WO2019037222A1 - Use of gpr31 inhibitor in drug preparation - Google Patents

Abstract

The invention provides the use of the GPR31 inhibitor in the preparation of the treatment of ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, cardiac inflammatory diseases, abnormal fat metabolism and related diseases, and provides a specific GPR31 interference targeting sequence. And shRNA sequences.

Description

Application of GPR31 inhibitor in pharmacy Technical field

The invention belongs to the field of biomedical technology, and particularly relates to the use of a GPR31 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, fat Metabolic abnormalities and related diseases.

Background technique

G protein-coupled receptor (GPCR) is a type of membrane protein with 7 transmembrane structures. It has more than 800 family members and is the largest membrane protein in mammalian genome. In humans, GPCRs are widely expressed in tissues and organs such as the cardiovascular system, immune system, and nervous system, and participate in growth and development and various pathophysiological processes. Due to its wide distribution and versatility, GPCR family molecules are regarded as the most potential drug development targets at present, and about 20-30% of FDA-approved marketed drugs use GPCR as a target.

The orphan receptor GPR31 is a GPCR family molecule, first discovered by Alessandra Zingoni et al. in 1997 (Zingoni, A. et al. Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor. Genomics 42,519-523 , doi: 10.106/geno. 1997.4754 (1997)). At present, the research on GPR31 is mainly concentrated in the field of cancer, and its function in other pathological processes is still unclear. GPR31 protein contains 319 amino acids with a molecular weight of 35KDa and is highly expressed in platelets, immune cells and various cancer cells, including bladder cancer cells, breast cancer cells, chronic lymphoblastic leukemia cells, etc. (Feinmark, SJet al. The Orphan Receptor GPR31 Is the Platelet and HUVEC Receptor for 12(S)-HETE.Blood 114(2008).). Studies have shown that GPR31 expression in clinical patients with colon cancer tissue is significantly higher than adjacent normal tissues, and is positively correlated with cancer metastasis rate, and negatively correlated with 5-year survival rate (Zou, Y. et al. [Expression and clinical significance Of G protein-coupled receptor 31 in colorectal cancer tissue].Zhonghua Wei Chang Wai Ke Za Zhi 18, 935-940 (2015).). GPR31 binds to RAS family molecules such as KRAS, HRAS, NRAS, etc., and regulates the cell membrane localization of KRAS, thereby participating in the development of cancer (Fehrenbacher N, et al. The G protein-coupled receptor GPR31 promotes membrane association of KRAS [J] .J Cell Biol, 2017: jcb.201609096.).

Ischemia-Reperfusion Injury (IRI) is the first concept proposed by Jennings in 1960. It refers to blood reperfusion after tissue and organ ischemia, which not only fails to restore the function of tissues and organs, but also increases the dysfunction and structure of tissues and organs. damage. Ischemia-reperfusion injury can occur in many important organs including heart, liver, lung, kidney, gastrointestinal tract, and the like.

Hepatic Ischemia Reperfusion Injury (HIRI) is a common pathological process in liver surgery. It is more common in pathological and physiological processes such as shock, liver surgery requiring liver blood flow, and liver transplantation. In recent years, with the development of clinical treatment technology, liver transplantation, thrombolytic therapy and hepatic occlusion surgery have been carried out more and more, although liver protection, surgical techniques and intraoperative monitoring are improving, but ischemia and reperfusion The liver injury is still the main cause of postoperative organ dysfunction, graft failure and even patient death. After the liver undergoes ischemia-reperfusion, liver tissue cells undergo a series of metabolic, structural and functional damages, which are easy to induce liver failure, which is one of the main factors affecting disease prognosis, surgical success rate and patient survival rate.

Acute coronary artery obstructive disease is one of the main causes of death of cardiovascular and cerebrovascular diseases. Despite significant advances in the treatment of bypass surgery, intervention, and thrombolysis, the mortality rate of patients with acute myocardial infarction is still high. One of the most important reasons is that there is no effective way to inhibit the blood flow of ischemic myocardium. Caused by ischemia-reperfusion injury. Myocardial reperfusion injury occurs in coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, and coronary artery bypass surgery. Myocardial reperfusion injury is mainly caused by free radicals, calcium overload, cell adhesion molecule-mediated neutrophil adhesion, aggregation, exudation, apoptosis, nitric oxide, complement system, renin-angiotensin. , nuclear factor-κB and so on.

The kidney is also a high perfusion organ, sensitive to ischemia and ischemia-reperfusion. Renal ischemia-reperfusion injury is an important injury link of ischemic acute renal failure, and also a limiting factor affecting early recovery of renal function in kidney transplantation. There are many related factors leading to ischemia-reperfusion injury. Among them, the inflammatory cascade caused by ischemia-reperfusion and reactive oxygen species oxidative damage are the two main factors of concern. At present, the prevention and treatment strategies for renal ischemia-reperfusion injury mainly include: inhibition of leukocyte activation and leukocyte-endothelial cell interaction, neutralization of reactive oxygen species, and anti-endothelin.

The mechanism of ischemic brain injury involves at least the following aspects: excitotoxicity, depolarization around the infarct, inflammation, and apoptosis.

Cardiac hypertrophy is an increase in the volume and weight of cardiomyocytes produced by the heart to accommodate various stimuli. His pathological changes include cardiomyocyte hypertrophy, myocardial stromal cell proliferation, and cardiac extracellular matrix alterations, ie, myocardial remodeling. The traditional view is that mature cardiomyocytes end differentiated, lose mitotic ability, and cannot enter the cell cycle; however, there is evidence that there are two processes of cardiomyocyte apoptosis and proliferation in cardiac development and pathology to maintain the steady state of cardiac function. . There are many diseases that cause cardiac hypertrophy in the clinic, such as primary or secondary hypertension, myocardial infarction, valvular disease, and congenital heart disease. Although early cardiac hypertrophy is conducive to maintaining normal heart function, but cardiac hypertrophy itself can increase myocardial oxygen consumption, reduce myocardial compliance, it will lead to heart failure for a long time, increase the incidence of sudden death. At present, it is believed that mechanical and neurohumoral factors induce cardiac hypertrophy, including renin-angiotensin system components, catecholamines, insulin-like growth factors, and nitric oxide synthesis systems. At the cellular and molecular level, the process of cardiomyocyte hypertrophy mainly includes four aspects: the appearance of stimulation signals, transmembrane signaling, immediate activation of early response genes, and expression of functional or structural proteins to the "embryonic" phenotype. The MAPK family signaling pathway, Ca ²⁺ and its dependent signaling pathway, phosphatidylinositol 3-kinase and its mediated signaling pathway, and JAK/STAT pathway were all involved in the signaling of cardiac hypertrophy, and between the pathways. There are also inextricably linked networks that form intricate signals (Dai Wenjian et al., Advances in Molecular Mechanisms of Cardiac Hypertrophy, Advances in Cardiovascular Diseases, Vol. 30, No. 1, 2009, 47-50).

The liver plays an important role in the body's fat metabolism. It participates in many important aspects of lipid metabolism, including fatty acid uptake and synthesis, lipid processing, storage, oxidative decomposition and export. When the amount of fatty acids obtained by the liver exceeds its processing capacity, lipids are deposited in the liver cells in the form of triglycerides, resulting in hepatic steatosis, becoming simple liver steatosis, and then developing into nonalcoholic steatohepatitis. Patients can progress to liver fibrosis, cirrhosis, and even liver cancer (Lu Ran et al., the pathogenesis of non-alcoholic fatty liver disease caused by lipid metabolism disorder, Journal of Clinical Hepatology, 2015, Vol. 31, No. 7, 1050-1054 ). In a similar situation, when the level of free fatty acids in the blood increases or the intracellular fat content increases, more than the storage capacity of adipose tissue and the oxidative capacity of various tissues for free fatty acids, excessive free fatty acids are deposited in the form of triglycerides in insulin. Target tissues such as adipose tissue, muscle and liver cause insulin resistance, which triggers a series of metabolic disorders and related diseases such as type II diabetes, metabolic syndrome, cardiovascular and cerebrovascular diseases, etc. (Chen Jinzhong, etc., lipid metabolism disorder and insulin Progress in related research on resistance, Chinese Practical Medicine, Vol. 3, No. 7, 2008, 147-149).

Summary of the invention

The experimental study of the present invention found that the table of GPR31 protein in tissues with the prolongation of tissue ischemia time There is a positive correlation between the amount of up-regulation and the severity of tissue ischemia-reperfusion injury, suggesting that GPR31 may be a novel regulator of ischemia-reperfusion injury in tissues or organs.

Overexpression of GPR31 aggravates the activity of hepatocytes, cardiomyocytes and kidney cells caused by hypoxia and reoxygenation, and promotes the inflammatory response of the corresponding cells, indicating that GPR31 can promote ischemia, reperfusion injury of liver, heart and kidney. And the development of other inflammatory reactions that occur in these organs.

In addition, the decrease of GPR31 expression in hepatocytes can inhibit cell lipid deposition induced by palmitate and oleic acid stimulation, indicating that GPR31 is expected to be a novel target for regulating hepatocyte fat accumulation, and is applied to abnormal fat metabolism and related diseases. During treatment.

Overexpression of GPR31 in cardiomyocytes aggravates cardiomyocyte hypertrophy caused by angiotensin II, indicating that GPR31 can promote the development of cardiac hypertrophy-related diseases.

On this basis, GPR31 can be used as a therapeutic target for ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, abnormal fat metabolism and related diseases.

The technical solution of the present invention is as follows:

A first aspect of the present invention provides a use of a GPR31 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and a related disease thereof, cardiac hypertrophy and a related disease thereof, an inflammatory disease of the heart, or Abnormal fat metabolism and related diseases.

According to the present invention, the ischemia-reperfusion injury and related diseases are selected from the group consisting of hepatic ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia-reperfusion injury and related diseases, And/or cerebral ischemia-reperfusion injury and related diseases. The ischemia-reperfusion injury may be caused by various reasons such as organ transplantation, partial or complete resection of tissue, and tissue ischemia caused by vascular embolization.

Inflammatory factors of hepatic ischemia-reperfusion injury and related diseases include, but are not limited to, liver cysts, liver transplantation, thrombolytic therapy, and hepatic occlusion.

Inflammatory factors of cardiac ischemia-reperfusion injury and related diseases include but are not limited to: myocardial infarction, myocardial infarction injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, coronary Arterial bypass.

Causes of renal ischemia-reperfusion injury and related diseases include, but are not limited to, kidney transplantation, renal cysts, and renal vascular surgery.

The triggering factors of cerebral ischemia-reperfusion injury and related diseases include but are not limited to: stroke, cerebrovascular surgery and the like.

Preferably, the ischemia-reperfusion injury and related diseases are hepatic ischemia-reperfusion injury, cardiac ischemia-reperfusion injury, renal ischemia-reperfusion injury, and/or cerebral ischemia-reperfusion injury.

According to the present invention, the cardiac hypertrophy and related diseases include, but are not limited to, cardiac hypertrophy, heart failure, arrhythmia, arterial embolism, coronary heart disease, angina pectoris, heart block, and the like.

There are many diseases that cause cardiac hypertrophy, such as primary or secondary hypertension, myocardial infarction, valvular disease, and congenital heart disease.

Preferably, the cardiac hypertrophy and related diseases are cardiac hypertrophy and heart failure.

Inflammatory diseases of the heart include, but are not limited to, myocarditis, endocarditis.

According to the present invention, the abnormalities in fat metabolism and related diseases include, but are not limited to, insulin resistance, metabolic syndrome, nonalcoholic fatty liver disease, obesity, diabetes, hyperglycemia, hyperlipemia, and the like.

The spectrum of nonalcoholic fatty liver disease includes: simple liver steatosis, nonalcoholic steatohepatitis, liver fibrosis, cirrhosis, and liver cancer.

Preferably, the abnormal fat metabolism and related diseases are nonalcoholic fatty liver disease, obesity, hyperlipemia, insulin resistance, and more preferably: simple liver steatosis, nonalcoholic steatohepatitis, obesity, hyperlipidemia.

According to the present invention, the GPR31 inhibitor may be an inhibitor that inhibits GPR31 protein activity or protein level, or an inhibitor that inhibits mRNA levels of GPR31. The inhibitory activity can be reversible or irreversible.

Inhibitors that inhibit GPR31 protein activity or protein levels include, but are not limited to, antibodies to GPR31, proteins, polypeptides, enzymes, natural compounds, synthetic compounds, organics, inorganics that inhibit GPR31 protein activity or protein levels. The inhibitor that inhibits GPR31 protein activity or protein level refers to a substance that binds to GPR31 but does not produce a biological response upon binding. The inhibitor can block, inhibit or attenuate the agonist-mediated response and can compete with the agonist for binding to GPR31.

The inhibitor that inhibits the mRNA level of GPR31 may be an antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA, or other protein, polypeptide, enzyme, or compound capable of inhibiting the mRNA level of GPR31.

According to the invention, the antibodies include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv) (including bispecific) scFv), single chain antibody, Fab fragment, F(ab') fragment, disulfide-linked Fv (sdFv) and any of the above epitope binding Fragment. In particular, antibodies useful in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecule used in the present invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class of immunoglobulin molecules (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) Or subclass. Preferably, the antibody is a human or humanized monoclonal antibody. As used herein, a "human" antibody includes an antibody having an amino acid sequence of a human immunoglobulin, and includes an antibody isolated from a human immunoglobulin library or from a mouse or other animal that expresses the antibody from a human gene.

In one embodiment of the present invention, the inhibitor is a shRNA of mRNA of GPR31, and the interference targeting sequence is CACTCTCCTGCCTTCAGTTTG.

Preferably, the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'.

According to the invention, the medicament further comprises a pharmaceutically acceptable adjuvant.

The pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrators, etc. .

The diluent is, for example, lactose, starch, cellulose derivative, inorganic calcium salt, sorbitol or the like. The binder is, for example, starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone or the like. The antioxidant is, for example, vitamin E, sodium hydrogen sulfite, sodium sulfite, butylated hydroxyanisole or the like. The pH adjusting agent is, for example, hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate or the like. The preservative is, for example, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, m-cresol, benzalkonium chloride or the like. The lubricant is, for example, magnesium stearate, finely divided silica gel, talc, or the like. The disintegrant is, for example, starch, methyl cellulose, xanthan gum, croscarmellose sodium or the like.

The dosage form of the medicament of the present invention may be in the form of an oral preparation, such as a tablet, a capsule, a pill, a powder, a granule, a suspension, a syrup, etc.; or a dosage form for injection administration, such as an injection solution, a powder injection, etc., Intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage form forms used are well known to those of ordinary skill in the pharmaceutical arts.

The medicament of the present invention can be administered to a subject by a route known in the art including, but not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal. Cheek, Sublingual, intranasal, transdermal, etc.

The dosage administered will depend on the age, health and weight of the recipient, the type of combination, the frequency of treatment, the route of administration, and the like. The drug can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three or four times daily. The dose can be administered one or more times, and the administration time can be from one day to several months or longer.

According to the present invention, the medicament can also be used in combination with other drugs which can ameliorate or inhibit diseases associated with ischemia-reperfusion injury.

According to the present invention, the medicament can also be used in combination with other drugs which can ameliorate or inhibit cardiac hypertrophy and related diseases.

According to the present invention, the medicament can also be used in combination with other drugs which can ameliorate or inhibit inflammatory diseases of the heart.

According to the present invention, the drug can also be used in combination with other drugs which can ameliorate or inhibit abnormalities in fat metabolism.

A second aspect of the present invention provides a use of a vector for expressing a shRNA targeting mRNA of GPR31 for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases thereof , inflammatory diseases of the heart, or abnormalities in fat metabolism and related diseases.

The disease is as defined above.

The target sequence for the shRNA interference is CACTCTCCTGCCTTCAGTTTG.

Preferably, the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'.

The vector may be an expression vector. A promoter and a transcription termination sequence operably linked to the above shRNA sequence may be included in the expression vector.

The expression vector can be a eukaryotic expression vector.

The eukaryotic expression vector can be a plasmid expression vector or a viral expression vector.

The plasmid expression vector may be, but not limited to, pcDNA3.1+/-, pcDNA4/HisMax B, pSecTag2 A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1(-)/myc-His A, pcDNA6-Myc/His B, pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-myc, pCMV-HA, pIRES-puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6 hygro, pSilencer3.1-H1hygro , pSilencer3.1-H1neo, pSilencer4.1-CMV neo.

The viral expression vector may be a lentiviral vector, an adenoviral vector, an adeno-associated virus expression vector or other types of viral vectors, including but not limited to pLKO.1, pLVX-IRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCV-MCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper, pAAV-LacZ, preferably pLKO.1 vector.

A third aspect of the present invention provides a use of a lentiviral vector comprising a shRNA targeting mRNA of GPR31 for the preparation of a medicament for treating ischemia-reperfusion injury and a related disease thereof, cardiac hypertrophy and Related diseases, inflammatory diseases of the heart, or abnormalities in fat metabolism and related diseases.

The disease is as defined above.

The shRNA-interfering targeting sequence is CACTCTCCTGCCTTCAGTTTG or other targeting sequences that can interfere with GPR31 expression.

Preferably, the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'.

Preferably, the lentiviral vector is a pLKO.1 vector.

A pharmaceutical carrier which can be used for the preparation of the medicament of the second and third aspects may be an injection vehicle conventionally used in the art, such as an isotonic NaCl solution, an isotonic glucose solution, or isotonicity. A solution containing a buffer system, such as a PBS solution or the like. It is also possible to selectively add a protective agent which is inactivated by preventing physical or chemical changes of the lentivirus, such as a divalent cation salt or a surfactant, depending on the needs of the preparation.

DRAWINGS

Figure 1: Western-blot detection of GPR31 protein expression in liver tissue at different ischemic times. In the figure, GAPDH is a control standard.

Figure 2: Identification of GPR31 protein expression after L02 cells were transfected with GFP and GPR31 overexpression lentivirus. In the figure, GAPDH is a control standard.

Figure 3: Statistical analysis of LDH release test results after L02 cells were overexpressed and normally expressed GPR31, respectively (n.s. represents P≥0.05, ** represents P<0.01).

Figure 4: Statistical analysis of RT-PCR results of mRNA levels of inflammatory factors Il-6, Tnf-α, and chemokine Cxcl2 after L02 cells were overexpressed and normally expressed GPR31, respectively. * represents 0.01 ≤ P < 0.05, ** represents P < 0.01).

Figure 5: Identification of GPR31 protein expression in H9C2 cells transfected with GFP and GPR31 overexpression lentivirus.

Figure 6: Statistical results of cell viability after hypoxia and reoxygenation in H9C2 cells under overexpression and normal expression of GPR31 (ns represent P≥0.05, * represents 0.01≤P<0.05, ** represents P <0.01).

Figure 7: Identification of GPR31 protein expression after HK2 cells transfected with GFP and GPR31 overexpression lentivirus.

Figure 8: Statistical analysis of the results of LDH release assay after hypoxia and reoxygenation in HK2 cells under overexpression and normal expression of GPR31 (n.s. represents P≥0.05, ** represents P<0.01).

Figure 9: Identification of GPR31 gene mRNA in L02 cells transfected with shRNA and shGPR31 lentivirus (** represents P<0.01).

Figure 10: Microscopic examination of hepatocyte oil red O staining after stimulation with palmitate (PA) and oleic acid (OA) (PA 0.5 mM + OA 1 mM) in L02 cells with low expression and normal expression of GPR31, respectively. Figure. "PA+OA" stands for palmitate and oleic acid stimulating groups.

Figure 11A: H9C2 cells were treated with angiotensin II in the presence of overexpressed and normally expressed GPR31, and then examined by microscopy.

Figure 11B: Statistical diagram of cell surface area after treatment with angiotensin II in H9C2 cells overexpressing and normal expression of GPR31 (n.s. represents P ≥ 0.05, ** represents P < 0.01).

Figure 11C: Statistical analysis of RT-PCR results of mRNA expression levels of cell hypertrophic marker genes Anp and Myh7 after treatment with angiotensin II in H9C2 cells overexpressing and normal expression of GPR31 (ns represent P≥0.05) , ** represents P <0.01).

Detailed ways

The invention will be further described below in conjunction with the embodiments. It should be noted that the embodiments are not intended to limit the scope of the invention, and those skilled in the art understand that any modifications and variations made on the basis of the invention are within the scope of the invention.

The chemical reagents used in the following examples are all conventional reagents and are commercially available. Experimental methods not specifically described are by conventional methods known in the art.

The animal model used in the following examples and the detection methods of each research index:

Experimental animals: Wild type mice (purchased from Beijing Huakangkang Biotechnology Co., Ltd.) of 8-10 weeks old, weighing 24 g-27 g, and background male C57BL/6 strain were selected as experimental subjects.

Animal feeding: All experimental mice were housed in the SPF laboratory animal center of Wuhan University. Breeding conditions: room temperature between 22-24 ° C, humidity between 40-70%, alternating light and dark lighting time is 12h, free to drink water.

HEK293T, human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu43.

L02, human hepatocyte cell line, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu6.

H9C2, rat cardiomyocytes, purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNR5.

HK2, human renal proximal tubule cells, purchased from the Chinese Academy of Sciences Cell Bank, catalog number SCSP-511.

The cells were cultured in DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin). Culture environment: 37 ° C, 5% CO ₂ .

Construction of mouse ischemic/reperfusion (I/R) injury model:

1) Fasting the mice 12 hours before surgery, free to drink water.

2) After successful anesthesia with 3% pentobarbital sodium before surgery, the patient was placed in a flat position, and the abdomen area of the mouse was shaved with a shaver, and treated with 10% iodine and 75% ethanol. District disinfection.

3) Take the midline incision into the abdomen and expose the liver pedicle of the left and middle leaves of the liver.

4) The portal vein and hepatic artery of the middle and left lobe were clamped with non-invasive vascular clamps, so that about 70% of the liver was ischemia. After 0.5 min, compared with the non-blocked right lobe, the blocking leaves turned white, indicating that the obstruction The success was successful and the ischemia was maintained for 60 min (the mice in the Sham group were operated in parallel with the mice in the surgery group, but no blood flow was blocked).

5) Remove the blood vessel clamp after 60 minutes of ischemia, restore the ischemic liver blood flow, close the abdominal cavity, and the mice after surgery Keep it alone and observe.

Materials: 1 hour after operation, sham operation group (Sham) and ischemia-reperfusion group mice, 3% pentobarbital sodium anesthesia, take the liver tissue of the ischemic area, immediately put into liquid nitrogen for more than 30min, then save It was used in RT-PCR and Western blot analysis in a -80 °C refrigerator.

RT-PCR

1) Extraction of RNA from cells

1 Collect the cells and wash them twice with PBS buffer. After completion, add 1ml of TRizol, blow evenly with a pipette, inhale into a 1.5ml centrifuge tube, shake the mixture for 30s, and let stand at room temperature for 5min to make the nuclear protein completely from the nucleic acid. Dissociation.

Centrifuge at 12000r/min for 24min at 24°C, take the supernatant, add 200μl of chloroform, vortex the mixer for 30s, and let stand on the ice box for 10min;

Centrifuge at 34 ° C 12000r / min for 15min, take the supernatant, add 0.5ml of isopropanol, mix well, let stand on the ice box for 10min, so that the RNA is fully precipitated;

Centrifuge at 44 ° C 12000 r / min for 15 min, discard the supernatant, add 1 ml of pre-cooled 75% ethanol, vortex mixer for 30s to wash the RNA precipitate;

Centrifuge at 54 ° C 12000 r / min for 5 min, discard the supernatant, and precipitate quickly and air dry. The extracted RNA was dissolved in an appropriate amount of DEPC deionized water.

2) Reverse transcription

Reverse transcription experiments were performed according to the kit instructions using the Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche, Basel, Switzerland) reverse transcription kit.

Western blot

1) Tissue protein extraction

1 Add 3-4 steel balls to the EP tube pre-cooled in dry ice, and add the tissue sample after weighing and quantification.

2 Add PMSF to the lysate, mix and add to the sample, and shake quickly.

3 The sample was ground in a -80 ° C pre-cooled grinder adapter with a grinding parameter set to 30 Hz/s for 90 s.

4 After the completion of the grinding, the ice was placed on the ice for 10 minutes, and the steel balls were taken out.

5 The ultrasonic pyrolyzer lysed the sample (5 KHz/time, 1 s each time, interval 1 s, repeated 10 times), and placed on ice for 10 min after completion of the ultrasound.

6 samples were placed in a 4 ° C pre-cooled centrifuge and centrifuged at 12000 rpm / min for 30 min.

7 The supernatant was transferred to a new EP tube and centrifuged at 14,000 rpm/min for 10 min at 4 °C.

8 Pipette the supernatant and transfer to a new EP tube and continue centrifugation. Centrifuge at 14000 rpm/min for 5 min at 4 °C.

9 Accurately aspirate the supernatant and quantify the protein using the BCA Protein Assay Kit (PierecTM, 23225).

2) Protein extraction in cells

The cells were added to the lysate, and after the completion of the lysis, the supernatant was centrifuged, and the protein sample was quantitatively collected using the BCA Protein Assay Kit.

3) Loading and electrophoresis

Configure the electrophoresis gel and add the electrophoresis solution to the electrophoresis tank. The protein sample was loaded into the SDS-PAGE gel well and the electrophoresis was started after the spotting was completed.

4) Transfer film

1 Prepare the transfer solution and pre-cool at 4 °C.

The 2PVDF membrane was immersed in methanol for 15 s before use, and then placed in a transfer solution for use.

3 The gel in the gel plate was taken out, and the gel was washed with a transfer liquid to cover the PVDF film.

4 The film voltage was set to 250V, the current was set to 0.2A, and the transfer was 1.5h.

5 After the transfer is completed, the PVDF membrane is taken out.

5) Closed

Place the protein film in the pre-prepared TBST and wash off the transfer solution on the membrane. The protein membrane was placed in a blocking solution and shaken slowly on a shaker at room temperature for 1-4 h.

6) Primary antibody incubation

1 Wash the protein membrane 3 times with TBST for 5 min each time.

2 Sealing machine seals the film into the hybrid bag, and adds a primary antibody to seal it.

3 Place the hybrid bag in a 4 ° C shaker overnight.

7) Secondary antibody incubation

1 The film was taken out and washed three times with TBST for 5 minutes each time, and the primary antibody was recovered.

2 Place the membrane in the corresponding secondary antibody dilution with secondary antibody and incubate for 1 h in the dark.

8) Protein detection

After incubation, wash 3 times with TBST for 5 min each time. The target band was detected using a Bio-Rad Chemi Doc XRS+ gel imaging system.

Construction of GPR31 overexpression plasmid:

1) PCR amplification of the GPR31 gene, the primers are:

Forward: 5'-ACACCGGCGGCCACGCGTATGCCATTCCCAAACTGCTC-3';

Reverse: 5'-GGAGGTACCTCCGGATCCTTACTTATCGTCGTCATCCTTG-3';

2) The PCR product is subjected to agarose gel electrophoresis, followed by recovery of the DNA fragment using a DNA gel recovery kit (Tiangen);

buffer或者

Green buffer、ddH₂O混合均匀(50μl体系)，置于37℃条件下反应。使用

AxyPrep^TMPCR Clean-Up Kit(Axygen)回收酶切产物。3) the resulting DNA product and restriction endonuclease FastDigest restriction enzymes (Thermo),

Buffer or

The Green buffer and ddH ₂ O were uniformly mixed (50 μl system) and placed at 37 ° C for reaction. use

^{AxyPrep TM PCR Clean-Up Kit (} Axygen) cleavage product recovered.

PCR一步定向克隆试剂盒(Novoprotein)，按照试剂盒说明书进行重组反应；4) use

PCR one-step cloning kit (Novoprotein), according to the kit instructions for recombination reaction;

5) Making E. coli competent cells, performing the transformation test on the above-mentioned ligation products, plating the plates, placing them in a 37 ° C incubator, and culturing overnight;

6) The overnight cultured plate was taken out from the 37 ° C incubator, and the colony was shaken and the colony PCR positive clone was detected.

7) 5-10 μl of the bacterial solution identified as positive by PCR was inoculated into 5 ml of LB (containing resistant) medium, and cultured overnight at 220 rpm, 37 ° C in a shaker.

8) The bacterial culture solution was taken out overnight, and the turbid bacterial liquid was subjected to plasmid extraction (Tiangen Plasmid DNA Mini Kit).

9) The extracted plasmid can be directly used for GPR31 transient transfer or construction of a lentiviral stable cell line.

GPR31 interference plasmid construction

1) GPR31 targeting interference sequence is CACTCTCCTGCCTTCAGTTTG, designing oligonucleotides suitable for pLKO.1 vector; forward oligonucleoside Acid: 5'CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG3'; reverse oligonucleotide: 5'AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG3'; negative control siRNA sequence: CAACAAGATGAAGAGCACCAA;

2) dissolving the above two oligonucleotides in half by adding sterile water to a final concentration of 100 mM for fusion;

3) according to the "GPR31 expression plasmid construction" step of enzymatic cleavage reaction, recovery of the enzyme digestion product, ligation reaction, transformation, single-collection and sequencing, extraction of plasmid;

4) The resulting plasmid can be used for lentiviral-mediated GPR31 knockdown cell line construction.

Lentiviral vector construction and packaging

1) The 293T cells were digested with trypsin and transferred to a 6-well plate at 1 × 10 ⁶ 293 T/well.

2) Transfection was started when the cell confluence reached 80%.

3) Take 1.5 ml of sterilized EP tube, add 2 packaging plasmids (pSpax and pMD2G) and 1 μg of each of the overexpressing or interference plasmids in 100 μl of serum-free medium. Gently mix and incubate for 5 min at room temperature.

4) A 1.5 ml sterile EP tube was taken, and 3 μl of PEI (1.6 μg/μl) was dissolved in 100 μl of serum-free medium. Gently mix and incubate for 5 min at room temperature.

5) Gently mix the DNA solution and the PEI solution. Incubate for 15 min at room temperature.

6) The above DNA-PEI mixture was added dropwise to a 6-well plate.

7) After 6 hours of transfection, replace with fresh medium.

8) The virus-containing supernatant was harvested 48-72 h after transfection. After centrifugation at 3000 rpm for 10 min, the precipitate was removed and filtered through a 0.45 μm filter.

9) The filtered virus can be used immediately for infection or storage at -80 °C.

Cell hypoxia reoxygenation (H/R):

1) The cells were cultured to log phase, washed twice with pre-warmed PBS, and discarded.

2) The cells were divided into normal control group and H/R experimental group. The control group was changed to complete medium, and cultured at 37 ° C, 5% CO ₂ . The experimental group was changed to glucose-free and serum-free DMEM medium, and O ₂ /CO was placed. ₂ Cell culture system in an incubator (37 ° C, 5% CO ₂ , 1% O ₂ ) hypoxia culture, after hypoxia for different time, the experimental group was replaced with complete medium reoxygenation culture.

3) After reoxygenation to a predetermined reoxygenation culture time, the supernatant was discarded, washed twice with PBS, and the collected cells were stored at -80 ° C until use.

LDH release and cell viability assay:

The amount of LDH released was measured using an LDH cytotoxic colorimetric test kit (G1782, Promega, Madison, WI, USA). Cell viability was measured using a non-radioactive CCK-8 kit (CK04; Dojindo, Kumamoto, Japan). Carry out relevant tests according to the instructions.

Oil red O staining:

1) The sample group and the control group were washed twice with 1×PBS, and fixed with 300 μl of 3% paraformaldehyde for 20 min;

2) After washing 2 times with 1×PBS, rinsing with 60% isopropyl alcohol for 10 s;

3) Washed twice with 1×PBS, and dried in a fume hood;

4) 500 μl per well was added with oil red O for 1 h;

5) Washed twice with 1×PBS, sorted by 60% isopropanol, and then washed twice with 1×PBS; photomicrographed and photographed.

Cardiomyocyte immunofluorescence staining:

1) The cells are fixed with 4% paraformaldehyde;

2) Wash PBS 3 times for 5 min each time;

3) Add 0.2% Triton and shake for 5 minutes on a shaker;

4) Wash PBS 3 times for 5 min each time;

5) Add 8% sheep serum and incubate in a wet box at room temperature for 60 min for blocking;

6) Discard the blocking solution, do not wash, add the primary antibody (Anti-α-Actinin, Millipore, #05-384) (1% sheep serum 1:100 dilution), 37 ° C 2h or 4 ° C overnight;

7) Discard the primary antibody and wash the PBS 4 times for 5 minutes each time;

488 donkey anti-mouse IgG，Invitrogen，A21202，用PBS按1:200比例稀释)，37℃孵育60min；8) Add 30 μl of diluted secondary antibody (Alexa

488 donkey anti-mouse IgG, Invitrogen, A21202, diluted 1:200 with PBS), incubated at 37 ° C for 60 min;

9) Discard the secondary antibody and wash 5 times with PBS for 5 minutes each time;

10) Take a slide with a SlowFade Gold antifade reagent with DAPI and observe the photograph under a fluoroscope.

Example 1 Expression changes of GPR31 in liver tissue at different time of ischemia

C57 mice were randomly divided into 6 groups, namely Sham group and operation group (divided into 5 different time points: ischemia 5 min, 10 min, 20 min, 40 min, 60 min). The liver tissues of the mice in the surgery group and the Sham group were taken. Western blot was used to detect the changes of GPR31 protein content in liver tissues of each group (three independent replicates). The primary antibody used for WB was: Anti-GPCR GPR31 antibody (ab75579; Abcam), and the secondary antibody was: Peroxidase AffiniPure goat anti-rabbit-IgG (H+L) (#111-035-003; Jackson Laboratory). A control standard with GAPDH as the expression level.

The results are shown in Fig. 1. The WB results of the Sham group showed almost no GPR31 band, and the GPR31 protein band became more and more obvious in the operation group with the prolongation of ischemia time. This result indicates a positive correlation between the expression of GPR31 protein and the severity of hepatic ischemia-reperfusion injury.

Example 2 Effect of overexpression of GPR31 on L02 cell injury and inflammatory response induced by H/R treatment

L02 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR31H/R group. The adherent L02 cells were transiently transferred to the corresponding plasmids, and H/R treatment was performed 24 hours later (hypoxia 6 h, reoxygenation 6 h). After plasmid transfection was completed, the total protein was extracted, and the overexpression of GPR31 was detected by Western blot (3 independent replicates, 3 replicates each time). The release of LDH in the culture medium was detected after H/R treatment (6 replicates per group) to evaluate the effect of GPR31 overexpression on H/R-induced hepatocyte injury; RNA was extracted for RT-PCR analysis (2 independent Repeat experiments, 3 replicates each time, to detect changes in inflammation-related cytokines and chemokine mRNA levels to evaluate the effect of GPR31 overexpression on H/R-induced hepatocyte inflammatory responses. The LDH release test results were calculated as GFP control group values of 1, and the ratios of the remaining groups were calculated.

The primer sequences used in RT-RCR are as follows:

gene Forward primer Reverse primer Il6 TCTGGATTCAATGAGGAGACTTG GTTGGGTCAGGGGTGGTTAT Tnfα TACTCCCAGGTCCTCTTCAAGG TTGATGGCAGAGAGGAGGTTG Cxcl2 GCGCCCAAACCGAAGTCATA TTTCTTAACCATGGGCGATGC

The results of GPR31 overexpression WB assay are shown in Figure 2. Compared to the GFP group, GPR31 overexpresses histones. The band was significantly enhanced, ie, GPR31 overexpression was significant in L02 cells.

The results of LDH release assay are shown in Figure 3. There was no significant difference in LDH release between the GPR31 control group and the GFP control group. When H/R treatment was performed, the release amount of LDH was significantly increased, and the release amount of LDH in the GPR31 overexpression group was significantly higher than that in the GFP group. This result indicates that overexpression of GPR31 aggravates hepatocyte injury and hepatotoxicity caused by H/R treatment.

Inflammatory factors and chemokine mRNA were detected with actin as a control standard. The results are shown in Figure 4. After H/R treatment, the inflammatory factors Il-6, Tnf-α, and chemokine Cxcl2 in the GPR31 overexpression group. The mRNA content was significantly increased. This result indicates that overexpression of GPR31 aggravates the hepatocyte inflammatory response caused by H/R treatment.

Example 3 Effect of GPR31 overexpression on H9C2 cell activity induced by H/R treatment

H9C2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR31 H/R group. The corresponding recombinant lentiviral virus solution was infected with cultured H9C2 cells, and H/R treatment was performed 24 hours later (anoxia 1 h, reoxygenation 6 h). After plasmid transfection was completed, total cellular protein was extracted, and overexpression of GPR31 was detected by Western blot (3 independent replicates). Cell viability was measured after completion of H/R (6 replicates per group). The test result of the GFP control group was 1, and the ratio of the remaining groups to the group was calculated.

The WB test results are shown in Fig. 5. Compared with the GFP group, the GPR31 overexpressing histone band was significantly enhanced, that is, the overexpression of GPR31 was significant in H9C2 cells.

The results of cell activity assays are shown in Figure 6. There was no significant difference in cell viability between the GPR31 control group and the GFP control group. When H/R treatment was performed, cell viability was significantly reduced compared to the control group. When GPR31 was overexpressed, the degree of cell viability in the GPR31 H/R group was significantly greater than that in the GFP H/R group. This result indicates that overexpression of GPR31 can significantly promote H/R-induced cardiomyocyte injury and decrease myocardial cell activity.

Example 4 Effect of overexpression of GPR31 on HK2 cell injury after H/R treatment

HK2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR31 H/R group. The corresponding lentiviral solution was infected with HK2 cells, and H/R treatment (hypoxia for 3 h, reoxygenation for 24 h) was performed after 24 h. The total protein was extracted after plasmid transfection, and the overexpression of GPR31 was detected by Western blot. 3 independent replicates). The amount of LDH released from the culture medium (6 replicates per group) was measured after completion of H/R to evaluate the effect of GPR31 overexpression on H/R-induced renal cell injury. LDH release assay in GFP control group The result of the test is 1, and the ratio of the remaining groups to the group is calculated.

The results of GPR31 overexpression in HK2 cells are shown in Figure 7. Compared with the GFP group, the GPR31 protein content of the GPR31 overexpression group was significantly increased.

The results of LDH release from kidney cells are shown in Figure 8. There was no significant difference in LDH release between the GPR31 control group and the GFP control group. When H/R treatment was performed, the release amount of LDH was significantly increased, and the release amount of LDH in the GPR31 overexpression group was significantly higher than that in the GFP group. This result indicates that GPR31 has the same effect in liver and cardiomyocytes, and the over-expression of GPR31 can aggravate renal cell damage caused by H/R treatment.

Example 5 Effect of GPR31 knockdown on hepatocyte fat accumulation

L02 cells were divided into 4 groups: shRNA control group, shGPR31 control group, shRNA experimental group, and shGPR31 experimental group. Adherent L02 cells were transiently transfected into corresponding plasmids. After 24 h, palmitate (PA) and oleic acid (OA) (PA 0.5 mM + OA 1 mM) were added to the two experimental groups. The same amount of BSA was added to the control group. After 12 h, oil red O staining was performed.

The primer sequences used in RT-PCR are as follows:

gene Forward primer Reverse primer GPR31 TCAACCTGCTGTCTCCTCAG GTGCTTCCTGCCAGATGATG

After the plasmid transfection was completed, the RNA was extracted, and the mRNA content of GPR31 gene was detected by RT-PCR (three independent experiments were repeated twice, each time), and the results are shown in FIG. The mRNA content of the GPR31 gene in the GPR31 knockdown group (shGPR31) was significantly lower than that in the shRNA control group.

The results of oil red O staining are shown in Fig. 10. The cells in the control group had no obvious red color, and when stimulated with PA+OA, the cells stained with oil red O were significantly increased compared with the control group, and the GPR31 knockdown group (shGPR31) In the experimental group, the increase in the staining area was smaller than that in the shRNA experimental group. This result indicates that a decrease in GPR31 expression can inhibit lipid deposition of PA-stimulated L02 cells.

Example 6 Effect of overexpression of GPR31 on cardiac hypertrophy

H9C2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP AngII group, GPR31 AngII group. The corresponding lentiviral fluids were infected with cultured H9C2 cells, and after 24 hours, they were stimulated with 1 μM angiotensin II (Ang II) or PBS (control group) for 48 h, and then subjected to immunofluorescence assay. Lentivirus transfection After the formation, the total protein was extracted, and the expression of GPR31 protein in H9C2 cells was detected by Western blot. After completion of the AngII stimulation, the cells were harvested for RT-PCR analysis (2 independent replicates, 3 technical replicates each) to detect mRNA levels of the hypertrophic marker genes Anp and Myh7. The cell surface area statistical results were calculated as the GFP control group value of 1, and the ratio of the remaining groups to the group was calculated.

The primer sequences used in RT-RCR are as follows:

gene Forward primer Reverse primer Anp ACCTGCTAGACCACCTGGAG CCTTGGCTGTTATCTTCGGTACCGG Myh7 CCGAGTCCCAGGTCAACAA CTTCACGGGCACCCTTGGA

The results of detection of GPR31 protein expression in H9C2 cells were the same as in Example 3.

The results of H9C2 cell hypertrophy and cardiac hypertrophy markers were shown in Figure 11. When treated with AngII, the cell surface area was significantly increased compared with the PBS control group, and the GPR31 overexpression group was significantly larger than GFP AngII. Group (Fig. 11A, B); consistent with the above results, mRNA analysis showed that the upregulation of cell hypertrophic marker genes Anp and Myh7 in GPR31 overexpression group was significantly higher than that in the control group after AngII treatment (Fig. 11C).

Claims (9)

Use of a GPR31 inhibitor for the preparation of a medicament for the treatment of ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, or abnormalities in fat metabolism and related diseases; Preferably, the ischemia-reperfusion injury and related diseases are selected from the group consisting of hepatic ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia-reperfusion injury and related diseases, and/or Cerebral ischemia-reperfusion injury and related diseases; Preferably, the hepatic ischemia-reperfusion injury and related factors are selected from the group consisting of liver cysts, liver transplantation, thrombolytic therapy, and/or hilar occlusion; Preferably, the cardiac ischemia-reperfusion injury and related factors are selected from the group consisting of myocardial infarction, myocardial infarction injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation and/or Or coronary artery bypass surgery; Preferably, the triggering factor of the renal ischemia-reperfusion injury and related diseases is selected from the group consisting of kidney transplantation, renal cyst and/or renal vascular surgery; Preferably, the priming factor of the cerebral ischemia-reperfusion injury and related diseases is selected from stroke and/or cerebrovascular surgery; Preferably, the ischemia-reperfusion injury and related diseases are hepatic ischemia-reperfusion injury, cardiac ischemia-reperfusion injury, renal ischemia-reperfusion injury, and/or cerebral ischemia-reperfusion injury; Preferably, the cardiac hypertrophy and related diseases are selected from the group consisting of cardiac hypertrophy, heart failure, arrhythmia, arterial embolism, coronary heart disease, angina pectoris and/or heart block; Preferably, the disease causing cardiac hypertrophy is selected from primary or secondary hypertension, myocardial infarction, valvular disease or congenital heart disease; Preferably, the cardiac hypertrophy and related diseases are cardiac hypertrophy and heart failure; Preferably, the inflammatory disease of the heart is selected from the group consisting of myocarditis and endocarditis; Preferably, the abnormal fat metabolism and related diseases are selected from the group consisting of insulin resistance, metabolic syndrome, nonalcoholic fatty liver disease, obesity, diabetes, hyperglycemia, hyperlipidemia; Preferably, the abnormal fat metabolism and related diseases are nonalcoholic fatty liver disease, obesity, hyperlipemia, insulin resistance; more preferably: simple liver steatosis, nonalcoholic steatohepatitis, obesity, hyperlipidemia. The use according to claim 1, wherein the GPR31 inhibitor inhibits GPR31 protein activity or An inhibitor of protein levels, or an inhibitor that inhibits mRNA levels of GPR31; Preferably, the inhibitor that inhibits GPR31 protein activity or protein level is selected from the group consisting of an antibody against GPR31, a protein, a polypeptide, an enzyme, a natural compound, a synthetic compound that inhibits GPR31 protein activity or protein level; Preferably, the inhibitor that inhibits the mRNA level of GPR31 is selected from the group consisting of an antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA, or a protein, polypeptide, enzyme, or compound capable of inhibiting the mRNA level of GPR31. The use according to claim 1 or 2, wherein the inhibitor is a shRNA of GPR31 mRNA, and the interference targeting sequence is CACTCTCCTGCCTTCAGTTTG; Preferably, the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'. The use according to any one of claims 1 to 3, further comprising a pharmaceutically acceptable excipient. Use of a vector for expressing a shRNA targeting mRNA of GPR31 for the preparation of a medicament for the treatment of ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, or fat metabolism Abnormalities and related diseases; Preferably, the targeting sequence of the shRNA interference is CACTCTCCTGCCTTCAGTTTG; Preferably, the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'. The use according to claim 5, wherein the vector is an expression vector comprising a promoter and a transcription termination sequence operably linked to the shRNA sequence; Preferably, the expression vector is a eukaryotic expression vector; Preferably, the eukaryotic expression vector is a plasmid expression vector or a viral expression vector; Preferably, the plasmid expression vector is selected from the group consisting of pcDNA3.1+/-, pcDNA4/HisMax B, pSecTag2 A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1(-)/myc-His A, pcDNA6-Myc/His B , pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-myc, pCMV-HA, pIRES-puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6 hygro, pSilencer3.1-H1 hygro, pSilencer3.1- H1 neo, pSilencer4.1-CMV neo; Preferably, the viral expression vector is selected from the group consisting of a lentiviral vector, an adenovirus vector, and an adeno-associated virus expression vector; further preferably pLKO.1, pLVX-IRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS- EF1-Puro, pCDH-MSCV-MCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP- 2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC, pHelper or pAAV-LacZ. Use of a lentiviral vector containing shRNA targeting GPR31 mRNA for the preparation of a medicament for the treatment of ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, or Abnormal fat metabolism and related diseases; Preferably, the targeting sequence of the shRNA interference is CACTCTCCTGCCTTCAGTTTG; Preferably, the shRNA sequence is: a forward oligonucleotide: 5'CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG 3'; a reverse oligonucleotide: 5'AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG 3'. The use according to claim 7, wherein the lentiviral vector is a pLKO.1 vector. The use according to any one of claims 5-8, wherein the medicament further comprises a pharmaceutically acceptable carrier; preferably the pharmaceutically acceptable carrier is an injection vehicle; preferably the injection carrier is selected from the group consisting of: isotonic NaCl solution, etc. A osmotic glucose solution, an isotonic solution containing a buffer system.

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