, the Cholesin protein is a mammalian Cholesin protein. Examples of mammalian Cholesin proteins include, but are not limited to, Cholesin proteins of humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cattle. In some embodiments, the Cholesin protein is a non-human primate Cholesin protein. In some embodiments, the Cholesin protein is a human Cholesin protein. In some embodiments, the human Cholesin protein comprises the amino acid sequence shown in SEQ ID NO: 1.

In other embodiments, the Cholesin protein is a mouse Cholesin protein. In some embodiments, the mouse Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:2. In other embodiments, the Cholesin protein is a macaque Cholesin protein. In some embodiments, the macaque Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:3. In other embodiments, the Cholesin protein is a bovine Cholesin protein. In some embodiments, the bovine Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:4. In other embodiments, the Cholesin protein is a Rattus norvegicus Cholesin protein. In some embodiments, the Rattus norvegicus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:5. In other embodiments, the Cholesin protein is a Gallus gallus Cholesin protein. In some embodiments, the Gallus gallus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the composition further comprises a second therapeutic agent. In a preferred embodiment, the second therapeutic agent is selected from a protein or peptide, a nucleic acid, and a small molecule drug.

In some embodiments, the second therapeutic agent is a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is selected from: HMGCR inhibitors, PCSK9 inhibitors, and cholesterol absorption inhibitors.

In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin. In a preferred embodiment, the second therapeutic agent is rosuvastatin.

In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe. In a preferred embodiment, the second therapeutic agent is ezetimibe.

Depending on the active agent used (such as Cholesin protein), the compositions of the present disclosure can be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, for example, in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. The processes known in the present invention for producing drugs are shown in the 22nd edition of Remington’s Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012) and may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. Compositions comprising, for example, the Cholesin protein described herein, an expression vector encoding the Cholesin protein, or comprising the nucleic acid, or an agent for enhancing the expression or activity of an endogenous Cholesin protein of a subject are generally provided in liquid form, and preferably contain a pharmaceutically acceptable buffer. The liquid preparation may be a solution or a suspension.

In some embodiments, the composition of the present invention is formulated as an injection preparation or an oral preparation. Examples of oral preparations include, but are not limited to, tablets, capsules, granules, suspensions. In some embodiments, the composition of the present invention It can be used for various routes of administration, such as parenteral administration. In some embodiments, the composition of the present invention can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally, etc. In some embodiments, the composition of the present invention can be administered orally.

In the compositions of the present invention, examples of carriers and excipients include, but are not limited to, fillers, adhesives, disintegrants, coating agents, adsorbents, antiadhesives, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, diluents, preservatives, emulsifiers, stabilizers and tension regulators. It is known to those skilled in the art that suitable excipients are selected to prepare the compositions of the present invention. Exemplary carriers used in the compositions of the present invention include saline, buffered saline, glucose and water. Typically, the selection of suitable excipients depends especially on the desired dosage form of the active agent used, the disease to be treated and the compositions.

In another aspect, the present invention provides the use of a composition as described herein in the preparation of a medicament for one or more of:

1) reducing plasma cholesterol levels in a subject;

2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject;

3) reducing plasma triglyceride levels in a subject;

4) inhibiting hepatic cholesterol synthesis in a subject;

5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and

6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

In yet another aspect, the present invention provides a method for treating a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of the composition of the present invention.

In another aspect, the present invention provides a method for diagnosing a disease associated with elevated plasma cholesterol levels in a subject, comprising the steps of:

a. obtaining a blood sample from the subject,

b. determining the level of Cholesin protein in the sample,

Wherein an increased level of Cholesin protein in the sample compared to a healthy control indicates that the subject is at risk for a disease associated with increased plasma cholesterol levels.

In an embodiment of the diagnostic method of the present invention, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones.

In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

In some embodiments, the blood sample is selected from blood, serum and plasma. In certain embodiments, the method is performed in vitro. In some embodiments, the method further comprises the step of isolating Cholesin protein from the sample. Methods for isolating Cholesin protein are known in the art.

Methods for determining the level of Cholesin protein in a sample are known in the art. For example, in step b, the level of Cholesin protein in the sample can be determined by a method selected from the following methods: mass spectrometry, fluorescence detection, chemiluminescence, ELISA, Western blotting, radioimmunoassay, immunohistochemical staining, multiple immunoassay and dot blot assay.

In some embodiments, the level of Cholesin protein in the sample can be determined by anti-Cholesin protein antibodies. The antibody is preferably a monoclonal antibody, but can also be Fab, Fab', (Fab') ₂ , Fv, scFv, bi-scFv, bispecific antibody, trispecific antibody, tetraspecific antibody, etc.

In yet another aspect, the present invention provides use of a reagent for determining the level of Cholesin protein in a blood sample from a subject in the preparation of a kit for diagnosing a disease associated with elevated plasma cholesterol levels in the subject.

In another aspect, the present invention provides a kit for diagnosing a disease associated with elevated plasma cholesterol levels in a subject, comprising reagents for determining the level of Cholesin protein in a blood sample from the subject.

In the embodiments of the diagnostic use and kit of the present invention, the reagent is used to determine the level of Cholesin protein in the sample in a method selected from the following: mass spectrometry, fluorescence detection, chemiluminescence, ELISA, Western blot, radioimmunoassay, immunohistochemical staining, multiple immunoassay and dot blot assay. In some embodiments, the reagent is an anti-Cholesin protein antibody. The antibody is preferably a monoclonal antibody, but can also be Fab, Fab', (Fab') ₂ , Fv, scFv, bi-scFv, bispecific antibody, trispecific antibody, tetraspecific antibody, etc.

In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones. In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

In some embodiments, the blood sample is selected from the group consisting of blood, serum, and plasma.

In another aspect, the present disclosure provides a method for preparing a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject. Use in a composition for reducing plasma cholesterol and/or triglyceride levels in a subject. In some embodiments, the plasma cholesterol is plasma total cholesterol or plasma LDL-cholesterol.

In yet another aspect, the present disclosure provides use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in the preparation of a composition for inhibiting liver cholesterol synthesis in a subject.

In yet another aspect, the present disclosure provides use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in the preparation of a composition for inhibiting hepatic VLDL-cholesterol secretion in a subject.

In yet another aspect, the present disclosure provides use of a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject in the preparation of a composition for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

In another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject, for use in reducing plasma cholesterol and/or triglyceride levels in a subject. In some embodiments, plasma cholesterol is plasma total cholesterol or plasma LDL-cholesterol.

In yet another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent that enhances the expression or activity of an endogenous Cholesin protein in a subject, for use in inhibiting liver cholesterol synthesis in a subject.

In yet another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject, for use in inhibiting hepatic VLDL-cholesterol secretion in a subject.

In another aspect, the present disclosure provides a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of an endogenous Cholesin protein in a subject, for use in treating or preventing a disease associated with elevated plasma cholesterol levels in a subject.

In embodiments of the use of the present invention, the Cholesin protein is isolated or purified. In some embodiments, the Cholesin protein is a mammalian Cholesin protein. Examples of mammalian Cholesin proteins include but are not limited to Cholesin proteins of humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cows. In some embodiments, the Cholesin protein is a non-human primate Cholesin protein. In some embodiments, the Cholesin protein is a human Cholesin protein. In some embodiments, the human Cholesin protein comprises an amino acid sequence as shown in SEQ ID NO:1.

In other embodiments, the Cholesin protein is a mouse Cholesin protein. The Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:2. In other embodiments, the Cholesin protein is a macaque Cholesin protein. In some embodiments, the macaque Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:3. In other embodiments, the Cholesin protein is a bovine Cholesin protein. In some embodiments, the bovine Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:4. In other embodiments, the Cholesin protein is a Rattus norvegicus Cholesin protein. In some embodiments, the Rattus norvegicus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:5. In other embodiments, the Cholesin protein is a Gallus gallus Cholesin protein. In some embodiments, the Gallus gallus Cholesin protein comprises the amino acid sequence shown in SEQ ID NO:6.

In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA.

In some embodiments, the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector, and an artificial chromosome.

In some embodiments, the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones. In some embodiments, the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. In some embodiments, the disease associated with elevated plasma cholesterol levels is atherosclerosis.

In some embodiments, the Cholesin protein described herein, the nucleic acid encoding the Cholesin protein, or the expression vector comprising the nucleic acid, or the agent that enhances the expression or activity of the subject's endogenous Cholesin protein is combined with a second therapeutic agent. In a preferred embodiment, the second therapeutic agent is selected from a protein or peptide, a nucleic acid, and a small molecule drug.

In some embodiments, the second therapeutic agent is a cholesterol-lowering drug. In some embodiments, the cholesterol-lowering drug is selected from: HMGCR inhibitors, PCSK9 inhibitors, and cholesterol absorption inhibitors.

In some embodiments, the HMGCR inhibitor is a statin. In some embodiments, the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin. In a preferred embodiment, the second therapeutic agent is rosuvastatin.

In some embodiments, the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran, and Tafolecimab.

In some embodiments, the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe. In a preferred embodiment, the second therapeutic agent is ezetimibe.

In some embodiments, the subject is a mammal. In a preferred embodiment, the subject is In some embodiments, the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, for example, the subject has an LDL receptor (LDLR) defect.

Example

The present invention is further described by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

Example 1: Cholesin is a cholesterol-induced intestinal secretory hormone

1.1 Discovery and identification of Cholesin

In this example, 10-12 week old wild-type male mice with a genetic background of C57BL/6J were selected. In order to find hormones that regulate cholesterol homeostasis, serum proteins of mice fed with a normal diet (RD, containing 0.02% cholesterol) and a Western diet (WD, containing 1.25% cholesterol) for 1 hour after fasting overnight were analyzed. The mice were randomly divided into 2 groups, 6 in each group. Among them, the first group of mice were fed a normal diet after starvation overnight (normal diet group (RD group)); the second group of mice were fed a Western diet after starvation overnight (Western diet group (WD group)). The blood of the two groups of mice was collected in anticoagulant tubes treated with lithium heparin, and centrifuged at 4°C and 4000rpm for 15 minutes. The supernatant was plasma, which was transferred to a new centrifuge tube. The plasma was treated with a column albumin/immunoglobulin removal kit (Bio-Rad) to remove IgG and albumin. Subsequently, the ProteoMiner Protein Enrichment Kit (BIO-RAD) was used to dilute high-abundance proteins and concentrate low-abundance proteins. The treated plasma was mixed with 5×SDS loading buffer (250mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-ME), boiled at 100°C for 10 minutes, and subjected to SDS-PAGE protein electrophoresis. Silver staining was performed after electrophoresis. The results showed that compared with the normal diet group, an enhanced band was observed at around 23kDa in the Western diet group (Figure 1).

By mass spectrometry identification of differential bands, it was determined that the differential bands were proteins encoded by 3110082I17Rik (mouse homologous gene of C7ORF50), which had not been reported in previous literature. Based on subsequent work, the inventors found that it had an inhibitory effect on cholesterol synthesis and named it Cholesin.

1.2 Cholesin is a secreted protein

In order to explore the localization of Cholesin in cells, an immunofluorescence experiment was performed. The specific method is as follows: 1) Place an appropriate amount of cell slides with a diameter of 12 mm in the well plate, and place HEK293T cells in the well plate at a ratio of 1:6. After the cells adhere to the well plate, overexpress human Cholesin (hCholesin) with a Flag tag at the C-terminus, mouse Cholesin (mCholesin) and a control empty plasmid; 2) After 36 hours of transfection, the culture medium was aspirated and the cells were washed once with PBS preheated at 37°C, and then fixed with a 4% paraformaldehyde/PBS solution equilibrated to room temperature for 20 minutes at room temperature; 3) Fix After the fixation, the 4% paraformaldehyde/PBS solution was aspirated and discarded, and the cells were washed once with PBS. Subsequently, the cells were treated with PBS solution containing 0.2% Triton X-100 at room temperature for 5-7 minutes to permeate the cell membrane; 4) The permeation solution was immediately aspirated and discarded, the cells were washed once with PBS, and the cells were blocked with 5% BSA/TBST solution for 30 minutes at room temperature; 5) The primary antibody was diluted with 5% BSA/TBST solution, and the primary antibody was incubated at room temperature for 1 hour after the blocking was completed; 6) The primary antibody was recovered, and the slides were washed with TBST for 3 times, each for 10 minutes; 7) The fluorescent secondary antibody and DAPI (final concentration 0.5 mg/ml) were diluted with 5% BSA/TBST solution, and stained at room temperature in the dark for 1 hour; 8) The secondary antibody was discarded, and the slides were washed with TBST for 3 times, each for 10 minutes; 9) The slides were sealed, and the results of Cholesin localization were photographed with a laser confocal microscope. The immunofluorescence results showed that Cholesin was mainly located in the cytoplasm (Figure 2A).

Subsequently, the secretion of Cholesin was explored by immunoblotting. Human Cholesin, mouse Cholesin and control empty plasmids with a Flag tag at the C-terminus were overexpressed in HEK293T cells. After 48 hours of transfection, the cells were replaced with serum-free medium and cultured overnight, and the culture medium was collected. 200 μl RIPA lysis buffer (25 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 100× protease lysis buffer, pH 7.4) was added to the adherent cells in each well of the 6-well plate. The cells were scraped from the bottom of the dish with a cell scraper and collected in an EP tube. Then, the cells were sonicated on ice according to the program of 6 seconds on, 6 seconds off, 18 seconds in total, and 40% power. The cells were centrifuged at the maximum speed of 4°C for 15 minutes, and the supernatant obtained by centrifugation was added with 5×SDS loading buffer. The samples were boiled at 100°C for 10 minutes to obtain the total protein extract. Cholesin in the culture medium and lysis supernatant was detected by immunoblotting. The results showed that the secretion of Cholesin could be detected in the culture medium of cells overexpressing human or mouse Cholesin (Figure 2B), indicating that Cholesin also has the ability to be secreted under in vitro conditions.

To characterize secreted Cholesin, a baculovirus overexpressing mouse Cholesin with a C-terminal Flag tag was constructed and used to infect insect Hi5 cells. The specific method is as follows: 1) culture 1L Hi5 cells to a density of 2-3×10 ⁶ cells/ml, add baculovirus at a ratio of 1:50, culture for 48-72 hours, collect the culture supernatant by centrifugation, and filter the culture supernatant with a 0.22 μm filter to remove cell debris; 2) use an equilibration buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, pH 7.4) to equilibrate a HisTrap excel (Cytiva) chromatography column for at least 5 column volumes; 3) use a pump head to load the filtered culture medium onto the column; 4) rinse with a wash buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 30 mM imidazole, pH 7.4) for at least 20 column volumes until no protein flows out; 5) use an elution buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 500 mM imidazole, pH 7.4) to wash the column for at least 20 column volumes until no protein flows out; 7.4) Perform gradient elution and collect the protein peak; 6) Use a concentrator tube containing a 10kDa NMWL filter to concentrate the protein peak to a total volume of less than 2ml, and use Superdex 200 Increase 10/300 GL molecular sieve to further separate and purify the sample based on the size and shape of the protein. Mass spectrometry identification of the purified protein sample shows that most of the sequence of Cholesin can be identified, except for the lysine and arginine-rich regions, indicating that Cholesin is secreted in its entirety.

These results indicate that Cholesin is a secretory protein that is secreted in its full-length form.

1.3 Cholesin is a hormone that responds to eating

In this example, 10-12 week old wild-type male mice with a genetic background of C57BL/6J were randomly divided into 9 groups, 8 mice in each group. Group 1 was starved overnight (fasting group); Groups 2 to 5 were starved overnight and then fed with a normal diet (RD) for 1, 2, 4, and 12 hours (normal diet group containing 0.02% cholesterol); Groups 6 to 9 were starved overnight and then fed with a Western diet (WD) for 1, 2, 4, and 12 hours (Western diet group containing 1.25% cholesterol). The 9 groups of mice were killed, and then plasma was collected and the Cholesin level in plasma was detected by enzyme-linked immunosorbent assay (ELISA).

The specific operation is as follows: 3 μl mouse plasma and 100 μl coating solution (0.05 mol/L Na2CO3/NaHCO3, pH 8.0) were added to the polystyrene microplate. The Cholesin whole body knockout mouse plasma was set as the control group for calibration in the 96-well microplate. The standard curve was set with the purified and quantified Cholesin protein standard. The concentration of the standard curve was 0, 12.5, 25, 50, 100, and 200 pmol. After the addition of the sample, it was incubated at 4°C overnight to allow the protein to adsorb to the bottom of the well. After overnight coating on a 4°C shaker, the microplate was removed, the liquid in the well was shaken dry, and 100 μl of 1× gelatin was added to each well to block the microplate for 2 hours at room temperature. The blocking solution was discarded, and 200 μl of TBST solution was added to each well to wash 4 times. The liquid was shaken dry as much as possible for the last time. The primary antibody for detection was prepared in TBST, 100 μl of primary antibody was added to each well, and incubated at 4°C shaker overnight. Discard the primary antibody, add 200μl TBST solution to each well and wash 4 times, and shake off the liquid as much as possible for the last time. Prepare the secondary antibody in TBST, add 100μl to each well, and incubate on a shaker at room temperature for 1 hour. Discard the secondary antibody, add 200μl TBST solution to each well and wash 4 times, and shake off the liquid as much as possible for the last time. Add 100μl TMB substrate to each well and incubate on a shaker at room temperature for 10 to 30 minutes. Then add 100μl stop solution (0.05mol/L H ₂ SO ₄ ) to each well, read the absorbance value of OD450nm with an enzyme reader within 30 minutes, and calculate the content of Cholesin in the sample to be tested based on the absorbance value of the standard curve.

The results showed that in mice that ate after starvation, the level of Cholesin in plasma increased rapidly and quickly reached a peak of about 1200pM 1 hour after eating a Western diet, then continued to decline and returned to the 0-hour level 12 hours after eating. The changes in Cholesin in plasma after feeding a normal diet also showed the same trend, but the changes were weaker than those in the Western diet group, with the peak of Cholesin only about 700pM 1 hour after eating (Figure 3A). These results indicate that eating a high-cholesterol diet promotes Cholesin secretion, suggesting that cholesterol may be an inducer of Cholesin secretion.

In order to observe whether the same secretion changes as in mice also exist in humans, clinical data were analyzed. The plasma Cholesin levels of 13 pairs of healthy people who were fasted overnight and fed for 1 hour were compared. The results showed that the plasma Cholesin content increased significantly after eating (Figure 3B).

These results suggest that cholesin is a hormone that is responsive to feeding and that cholesterol is a possible inducer.

1.4 Tissue distribution of Cholesin

In this example, five wild-type (WT) and Cholesin knockout (KO) male mice of 10-12 weeks old with a genetic background of C57BL/6J were selected, and the brain, stomach, small intestine, colon, liver, kidney, and brown fat tissues of the mice were collected. RIPA lysis buffer (25mM Tris, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.4) was prepared, and protease inhibitors and phosphatase inhibitors were added before use according to the volume of the lysis buffer to prepare tissue protein extract. Weigh an appropriate amount of tissue, add 500μl RIPA lysis buffer and magnetic beads, and grind at 60Hz for 1 minute in a tissue grinder. After grinding, centrifuge at 15000rpm for 15 minutes in a 4℃ precooled centrifuge. When the lipid content in the tissue is high, transfer the supernatant of the first centrifugation to a new centrifuge tube and centrifuge for 15 minutes under the same parameters. The supernatant was taken for protein quantification using the BCA method, 5× SDS loading buffer was added and mixed thoroughly, and then heated in a 100°C metal bath for 10 minutes to obtain a tissue protein sample. Then 25 μg of total protein was taken for SDS-PAGE electrophoresis to determine the tissue distribution of Cholesin at the protein level.

The results are shown in FIG4 . The size of the endogenous Cholesin protein is slightly less than 25 kDa, and its expression level is highest in the small intestine, followed by the colon and stomach, and its expression level in other tissues is low, showing strong tissue specificity.

1.5 Cholesin is an intestinal secretory hormone that responds to cholesterol absorption

Since Cholesin is expressed most highly in the intestine and the small intestine is the main organ for exogenous cholesterol absorption, it is speculated that Cholesin may be secreted by the small intestine to regulate cholesterol homeostasis. To verify this hypothesis, Cholesin intestinal-specific knockout (Cholesin IKO) mice were constructed as a research tool.

In this example, WT and IKO mice were randomly divided into 2 groups, each with 8 mice. The first group of mice were starved overnight, and the second group of mice were starved overnight and then fed with a Western diet (WD) containing 1.25% cholesterol for 1 hour. Then the plasma and small intestine of the mice were collected respectively, and the content of total cholesterol (TC) in the small intestine and the content of cholesin, TC and triglyceride (TG) in plasma were measured. The results are shown in Figures 5A and 5B.

To further confirm the role of cholesterol in promoting Cholesin secretion, WT and IKO mice were randomly divided into 2 groups, 7 mice in each group, and the mice were starved overnight. The first group of mice were gavaged with 150 μl corn oil containing 20% anhydrous ethanol (chol-), and the second group of mice were gavaged with 150 μl 200 mg kg ^-1 body weight of cholesterol (chol+, where cholesterol is dissolved in corn oil containing 20% anhydrous ethanol). Plasma and small intestinal tissues were collected from mice 1 hour after gavage. The content of TC in the small intestine and the content of Cholesin, TC and TG in plasma were measured. The results are shown in Figures 5C and 5D.

The results of Figure 5A show that the Cholesin content in the plasma of WT mice increased significantly after feeding WD, while the Cholesin content in the plasma of IKO mice hardly increased, and the Cholesin content in the plasma of IKO mice was significantly lower than that of WT mice under starvation. These results indicate that the small intestine may be the main organ that secretes Cholesin under the stimulation of a Western diet. The results of Figure 5A also show that the total cholesterol content in the small intestine increased after feeding WD, but there was no significant difference between the WT and IKO groups in both the starving and fed states. This result suggests that there is no difference in the cholesterol absorption capacity of the small intestine of WT and IKO group mice, and their response to the Western diet is not due to different absorption capacities of the small intestine.

The results in Figure 5B show that there was no significant change in plasma TC of mice before and after eating, but the plasma TC of IKO mice was higher than that of the WT group before and after eating. It has not yet entered the blood circulation, so plasma TC will not change with eating; the higher plasma TC in the IKO group than in the WT group suggests that Cholesin may negatively regulate the plasma TC level. Figure 5B also shows that the plasma TG content of mice decreased significantly before and after eating, and there was no significant difference in plasma TG between WT and IKO mice under starvation conditions. After eating, the plasma TG of IKO mice was slightly higher than that of the WT group, indicating that eating would reduce the total triglycerides circulating in the blood, and the difference in plasma TG between the WT and IKO groups also suggests that Cholesin may have an effect on plasma TG.

Similar to the effect of WD feeding, the results of Figure 5C show that the Cholesin content in the plasma of WT mice increased significantly after oral administration of cholesterol, while IKO mice basically did not respond to the stimulation of cholesterol, and the absorption of cholesterol by IKO mice was also unaffected. The results of Figure 5D show that there was no significant change in the plasma TC content of mice after oral administration of cholesterol, but the plasma TC of IKO mice was higher than that of the WT group in both the control group and the cholesterol-treated group. The total TG content of mouse plasma increased after oral administration of cholesterol, and there was no significant difference in the plasma TG of WT and IKO mice in the experimental group and the control group. This is because cholesterol is dissolved in corn oil, so the TG in plasma will increase after oral administration.

These results demonstrate that Cholesin is a hormone secreted by the small intestine in response to cholesterol absorption.

Example 2: NPC1L1-mediated cholesterol absorption promotes Cholesin secretion

2.1 Cholesin expression in absorptive enterocytes

Cholesterol absorption is currently believed to be mediated by NPC1L1 on the apical surface of absorptive enterocytes in the intestine. Since cholesterol absorption can promote the secretion of Cholesin, we speculated that Cholesin may be mainly expressed in absorptive enterocytes. To verify the above hypothesis, Cholesin-GFP and Apoa1-mCherry (APOA1 is a marker protein for absorptive enterocytes) double gene knock-in mice were constructed for immunofluorescence experiments to determine the cell type expressing Cholesin. The specific experimental steps are as follows: 1) Collect the mouse small intestine tissue, rinse the intestinal feces with PBS and immediately place it in a 4% paraformaldehyde solution prepared in PBS and fix it overnight at 4°C; 2) Transfer the fixed tissue to a PBS solution containing 30% sucrose and dehydrate it overnight at 4°C; 3) After dehydration, the tissue should sink to the bottom of the sucrose solution. Take the tissue out of the sucrose solution, try to dry the liquid on the surface of the tissue, add OCT to the embedding box and put the tissue block in, and freeze the embedded tissue in a -80°C refrigerator for 6 hours; 4) Before slicing, place the tissue block in a -20°C refrigerator for equilibrium, cut slices with a thickness of 5-8μm with a freezing microtome, and stick the tissue slices on a cationic anti-shedding film; 5) Before staining, take out the slices and equilibrate them at room temperature for 20 minutes, then place them in a 55°C oven for 10 minutes to prevent the tissue from falling off the slide, and circle the tissue on the slide with a tissue pen; 6) Wash the tissue 3 times with TBST to wash off the OCT, and then use 0.2% Triton X-100/PBS penetrates the tissue for 5-7 minutes; 7) After penetration, immediately remove 0.2% Triton X-100/PBS, rinse once with TBST, and then block with 5% BSA/TBST at room temperature for 30 minutes; 8) Dilute the primary antibody with 5% BSA/TBST, discard the blocking solution after blocking, add GFP primary antibody, each tissue block requires 80-100μl of primary antibody, add double distilled water to the wet box, and incubate the slices at 4℃ overnight; 9) Discard the primary antibody, wash the slices 3 times with TBST at room temperature, 5 minutes each time; 10) Dilute the fluorescent secondary antibody with 5% BSA/TBST 11) discard the secondary antibody and DAPI, wash the slides 3 times with TBST at room temperature, 5 minutes each time; 12) seal the slides, and use a laser confocal microscope to photograph the Cholesin localization results.

As shown in Figure 6, colocalization of Cholesin-GFP and Apoa1-mCherry was observed, indicating that Cholesin is expressed in absorptive enterocytes.

2.2 Cholesin levels increased in a dose-dependent manner with increasing cholesterol concentration

In order to study whether cholesterol directly affects the secretion of Cholesin, HCT116 cells (human colorectal cancer cells) that stably overexpress Cholesin were constructed. The HCT116 stably transfected cell line overexpressing Cholesin was plated in a 24-well plate one day before the experiment, and the cells should be in the logarithmic growth phase during the experiment. Wash the cells once with preheated serum-free RPMI1640 medium before treating the cells. Dilute exogenous cholesterol with serum-free RPMI1640 to a final concentration of 0μM, 2.5μM, 5μM, 10μM, 20μM and 40μM. Add 500μl of serum-free RPMI1640 medium containing the final concentration of cholesterol to the wells of the 24-well plate and culture in a 37°C incubator for 1 hour. After the culture is completed, aspirate 400μl of culture medium into a 1.5ml EP tube and centrifuge at a maximum speed of 4°C for 5 minutes to remove cell debris. The culture medium collected from each well of cells was spotted into the microplate three times in parallel, with 100 μl in each well, and ELISA was performed to detect the secretion of Cholesin in the culture medium.

The total cholesterol level in the cells was also detected, and the specific method was as follows: 1) After the cells absorbed the culture medium to detect the amount of Cholesin secretion, the remaining cells were digested with Trypsin, and the cells were resuspended in PBS and counted; 2) The supernatant was discarded after high-speed centrifugation, 150 μl of isopropanol was added to each sample, and the cells were ultrasonically disrupted on ice (40% intensity, ultrasonic for 2 seconds, interval of 3 seconds, total time of 3 minutes); 3) After the end of ultrasonication, centrifuge at 10000g and 4°C for 10 minutes, and the supernatant was the total cholesterol extract of the cells; 4) The total cholesterol concentration in the extract was determined using the CHOD-PAP cholesterol determination kit (Sino-Biotech Holdings), and the total cholesterol amount was calculated based on the volume of the extract, and finally the total cholesterol amount was divided by the total number of cells to convert the cholesterol content per 106 cells.

The results are shown in Figure 7. The results show that as the cholesterol concentration increases, the level of Cholesin in the culture medium increases in a dose-dependent manner. In addition, as the cholesterol treatment dose increases, the total amount of cholesterol in the cells also gradually increases.

NPC1L1 knockdown in HCT116 cells disrupts cholesterol absorption and Cholesin secretion

In order to explore the role of NPC1L1 in cholesterol-induced Cholesin secretion at the cellular level, NPC1L1 was knocked down (Npc1l1 KD) based on the HCT116 stable cell line overexpressing Cholesin. Subsequently, Western blotting confirmed that NPC1L1 knockdown had no effect on Cholesin expression at the protein level (Figure 8A).

According to the method in 2.2, non-target knockdown control cells (NT) and NPC1L1 knockdown cells (Npc1l1 KD) were plated in 24-well plates, and then cultured with serum-free RPMI1640 containing 0μM and 10μM cholesterol for 1 hour. After the culture, the culture medium was collected and the secretion of Cholesin was detected by ELISA. Total cholesterol was extracted by ultrasonication with propanol, and the total cholesterol concentration in the extract was determined using the CHOD-PAP cholesterol assay kit (Zhongsheng Beikong). The total cholesterol amount was calculated based on the volume of the extract, and finally the total cholesterol amount was divided by the total number of cells to convert the cholesterol content per 10 ⁶ cells. The results showed that knocking down NPC1L1 in HCT116 cells impaired cholesterol absorption and Cholesin secretion (Figure 8B).

The results of Examples 2.2 and 2.3 together indicate that NPC1L1-mediated cholesterol absorption stimulates the secretion of Cholesin.

NPC1L1 inhibitor treatment and Npc1l1 gene knockout in mice effectively blocked small intestinal cholesterol absorption and cholesin secretion

To further verify whether Cholesin secretion in mice is dependent on NPC1L1-mediated cholesterol absorption, two methods were used: treating mice with the NPC1L1 inhibitor Ezetimibe and directly using Npc1l1 gene knockout mice.

NPC1L1 inhibitor treatment

Male mice aged 10-12 weeks were fasted overnight, and then gavaged with ezetimibe (Ezetimlbe+) at a dose of 10 mg kg ^-1 , and gavage with corn oil solvent as a control (Ezetimlbe-). One hour after gavage, the mice were gavaged with 150 μl corn oil solvent control (chol-) or cholesterol (chol+, 200 mg kg ^-1 body weight). One hour after gavage, plasma was collected to detect total cholesterol (TC) and Cholesin levels in plasma, and small intestinal tissues of mice were collected to determine the total cholesterol content in the small intestine. The results are shown in Figures 9A-9B.

The results showed that the level of Cholesin in the plasma of control mice (Chol-) that had fasted overnight was low, with a plasma content of about 500 pM. The level of Cholesin in the plasma of mice that were not treated with ezetimibe increased significantly after oral administration of cholesterol, while mice treated with ezetimibe no longer responded to the stimulation of cholesterol administration, and the level of Cholesin in the plasma was basically the same as that in the starvation state (Figure 9A). At the same time, the total cholesterol content in the small intestine indicated that the treatment with ezetimibe inhibited the absorption of cholesterol in the small intestine (Figure 9A). The determination of the plasma TC content of mice showed that short-term administration stimulation did not cause changes in plasma TC levels (Figure 9B).

Npc1l1 knockout

To further explore the role of NPC1L1 in cholesterol-induced Cholesin secretion, Npc1l1 knockout (Npc1l1 ^-/- ) mice were constructed. Immunoblotting confirmed that the absence of Npc1l1 did not affect the expression of Cholesin (Figure 9C). Wild-type (Npc1l1 ^+/+ ) and Npc1l1 knockout (Npc1l1 ^-/- ) mice were then fasted overnight, and then orally gavaged with 150 μl corn oil solvent control (chol-) or cholesterol (chol+, 200 mg kg ^-1 body weight). One hour later, whole blood was collected from the mice to separate plasma to detect plasma Cholesin and TC levels, and small intestinal tissue was collected to determine TC content. The results are shown in Figures 9D-9E.

The results showed that Npc1l1 deficiency could significantly inhibit the secretion of Cholesin and the absorption of cholesterol in the small intestine of mice. (Fig. 9D) The measurement of plasma TC content in mice showed that short-term cholesterol treatment did not cause changes in plasma TC levels (Fig. 9E).

The above in vitro and in vivo results of Example 2 indicate that NPC1L1-mediated cholesterol absorption promotes the secretion of Cholesin.

Example 3: Cholesin regulates cholesterol metabolism in humans

In order to explore whether Cholesin has a similar regulatory effect on cholesterol in humans, 600 plasma samples were collected from humans 1 hour after eating for ELISA assay to detect the levels of Cholesin, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in plasma samples and conduct correlation analysis. The specific operation is as follows: 3μl human plasma and 100μl coating solution (0.05mol/L Na2CO3/NaHCO3, pH8.0) were added to the polystyrene microplate, and the plasma of Cholesin knockout mice was set as the control group in the 96-well microplate for calibration. The standard curve was set with purified and quantified Cholesin protein standards, and the concentrations of the standard curve were 0, 12.5, 25, 50, 100, and 200pmol. After the addition of samples, the samples were incubated at 4℃ overnight to allow the protein to adsorb to the bottom of the wells. After overnight coating on a 4℃ shaker, the microplate was removed, the liquid in the wells was dried, and 100μl of 1×gelatin was added to each well to block the microplate at room temperature for 2 hours. Discard the blocking solution, add 200μl TBST solution to each well and wash 4 times, and shake off the liquid as much as possible for the last time. Prepare the primary antibody for detection in TBST, add 100μl primary antibody to each well, and incubate overnight at 4℃ on a shaker. Discard the primary antibody for detection, add 200μl TBST solution to each well and wash 4 times, and shake off the liquid as much as possible for the last time. Prepare the secondary antibody in TBST, add 100μl to each well, and incubate at room temperature on a shaker for 1 hour. Discard the secondary antibody, add 200μl TBST solution to each well and wash 4 times, and shake off the liquid as much as possible for the last time. Add 100μl TMB substrate to each well and incubate at room temperature on a shaker for 10 to 30 minutes. Then add 100μl stop solution (0.05mol/L H ₂ SO ₄ ) to each well, read the absorbance value of OD450nm with an enzyme reader within 30 minutes, and calculate the content of Cholesin in the sample to be tested based on the absorbance value of the standard curve. The data were subjected to Pearson correlation analysis using Excel, and the p value and r ² were calculated.

Correlation analysis results showed that Cholesin levels were negatively correlated with TC and LDL-C levels (Figures 10A and 10B), indicating that Cholesin has a negative regulatory effect on cholesterol levels.

Example 4: Cholesin knockout increases liver cholesterol synthesis and VLDL secretion

4.1 Determination of plasma total cholesterol, HDL, LDL, VLDL and triglyceride levels and liver cholesterol and triglyceride levels in Cholesin IKO mice

In this example, 12-14 week old wild-type (WT) and intestinal Cholesin-specific knockout (Cholesin IKO) male and female mice with a genetic background of C57BL/6J were selected and fed with a normal diet (RD) containing 0.02% cholesterol and a Western diet (WD) containing 0.2% cholesterol for 8 weeks. After the mice were starved overnight and then fed for 6 hours, the mouse plasma was collected to detect the plasma total cholesterol (TC) and triglyceride (TG) levels. The mouse liver tissue was also collected to detect the level of cholesterol. The levels of TC and TG in the liver were detected. The results are shown in Figures 11A and 11B.

Since cholesterol exists mainly in the form of lipoproteins (such as HDL, LDL and VLDL) in the blood, fast protein liquid chromatography was used to analyze the content of each lipoprotein in plasma. The experimental method is as follows: the plasma of all mice in each group is combined, mixed and placed on ice for loading. The Superose 6 Increase 10/300 GL column was connected to the AKTA purification system, and the column was balanced with PBS. The sample loop accurately injected 1 ml of total plasma. After complete injection, the eluted components can be collected without waiting for the peak. Each component is sampled 400 μl, and the sampling is stopped when the salt peak appears. The total cholesterol content of each component was determined by enzymatic method, and the FPLC curve was drawn. According to the area under the FPLC curve, the relative levels of very low density lipoprotein (VLDL), intermediate/low density lipoprotein cholesterol (IDL/LDL) and high density lipoprotein cholesterol (HDL) were calculated. The results are shown in Figure 11C.

Figure 11A shows that when wild-type mice were fed with a normal diet, the plasma TC of male mice was about 100 mg/dL, and that of female mice was slightly lower than that of male mice; while under the WD feeding condition, the plasma TC of male mice reached about 170 mg/dL, and that of female mice was 140 mg/dL. After IKO male and female mice were fed with a normal diet or a Western diet, the plasma TC levels were about 20% higher than those of wild-type mice. The TG content in mouse plasma was slightly increased in IKO mice compared with the WT group in male and female mice fed with RD or WD.

Figure 11B shows that in male and female mice fed RD or WD, the liver TC content of IKO mice was higher than that of WT group. There was no significant difference in liver TC content between male and female mice, and the difference in liver TC between WT and IKO was amplified under WD feeding conditions. There was no significant difference in liver TG between WT and IKO groups under RD conditions, but under the induction of WD, the liver TG of IKO group was slightly higher than that of WT group.

Figure 11C shows that the levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) were increased in Cholesin IKO mice.

These results suggest that Cholesin deficiency leads to elevated plasma cholesterol and triglyceride levels.

4.2 Elevated plasma cholesterol levels in Cholesin IKO mice are not caused by enhanced cholesterol absorption in the small intestine

According to previous reports, the increase in plasma cholesterol levels may be caused by enhanced intestinal cholesterol absorption, increased hepatic cholesterol synthesis and VLDL secretion, or decreased cholesterol excretion. To explore whether the elevated plasma cholesterol levels in IKO mice were caused by enhanced intestinal cholesterol absorption, the body weight, body fat percentage, food and water intake, small intestinal total cholesterol and triglyceride levels, and chylomicron decomposition rate of the mice in Example 4.1 were measured.

The mice in the random feeding state were weighed and their body fat was measured by EchoMRI awake animal body composition analyzer, and the measurement was repeated 3 times for each mouse. The body fat percentage of mice was calculated according to the following formula: body fat percentage = mouse body fat/mouse weight × 100%. The results are shown in Figures 12A and 12B.

Food intake was measured in individually housed mice using the PhenoMaster system from TSE Systems. Mice were allowed at least 24 hours of acclimatization before measurement. Food and water were freely available in appropriate apparatus and The data of free food and water intake were collected continuously for 48 hours by the built-in automatic instrument. The results are shown in Figures 12C and 12D.

In order to determine the total cholesterol and triglyceride content in the small intestine of mice, the small intestine tissues of the mice that were starved overnight and then fed for 6 hours in Example 4.1 were collected. The determination results are shown in FIG. 12E .

The lipid tolerance test was performed by orally administering a certain dose of olive oil to mice to evaluate the rate of chylomicron decomposition in the small intestine of mice. The mice of Example 4.1 were fasted for 6 hours to synchronize the intestinal state of the mice, and then 200 μl of olive oil was orally administered. Blood was collected from the tail vein at different time points (0, 1, 2, 3, and 4 hours) before and after oral administration, and then plasma was separated. The triglyceride level in the plasma was determined and the triglyceride secretion rate (△TG: plasma TG at 1, 2, 3, and 4 hours of each mouse minus the plasma TG at 0 of the mouse) was calculated to evaluate the rate of chylomicron decomposition. The results are shown in Figure 12F.

Figure 12A shows that there was no significant difference in body weight between WT and IKO groups of male and female mice under the two dietary treatments. Figure 12B shows that there was no significant difference in body fat percentage between WT and IKO groups of male and female mice under the two dietary treatments. Figures 12C and 12D show that there was no significant difference in food intake and water intake between WT and IKO groups of male and female mice under the two dietary treatments. Figure 12E shows that there was no significant difference in total cholesterol and triglyceride content in the small intestine between IKO mice and WT mice in male and female mice fed a normal diet or a Western diet. Figure 12F shows that the rate of chylomicron decomposition in IKO mice was not affected in male and female mice fed a normal diet or a Western diet.

These results suggest that intestinal absorption is similar in IKO and WT mice and that the elevated plasma cholesterol levels in IKO mice are not due to enhanced cholesterol absorption in the small intestine.

4.3 Elevated plasma cholesterol levels in Cholesin IKO mice are not caused by decreased cholesterol excretion

To investigate whether the elevated plasma cholesterol levels in IKO mice were caused by decreased cholesterol excretion, the bile cholesterol, bile acid levels, and fecal cholesterol levels of the mice of Example 4.1 were measured.

Cholesterol in mice is generally excreted through bile acid or feces. The cholesterol content in bile acid and feces was measured to explore whether the plasma TC level of IKO mice was affected by changes in cholesterol excretion. Mouse bile is excreted from the gallbladder during eating and is mainly stored in the gallbladder when hungry. The mice were starved overnight, and the bile in the gallbladder was collected with a syringe. The volume of the gallbladder was then weighed, and the concentrations of total bile cholesterol (cholesterol determination kit (CHOD-PAP method, Zhongsheng Beikong) and bile acid (total bile acid (TBA) kit, Nanjing Jiancheng) were measured using kits. The results are shown in Figure 13A.

To measure the cholesterol content in feces, the feces of mice were collected and the total cholesterol and triglycerides in the feces were extracted by the following method: 40 mg of feces were weighed and dried in a 55°C oven, and then the dried and crushed feces were suspended in 1200 μl of a 2:1 chloroform-methanol (v/v) mixture. The suspension was then mixed with 300 μl of water and vortexed vigorously for 60 seconds. Then, it was centrifuged at 4000 rpm for 10 minutes at 4°C, and the organic layer was carefully transferred to a fresh The dried lipids were then resuspended in 100 μl of a 9:1 isopropanol-Triton X-100 (v/v) solution, and the resulting fecal extract was used for enzymatic assays to detect total cholesterol and triglyceride levels. The results are shown in FIG13B .

Figure 13A shows that there were no significant differences in the total bile volume, bile cholesterol level and bile acid level between WT and IKO mice under the two dietary treatments for male and female mice. Figure 13B shows that there were no significant differences in the total cholesterol content and triglyceride content in the feces between WT and IKO mice under the two dietary treatments for male and female mice.

These results indicate that the elevated plasma cholesterol levels in IKO mice are not caused by decreased cholesterol excretion.

4.4 Elevated plasma cholesterol levels in Cholesin IKO mice are caused by increased cholesterol synthesis in the liver.

To investigate whether the elevated plasma cholesterol levels in IKO mice were caused by enhanced cholesterol synthesis in the liver, the expression of cholesterol synthesis genes in the liver was examined at the mRNA and protein levels.

qPCR was used to detect the mRNA levels of cholesterol synthesis-related genes in the liver. The specific experimental procedures are as follows: 1) About 20 mg of liver tissue was weighed and the total liver RNA was extracted using HP total RNA Kit (Omega); 2) 1 μg RNA was taken and reverse transcribed using REVERTAID 1ST CDNA SYNTH KIT (thermo); 3) The cDNA obtained by reverse transcription was subjected to qPCR. The results are shown in Figure 14A.

Immunoblotting was used to detect the protein expression level of cholesterol synthesis genes in the liver. The specific operation is as follows: 1) Weigh 20 mg of liver tissue, add 500 μl RIPA lysis buffer and magnetic beads, and grind at 60 Hz for 1 minute in a tissue grinder; 2) Centrifuge at maximum speed at 4°C for 10 minutes; 3) Transfer the supernatant to a new EP tube, be careful not to absorb the fat layer, add 5× loading buffer, mix well, and boil the sample at 100°C for 10 minutes; 4) Use the BCA method for protein quantification; 5) Detect the expression levels of SREBP2 (including pSREBP2 and mSREBP2), HMGCR, HMGCS1 and LDLR in the tissue by immunoblotting. The results are shown in Figure 14B.

Figures 14A and 14B show that compared with wild-type mice, the expression levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, Pmvk, sqle in the liver of IKO mice fed with a normal diet and a Western diet, as well as the transcription factor SREBP2 that regulates the expression of these cholesterol synthesis-related genes and its downstream regulated LDLR were significantly increased. It is worth noting that the expression of SREBP2 and cholesterol synthesis-related genes was lower in mice fed with a Western diet, indicating that the absorption of intestinal cholesterol inhibits the synthesis of cholesterol in the liver.

4.5 Elevated plasma cholesterol levels in Cholesin IKO mice are caused by enhanced VLDL secretion in the liver.

To investigate whether the elevated plasma cholesterol levels in IKO mice were caused by enhanced VLDL secretion from the liver, mice were injected with poloxamer 407, an inhibitor of lipoprotein lipase and VLDL metabolism, and plasma cholesterol and triglyceride levels were monitored to assess hepatic VLDL secretion.

The mice were fasted for 4 hours and then intraperitoneally injected with 1 g kg-1 body weight of Poloxamer 407. Blood samples were collected from the tail vein before, 1 hour and 2 hours after the injection of Poloxamer 407. Plasma total bile duct was measured at each time point. The levels of sterol (TC) and triglyceride (TG) were measured and the secretion rates of TC and TG were calculated (the calculation method was the TC or TG level of each mouse at 1 or 2 hours minus the TC or TG level of the mouse at 0 hours). The results are shown in FIG15 .

The results in Figure 15 showed that the secretion rates of TC and TG in male and female IKO mice fed with a normal diet and a Western diet were significantly increased compared with those in WT mice, indicating that Cholesin deficiency increases the secretion of hepatic VLDL, thereby leading to increased plasma cholesterol levels.

The above results of Example 4 show that knockout of Cholesin increases cholesterol synthesis and VLDL secretion in the liver, thereby increasing plasma cholesterol and triglyceride levels. This suggests that administration of Cholesin can reduce cholesterol synthesis and VLDL secretion in the liver, thereby reducing plasma cholesterol and triglyceride levels.

Example 5: GPR146 is a receptor for Cholesin

5.1 Target tissues of Cholesin

In order to identify the target tissues of Cholesin, 8-10 week old wild-type (Gpr146 ^+/+ ) mice with a genetic background of C57BL/6J were selected, and purified GST-Cholesin was incubated with different tissues of mice for in vitro binding experiments on frozen tissue sections. The specific process is as follows: 1) Collect mouse tissues: According to the experimental design, the mouse adipose tissue (epididymal white adipose tissue (eWAT) and brown adipose tissue (BAT)), muscle, liver, pancreas, small intestine and kidney tissues were collected and immediately placed in a 4% paraformaldehyde solution prepared in PBS and fixed overnight at 4°C; 2) Tissue dehydration and embedding: Different dehydration treatments were performed according to the different OCT and paraffin embedding agents. Except for white adipose tissue used for paraffin embedding, other tissues were subsequently embedded in OCT; 3) Tissue sectioning: The thickness of the sections was 10 μm using a freezing microtome, and the thickness of the paraffin sections was 5 μm; 4) The frozen sections were placed in an immunofluorescence humidified box and thawed at room temperature. The paraffin sections required antigen repair. The pre-treated sections were drawn with an immunohistochemistry pen to draw a closed circle around the tissue and washed twice with PBS; 5) Protein binding: Prepare binding proteins (GST-Cholesin 200nM; GST 200nM), incubate at room temperature for 30 minutes; 6) After the protein incubation, wash twice with PBS, and block with 5% BSA blocking solution prepared in PBS at room temperature for 1 hour; 7) Discard the blocking solution, incubate with primary antibody diluted in blocking solution at 4°C overnight; 8) Discard the primary antibody, wash three times with TBST, and incubate with secondary antibody diluted in blocking solution and DAPI with a final concentration of 0.5mg/ml at room temperature for 1h; 10) Discard the secondary antibody and DAPI, and wash three times with TBST; 11) Try to dry the solution on the surface of the tissue, add an appropriate amount of mounting medium on the surface of the slide according to the size of the tissue, carefully cover with a coverslip to avoid bubbles, and place at room temperature away from light until the mounting medium solidifies, and then place at room temperature away from light for imaging observation.

From the results of immunofluorescence, it can be seen that GST-Cholesin has binding signals in muscle, liver, kidney and adipose tissue, and the binding signals in muscle, liver, kidney and adipose tissue are relatively stronger (Figure 16A). This indicates that muscle, liver, kidney and adipose tissue are the target tissues of Cholesin.

5.2 Identification of Cholesin Receptors

To identify the receptor for Cholesin, a genome-wide screening was performed using a synthetic viral library containing 65,383 sgRNAs targeting 19,050 human protein-coding genes, 21,864 sgRNAs targeting miRNAs, and 3,000 non-targeting control sgRNAs. Based on the screening results, GPR146 was identified as a candidate receptor for Cholesin. GPR146 is a conserved orphan G protein-coupled receptor involved in the regulation of cholesterol synthesis.

In order to study whether GPR146 is a receptor for Cholesin, 8-10 week old Gpr146 knockout (Gpr146 ^-/- ) mice with a genetic background of C57BL/6J were selected to perform in vitro binding experiments as described in Example 5.1. The immunofluorescence results are shown in FIG16A .

In order to prove that GPR146 is a specific receptor for Cholesin, mutants of GPR146 (R90A, H101A, H169A, R179A, D187A, E265A) were designed based on the conservation and acid-base properties of the amino acids in the extracellular region of GPR146. Since the liver is the main target tissue of Cholesin, primary hepatocytes from wild-type (Gpr146 ^+/+ ) and Gpr146 knockout (Gpr146 ^-/- ) mice were isolated for binding staining. For primary hepatocytes of Gpr146 ^-/- mice, plasmids were transfected to overexpress wild-type GPR146 (WT) and mutant GPR146 (Mut). The specific experimental method is as follows: 1) According to the experimental needs, a suitable number of cell slides with a diameter of 12 mm were spread in a 6-well plate in advance, and primary hepatocytes of Gpr146 ^+/+ and Gpr146 ^-/- mice were isolated at the same time, and the cells were diluted to a suitable density and spread in a 6-well plate; 2) After the cells of Gpr146 ^-/- mice adhered for 6 hours, plasmids were transfected to overexpress wild-type GPR146 (WT) and mutant GPR146 (Mut); 3) After 36 to 48 hours of cell culture, the cell slides in the 6-well plate were transferred to a 24-well plate and washed once with PBS buffer. GST and GST-Cholesin were diluted to 200nM with fresh culture medium, and Cholesin-His was diluted to 20μM. WT cells were divided into 3 groups: GST negative control group, GST-Cholesin binding group, and GST-Cholesin and Cholesin-His co-incubation group. Gpr146 ^-/- cells were only co-incubated with 200nM GST-Cholesin. 200μl of ligand-containing culture medium was added to each well and placed in a 37°C constant temperature incubator for 30min; 4) After incubation, the culture medium was aspirated and washed three times with PBS buffer; 5) 250μl of 4% paraformaldehyde/PBS solution was added to each well and fixed at room temperature for 30min on a horizontal shaker; 6) The paraformaldehyde solution was aspirated, washed once with PBS, and then 250μl of 5% BSA/PBS solution was added to block at room temperature for 30min on a horizontal shaker; 7) The primary antibody was diluted in a 5% BSA/PBS solution according to an appropriate ratio, the blocking solution was aspirated and 200μl of the primary antibody solution was added to incubate at room temperature for 1h; 8) The primary antibody was recovered and stored at 4°C, and washed three times with PBS solution, each for 10min; 9) 5% Dilute the fluorescent secondary antibody and DAPI with BSA/PBS, add 200 μl of secondary antibody and DAPI solution to each well, and incubate at room temperature in the dark on a horizontal shaker for 1 hour; 10) Aspirate and discard the secondary antibody and DAPI solution, wash three times with PBS solution, 10 minutes each time; 11) Wipe the slide with dust-free paper and mark the experimental conditions, drop an appropriate amount of mounting medium on the slide, clamp the cell slide and gently absorb the remaining liquid along the edge of the slide on the absorbent paper, slowly press the side with cells toward the mounting medium and avoid bubbles, place at room temperature in the dark overnight, and wait for the mounting medium to solidify before imaging observation. The experimental results are shown in Figure 16B.

In addition, primary culture samples of 8-10 week-old wild-type mice and Gpr146 ^-/- mice with a genetic background of C57BL/6J were collected. Saturation binding experiments were performed on hepatocytes. The specific process is as follows: 1) Primary hepatocytes from WT and Gpr146 ^-/- mice were separated and plated on 12-well plates, and 4 groups were set up: WT cells, Gpr146 ^-/- cells, Gpr146 ^-/- complemented wild-type GPR146 cells, and Gpr146 ^-/- complemented mutant GPR146 cells. 1) After 6 hours of cell attachment, adenovirus overexpressing wild-type and mutant GPR146 was added to Gpr146 ^-/- cells; 2) the purified Cholesin was labeled with a biotin labeling kit, and the concentration of biotin-labeled Cholesin was measured; 3) the culture medium of cells infected for 24 hours was discarded, 5% BSA (prepared with M199 culture medium) was added, and incubated for 30 minutes for blocking; 4) the blocking solution was discarded, different gradients of Cholesin-biotin protein were added, and added to 4 groups of primary hepatocytes, with 3 replicates for each experimental group, and incubated at room temperature for 30 minutes; 5) after incubation, the culture medium was discarded, the cells were washed 3 times with PBS, and high-sensitivity-HRP-streptavidin antibody (1:8000) prepared with 1% BSA in PBS buffer was added, and incubated at room temperature for 1 hour; 6) the culture medium was discarded, PBS buffer was added for washing three times, and 200ul of cell lysis buffer (50mM HEPES pH7.4, 150mM NaCl, 1% TritonX-100) was used to lyse the cells, 10ul was taken for protein quantification, 30ul was transferred to a 96-well plate, 100ul of soluble single-component TMB substrate solution was added, and the plate was incubated at room temperature in the dark for color development. After the color development reached an appropriate degree, 100ul of 0.1mol/L sulfuric acid solution was added to terminate the reaction, and the plate was placed in an ELISA instrument to detect the OD450nm absorbance. The ratio of the absorbance value to the protein amount represents the binding of a specific concentration of Cholesin to the cells. The relationship between the binding amount of different concentrations of Cholesin and primary hepatocytes was analyzed by Graphpad software to obtain a saturation binding curve. The experimental results are shown in Figure 16C.

The results of Figure 16A show that the binding signals of GST-Cholesin in the muscle, liver, kidney and adipose tissue of Gpr146 ^{-/ - mice are significantly weakened compared with those of wild-type mice. As shown in the immunofluorescence results of Figure 16B, GST-Cholesin can bind to the cell membrane of WT hepatocytes, and this binding can be competed out by Cholesin-His; the binding of GST-Cholesin to Gpr146 -/-} cells is significantly weakened, and cells complemented with wild-type GPR146 have restored the binding to GST-Cholesin, while cells complemented with mutant GPR146 still cannot bind to GST-Cholesin. As shown in the saturation curve of Figure 16C, the saturation binding degree of Gpr146 ^-/- cells is significantly reduced, and cells complemented with wild-type GPR146 have restored the binding to Cholesin, while cells complemented with mutant GPR146 still cannot bind to Cholesin.

These in vitro and in vivo binding assay results indicate that GPR146 is a receptor for Cholesin.

5.3 Determination of the binding affinity of Cholesin to GPR146

Microthermophoresis is a technique that quantifies the interactions between biomacromolecules based on the thermophoresis properties of molecules. This technique was used in this example to measure the binding affinity of Cholesin to wild-type (WT) and mutant (Mut) GPR146. The specific experimental steps are as follows: 1) Receptor fluorescence labeling: We purified wild-type and mutant GPR146 proteins from Hi5 cells and concentrated them to about 100ul with a concentration of about 0.4ug/ul. We took 90ul of protein sample and used the RED-NHS protein amino labeling kit to fluorescently label it. After labeling, its concentration was 1/5 of that before labeling. The volume was expanded to 450ul, and the labeled sample was subjected to fluorescence signal detection. According to the intensity of the fluorescence signal, the final receptor concentration used in the MST experiment was determined to be 50nM; 2) The Cholesin protein was purified by Superdex 200Incresae 5/15GL molecular sieve chromatography column. Replace with HEPES-NaCl buffer containing DDM and CHS, dilute Cholesin to 6uM with buffer, and then dilute it with buffer in a 1:1 gradient to form 16 concentration gradients, take 10ul 50nM labeled wild-type and mutant GPR146 and mix them thoroughly with 10ul Cholesin of different concentrations, and let it stand at room temperature for 20min; 3) Use Monolith NT.115 capillary to absorb samples, place them on the sample stage in order from high to low Cholesin concentration, put them into micro-thermophoresis instrument for measurement, set the measurement parameters to 10% LED power and 40% MST power, use NanoTemper analysis software to analyze and calculate the experimental results to obtain the equilibrium dissociation constant kd value between Cholesin and wild-type and mutant GPR146 proteins.

The results are shown in FIG17 , which show that Cholesin has a high binding affinity to wild-type GPR146, with a Kd value of 21.34±0.98 nM, while Cholesin has a weakened binding to mutant GPR146, with a Kd value of 971.0±91.5 nM.

Example 6: Cholesin inhibits cAMP production via the GPR146-coupled Gαi signaling pathway

6.1 Cholesin inhibits the interaction between GPR146 and GNAI1

Having confirmed the interaction between Cholesin and GPR146, the downstream signaling of GPR146 was further investigated in mouse primary hepatocytes. GPR146 is a GPCR, and the G protein associated with the GPCR is a heterotrimer containing three different subunits: α, β, and γ. The binding of ligands to the GPCR causes a conformational change in the receptor, which in turn changes and binds to and activates the G protein. The active form of the G protein is then released from the receptor surface, dissociating into its α and β/γ subunits. Both subunits will then activate their specific effectors, which release the second messenger. These messengers are recognized by protein kinases, leading to their activation and triggering a signaling cascade toward cellular events. The Gαs and Gαi isoforms activate or inactivate adenylate cyclase, respectively, which converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), releasing inorganic pyrophosphate in the process. Guanine nucleotide binding protein G(i) subunit α1 (GNAI1) is a G protein inhibitory subunit, and the dissociation of Gαi inhibits the downstream cAMP signal. It is speculated that Cholesin promotes the dissociation of Gαi after binding to GPR146, thereby inhibiting downstream cholesterol synthesis by inhibiting cAMP. In order to verify the above hypothesis, an immunoprecipitation experiment was performed.

Primary hepatocytes from wild-type mice were isolated and adenovirus overexpressing GNAI1 with HA tag was added after the cells were attached to the wall. The cells were then divided into three groups, with empty adenovirus, wild-type GPR146 (WT) adenovirus with Flag tag, and mutant GPR146 (Mut) adenovirus with Flag tag added. 24 hours after adenovirus infection, each group was treated with 0nM and 200nM Cholesin for 10 minutes, and then immediately added RIPA lysis buffer to lyse the cells. 40μl of the lysed sample was added to 5×SDS loading buffer and boiled at 100℃ for 10min as the input sample, and the Flag-adsorbed beads were added to the cell lysate and incubated overnight on a 4℃ rotating mixer. After binding, centrifuge at 5000 rpm and 4°C for 3 min, discard the supernatant, add 600 μl of pre-cooled RIPA lysis buffer and vortex to remove the remaining liquid sample and proteins non-specifically bound to the beads, centrifuge and discard the supernatant, repeat washing 6 times to fully remove the residual liquid. 40 μl 1×SDS loading buffer was added and the sample was boiled at 100°C for 10 min, and then subjected to SDS-PAGE and immunoblotting. The immunoprecipitation results are shown in FIG18 .

The results showed that Cholesin treatment disrupted the interaction between wild-type GPR146 and GNAI1, while mutant GPR146 had no such effect, indicating that Cholesin disrupts the interaction between GPR146 and GNAI1.

6.2 Cholesin treatment reduces cAMP levels in wild-type primary hepatocytes

Primary hepatocytes from wild-type (Gpr146 ^+/+ ) and Gpr146 knockout (Gpr146 ^-/- ) mice were isolated and placed in 6-well plates. Four groups were set up: Gpr146 ^+/+ cells, Gpr146 ^-/- complemented empty vector (Vec) cells, Gpr146 ^-/- complemented wild-type GPR146 (WT) cells, and Gpr146 ^-/- complemented mutant GPR146 (Mut) cells. After primary hepatocytes adhered to the wall, adenoviruses complemented with wild-type and mutant GPR146 were added. After 24 hours of infection, each group was treated with 0nM or 200nM Cholesin for 10 minutes, and then the cell culture medium was immediately aspirated and the cAMP level in the cells was detected using Cayman's Cyclic AMP ELISA Kit. The experimental results are shown in Figure 19.

The results showed that cAMP levels in wild-type mouse primary hepatocytes decreased after Cholesin treatment, while Gpr146 ^-/- primary hepatocytes no longer responded to Cholesin treatment. After wild-type GPR146 was supplemented, Gpr146 ^-/-' s response to Cholesin was restored, while supplementation of mutant GPR146 still did not respond to Cholesin's inhibitory effect on cAMP. This indicates that Cholesin treatment reduces cAMP levels in primary hepatocytes.

The above results of Example 6 indicate that Cholesin inhibits the production of cAMP via the GPR146-coupled Gαi signaling pathway.

Example 7: Intraperitoneal injection of Cholesin reduces Hmgcr mRNA levels and plasma and liver cholesterol levels

7.1 Dose-dependent inhibition of Hmgcr mRNA levels in mouse liver by intraperitoneal injection of Cholesin

In this example, 10-12 week old wild-type male mice with a genetic background of C57BL/6J were randomly divided into 4 groups, with 10 mice in each group. The mice were fasted overnight, and injected with 0 mg/kg, 1 mg/kg, 5 mg/kg and 10 mg/kg of eukaryotic purified Cholesin-his protein on the second day. The mouse liver tissue was collected 6 hours after the mice were fed. Total RNA of mouse liver tissue was extracted, and qPCR was performed to detect the expression level of Hmgcr, and statistical analysis was performed with Rpl32 as an internal reference.

The results are shown in Figure 20. Injection of exogenous Cholesin can inhibit the expression of liver Hmgcr, and this inhibitory effect is dose-dependent.

7.2 Effects of intraperitoneal injection of Cholesin on Hmgcr mRNA levels in mouse liver and TC content in plasma and liver

Eight-week-old wild-type male mice with a genetic background of C57BL/6J were selected and divided into two groups. They were injected with 0 mg/kg and 5 mg/kg of eukaryotic purified Cholesin-his protein, respectively. The injections were given twice a week for two weeks. When the mice were 10 weeks old, the liver tissue and whole blood were collected after the mice were starved and fed for 6 hours. The operation process is shown in Figure 21A. Liver, total RNA of mouse liver tissue was extracted for qPCR to detect the expression level of Hmgcr; total cholesterol (TC) of the liver was also extracted to detect the level of TC in the extract. For whole blood of mice, plasma was separated by low-speed centrifugation, and the level of TC in plasma was detected. The Hmgcr mRNA level in mouse liver, the TC level in mouse plasma and liver are shown in Figures 21B, 21C and 21D, respectively.

The results showed that the injection of Cholesin could inhibit the expression of Hmgcr in mouse liver ( FIG. 21B ). The plasma TC level ( FIG. 21C ) and liver TC level ( FIG. 21D ) of mice decreased after the injection of Cholesin.

Example 8: Cholesin inhibits cholesterol synthesis via GPR146

In order to explore whether Cholesin inhibits cholesterol synthesis in mice through GPR146, 10-week-old wild-type (Gpr146 ^fl/fl ) mice and Gpr146 knockout (Gpr146 LKO) mice with a genetic background of C57BL/6J were selected for the experiment. Gpr146 LKO mice were divided into 3 groups at 10-12 weeks, with 12 mice in each group. One group was injected with adenovirus control (Vec) through the tail vein, one group was injected with adenovirus expressing wild-type GPR146 (WT), and the other group was injected with adenovirus expressing mutant GPR146 (Mut). The total amount of adenovirus injected into each mouse through the tail vein was 5×10^8. Each group was then divided into two small groups, with 6 mice in each group, and 0 mg/kg or 5 mg/kg Cholesin was injected twice a week for 4 consecutive times. The liver tissue and whole blood were collected after the mice were starved and fed for 6 hours at 14.5 weeks. The operation process is shown in Figure 22A.

For mouse liver, total liver RNA was extracted and then qPCR was performed to detect the expression levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk and Pmvk, and the results are shown in Figure 22B. At the same time, total liver protein was extracted and immunoblotted with antibodies to SREBP2, HMGCR, HMGCS1, LDLR, pKA Sub and HSP90, with HSP90 as the internal reference, and the results are shown in Figure 22C. Total cholesterol (TC) in the liver was also extracted to detect the level of TC in the extract, and the results are shown in Figure 22D. For mouse whole blood, low-speed centrifugation was performed to separate plasma, and the level of TC in plasma was detected, and the results are shown in Figure 22E.

As shown in the results of Figures 22B and 22C, Cholesin inhibits the expression of PKA and SREBP2 in wild-type mice, thereby reducing the expression of cholesterol synthesis-related genes and reducing liver and plasma TC levels. However, in GPR146 knockout mice, this effect of Cholesin was eliminated, and the effect of Cholesin was restored after adding wild-type GPR146, but the effect of Cholesin could not be restored after adding mutant GPR146.

These results suggest that Cholesin regulates cholesterol homeostasis by inhibiting hepatic SREBP2 expression through GPR146.

Example 9: Cholesin can prevent hypercholesterolemia and atherosclerosis

Ldlr ^-/- (Ldlr gene knockout) mice are currently a commonly used disease model for studying ^{atherosclerosis} . The plasma cholesterol of Ldlr-/- mice was significantly higher than that of wild-type mice, and atherosclerotic plaques were formed under the induction of Western diet (WD). Five-week-old Ldlr ^-/- mice were first fed a Western diet for 4 weeks, and then the mice were divided into four groups, with 6-7 mice in each group. The first group was administered with Cholesin alone by intraperitoneal injection (5 mg kg ^-1 , twice a week); the second group was administered with rosuvastatin (Ros) alone by drinking water (10 mg kg ^- 1day ^-1 ); the third group was administered with Cholesin (5 mg kg ^-1 , twice a week) and rosuvastatin (10 mg kg ^- 1day ^-1 ) in combination (Cholesin+Ros); the fourth group was the vehicle control group (Veh), and the specific operation is shown in Figure 23. During the period, the tail vein blood of mice was collected every two weeks to detect the plasma total cholesterol (TC) and triglyceride (TG) levels, and the weight and food intake of mice were detected. At the same time, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse plasma were also detected after 8 weeks of administration. After 8 weeks of continuous administration, the mice were killed, and the liver tissues were collected to detect the liver TC and TG levels, the expression levels of cholesterol synthesis-related genes in the liver, and the liver tissues were stained with HE to observe the lipid accumulation in the mouse liver. In addition, the whole aorta of the mice after 8 weeks of administration was stained with oil red to observe the changes in atherosclerotic plaques.

Plasma and liver TC and TG levels in mice

The changes in mouse plasma TC levels are shown in Figure 24A. The mice were able to significantly reduce the TC levels in the plasma of Ldlr ^-/- mice when they were given rosuvastatin or Cholesin alone. Among them, rosuvastatin was observed to slightly increase the plasma TC level when the drug dosage remained unchanged after 4 weeks of administration, indicating that there was a drug resistance problem in the later stage of rosuvastatin administration. The combined administration of rosuvastatin and Cholesin had the best effect on reducing plasma TC, which was better than the single administration of rosuvastatin or Cholesin. The combined administration could achieve a good inhibitory effect in the early stage of administration and maintain the inhibitory effect for a long time. The area under the curve (AUC) of the changes in plasma TC within 8 weeks was statistically analyzed in order to more intuitively compare the effects of the three treatment regimens. Among them, the single treatment of Cholesin and rosuvastatin could significantly reduce the TC level in plasma, and the inhibitory effect of the combined treatment of the two was more prominent, achieving a synergistic effect.

The TC level in the liver of mice after 8 weeks of administration is shown in Figure 24B. The TC level in the liver of mice was significantly reduced after administration of rosuvastatin or Cholesin. The combined administration of rosuvastatin and Cholesin had the best effect on reducing liver TC, which was better than the single administration of rosuvastatin or Cholesin, achieving a synergistic effect.

The TG levels in mouse plasma and liver are shown in Figures 24C and 24D. Cholesin and rosuvastatin alone can reduce the TG levels in plasma or liver, and the combined use of the two has the most significant inhibitory effect on TG in plasma or liver, indicating that the combination of the two achieves a synergistic effect.

Expression levels of cholesterol synthesis-related genes in mouse liver

The reduction in total cholesterol may be due to the inhibition of cholesterol synthesis, so the expression levels of cholesterol synthesis-related genes Hmgcs1, Hmgcr, Mvd, Mvk, Pmvk and Sqle in mouse liver and the transcription factor SREBP2 that regulates the expression of these genes were detected by qPCR and immunoblotting. The results are shown in Figures 25A and 25B.

The results showed that after 8 weeks of administration, the cholesterol synthesis-related genes and transcription factors in the liver of rosuvastatin-treated mice were significantly upregulated. The expression of SREBP2 was significantly upregulated in both groups, while the injection of Cholesin could significantly inhibit the expression of cholesterol synthesis-related genes and transcription factor SREBP2. When Cholesin and rosuvastatin were used in combination, the upregulation of cholesterol synthesis genes caused by statins was significantly alleviated, and the expression level of cholesterol synthesis-related genes was suppressed to a lower level.

Oil red staining of whole mouse aorta

In order to observe the overall pathological changes of mouse arteries, the whole aorta of mice was stained with Oil Red O. Oil Red O is a strong lipid staining agent that can specifically stain neutral triglycerides, lipids and lipoproteins red. Since atherosclerotic plaques are rich in lipids, the plaque part can be stained red. The proportion of the stained plaque area to the entire aorta is calculated to evaluate the pathological condition.

The specific experimental steps are as follows: 1) Anesthetize the mouse, fix it in supine position on a foam board, open the chest cavity to expose the heart, draw 20 ml of PBS into the syringe, insert the syringe needle into the left ventricle from the apex of the heart, cut a small hole in the right atrium, and manually push PBS for systemic perfusion. It can be seen that the color of the liver changes from red to yellow after perfusion. Leave the needle in place and draw 10 ml of 4% paraformaldehyde/PBS into the syringe to continue perfusion. After the fixative is injected, the mouse tail can be seen swinging slightly, indicating that the perfusion fixation effect is good; 2) Remove the mouse viscera, expose the spine, carefully peel off the heart and the entire aorta under a stereo microscope, cut the aorta below the bifurcation of the iliac artery, and cut the ascending aorta near the heart. After the entire aorta is peeled off, the fat tissue on the adventitia of the blood vessels must be carefully removed. Blunt peeling is mainly used to avoid damaging the blood vessels. The processed whole aorta is stored in 4% paraformaldehyde/PBS and wait for staining; 3) Take out the fully fixed aorta, use Vannas scissors to extend into the blood vessels from the fracture of the ascending aorta under a stereo microscope, and longitudinally dissect the aortic arch along the greater curvature of the aortic arch to the distal end, leaving the three branches untreated. The good aortic intima was unfolded upwards and fixed on a black-bottomed plastic plate with the smallest insect pin; 4) Prepare Oil Red O dye solution: weigh 0.5g of Oil Red O powder, add 100ml of isopropanol, and place in a water bath at 90℃ for 1 hour. After filtering, a saturated Oil Red dye solution was obtained. When using, the saturated solution and double distilled water were mixed in a ratio of 3:2, and then filtered again after a water bath at 42℃ for 10 minutes. This is the Oil Red working solution. Both the saturated solution and the working solution need to be stored at low temperature and away from light; 5) When staining, the sample was first pretreated in 60% isopropanol for 10 minutes, and then stained in the Oil Red working solution for 15 minutes. After staining, the sample was decolorized in 60% isopropanol for 5 minutes to reduce the background. The stained sample can be stored in the fixative for a long time; 6) Then, a Zeiss Stemi 508 stereo microscope was used to take images of the stained aorta. Image J software was used to quantify the lesion area of the thoracic aorta. ImageJ was used to measure the plaque area and the entire aortic intima area, and then the plaque area was divided by the entire intima area × 100% to obtain the relative percentage of plaques. Dividing by the intima area was mainly to eliminate the effects of differences in mouse body size.

The experimental results are shown in FIG26 . Obvious plaques can be seen in the vehicle control group. Rosuvastatin and Cholesin can both effectively inhibit arterial lesions when administered alone, and their combined use can achieve better therapeutic effects and realize a synergistic effect.

Mouse body weight and food intake

The changes in body weight and food intake of mice during the administration process are shown in Figures 27A and 27B, respectively. The results showed that there was no significant difference in body weight between the rosuvastatin group and the control group, while Cholesin alone and Cholesin combined with rosuvastatin Vastatin combined treatment can inhibit the increase of mouse body weight (Figure 27A). In addition, the food intake of mice was not affected during the administration process, and there was no significant difference among the four groups (Figure 27B), which also indicates that the weight change of mice is not caused by the difference in food intake of mice.

HE staining of mouse liver tissue

The accumulation of lipids in the liver was observed by HE staining of liver tissue. The specific operation was as follows: 1) A piece of mouse liver tissue was taken and fixed in 4% paraformaldehyde overnight; 2) Alcohol was used as a dehydrating agent from low concentration to high concentration to gradually remove the water in the tissue block, and then the tissue block was placed in xylene to make it transparent, and the alcohol in the tissue block was replaced with xylene, and then immersed in wax and embedded; 3) The transparent tissue block was placed in the melted paraffin, placed in a wax melting box for insulation, and embedded after the paraffin was completely immersed in the tissue block; 4) The embedded wax block was fixed on a microtome and cut into thin slices, generally 5μm, and placed in a 45℃ constant temperature box for drying after pasting; 5) The slices were placed in xylene I for 10min-xylene II for 10min-anhydrous ethanol I for 5min-anhydrous ethanol I for 5min Ethanol II 5min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5min-wash with distilled water; 6) Stain the slices with Harris hematoxylin for 3-8min, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, blue with 0.6% ammonia water, and rinse with running water; 7) Stain the slices with eosin staining solution for 1-3min; 8) Dehydrate and make transparent the slices in 95% alcohol I 5min-95% alcohol II 5min-anhydrous ethanol I 5min-anhydrous ethanol II 5min-xylene I 5min-xylene II 5min, take the slices out of xylene and dry them slightly, and seal them with neutral gum; 9) Examine under a microscope, collect and analyze images.

The HE staining results are shown in Figure 28. The white area in the cells is lipid accumulation. It can be observed that the control group has the most white lipid droplets in the cells, while the experimental groups have significantly weakened them, indicating that the lipid accumulation in the liver of mice after Cholesin administration is significantly reduced.

ALT and AST levels in mouse plasma

To evaluate the liver damage in mice after administration, the AST and ALT levels in the plasma of the four groups of mice were detected using an alanine aminotransferase (alanine aminotransferase/ALT/GPT) kit (Nanjing Jiancheng) and an aspartate aminotransferase (aspartate aminotransferase/AST/GOT) kit (Nanjing Jiancheng). The results are shown in Figures 29A and 29B.

The results showed that AST and ALT levels were significantly reduced after Cholesin was administered alone or in combination with rosuvastatin, indicating that Cholesin has an alleviating effect on liver damage.

The above results of Example 9 indicate that Cholesin, whether used alone or in combination with rosuvastatin, has a significant protective effect on hypercholesterolemia and atherosclerosis.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If any incorporated reference conflicts with this specification, the present specification shall prevail. In addition, any specific embodiment of the present invention that falls within the scope of the prior art may be specifically identified from any one or more of the claims. Because the embodiments are considered to be known to those skilled in the art, they may be excluded even if the exclusion is not explicitly listed in the present application. Any specific embodiment of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. In addition, many modifications may be made to adapt specific situations, materials, compositions, methods, process steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims.

Claims (33)

A method for reducing plasma cholesterol level and/or triglyceride level in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein. The method according to claim 1, wherein the plasma cholesterol is selected from the group consisting of plasma total cholesterol and plasma LDL-cholesterol. A method for inhibiting liver cholesterol synthesis in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein. The method according to claim 3, wherein the method inhibits the expression of genes related to cholesterol synthesis or uptake, and the genes are selected from Hmgcr, Hmgcs1, Mvd, Mvk, Pmvk, Sqle and Ldlr. The method according to claim 3 or 4, wherein the method inhibits the expression of a transcription factor that promotes the expression of a gene related to cholesterol synthesis or uptake, and the transcription factor is SREBP2. A method for inhibiting hepatic VLDL-cholesterol secretion in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein. A method for treating or preventing a disease associated with elevated plasma cholesterol levels in a subject, comprising the step of administering to the subject an effective amount of a Cholesin protein, a nucleic acid encoding the Cholesin protein, or an expression vector comprising the nucleic acid, or the step of enhancing the expression or activity of the subject's endogenous Cholesin protein. The method according to any one of claims 1 to 7, wherein the Cholesin protein is human Cholesin protein. The method according to any one of claims 1 to 8, wherein the nucleic acid is DNA or RNA. The method according to any one of claims 1 to 9, wherein the expression vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a plasmid, a DNA vector, an mRNA vector, a transposon-based vector and an artificial chromosome. The method according to any one of claims 7 to 10, wherein the disease associated with elevated plasma cholesterol levels is selected from hyperlipidemia (e.g., hypercholesterolemia, hypertriglyceridemia), atherosclerosis, cardiovascular disease (e.g., myocardial infarction, coronary heart disease), cerebrovascular disease (e.g., cerebral thrombosis, cerebral hemorrhage, cerebral infarction), hyperglycemia, diabetes, obesity, hypertension, fatty liver, cirrhosis, nephrotic syndrome and gallstones. The method according to claim 11, wherein the disease associated with elevated plasma cholesterol levels is hypercholesterolemia. The method according to claim 11, wherein the disease associated with elevated plasma cholesterol levels is atherosclerosis. The method according to any one of claims 1 to 13, wherein the method further comprises the step of administering a second therapeutic agent, preferably, the second therapeutic agent is selected from proteins or peptides, nucleic acids and small molecule drugs. The method of claim 14, wherein the second therapeutic agent is a cholesterol-lowering drug selected from the group consisting of an HMGCR inhibitor, a PCSK9 inhibitor, and a cholesterol absorption inhibitor. The method of claim 15, wherein the HMGCR inhibitor is a statin. The method according to claim 16, wherein the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin. The method according to claim 15, wherein the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran and Tafolecimab. The method according to claim 15, wherein the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably Choose ezetimibe or hebomibe. The method according to any one of claims 14-19, wherein the second therapeutic agent is administered before, after or simultaneously with the Cholesin protein, the nucleic acid or the expression vector. The method according to any one of claims 1-20, wherein the subject is a human. The method according to any one of claims 1-14 and 20-21, wherein the subject is unresponsive or intolerant to one or more of a cholesterol absorption inhibitor, an HMGCR inhibitor, and a PCSK9 inhibitor, e.g., the subject has an LDL receptor (LDLR) defect. The composition comprises a Cholesin protein, a nucleic acid encoding the Cholesin protein, an expression vector comprising the nucleic acid, or an agent for enhancing the expression or activity of human endogenous Cholesin protein, and a pharmaceutically acceptable carrier or excipient. The composition according to claim 23, wherein the composition is used for one or more of the following: 1) reducing plasma cholesterol levels in a subject; 2) reducing plasma total cholesterol or plasma LDL-cholesterol levels in a subject; 3) reducing plasma triglyceride levels in a subject; 4) inhibiting hepatic cholesterol synthesis in a subject; 5) inhibiting hepatic VLDL-cholesterol secretion in a subject; and 6) Treating or preventing a disease associated with elevated plasma cholesterol levels in a subject. The composition according to claim 23 or 24, wherein the Cholesin protein is human Cholesin protein. The composition according to any one of claims 23-25, wherein the composition further comprises a second therapeutic agent, preferably, the second therapeutic agent is selected from proteins or peptides, nucleic acids and small molecule drugs. The composition of claim 26, wherein the second therapeutic agent is selected from cholesterol-lowering drugs. The composition according to claim 27, wherein the cholesterol-lowering drug is selected from HMGCR inhibitors, PCSK9 inhibitors and cholesterol absorption inhibitors. The composition of claim 28, wherein the HMGCR inhibitor is a statin. The composition according to claim 29, wherein the statin is selected from rosuvastatin, lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, cerivastatin and pitavastatin, preferably rosuvastatin. The composition according to claim 28, wherein the PCSK9 inhibitor is selected from Evolocumab, Alirocumab, Inclisiran and Tafolecimab. The composition according to claim 28, wherein the cholesterol absorption inhibitor is an NPC1L1 inhibitor, preferably ezetimibe or hebomibe. The composition according to any one of claims 23 to 32, wherein the composition is formulated as an injection preparation or an oral preparation.