CN114903905A - Application of glycodeoxycholic acid in preparing medicine for treating intrahepatic cholestasis and its pharmaceutical composition - Google Patents

Abstract

The present invention discloses the application of glycodeoxycholic acid or its stereoisomer or its derivative in the preparation of a medicine for preventing and/or treating intrahepatic cholestasis in humans or mammals. The invention also discloses a pharmaceutical composition as an FXR agonist. Beneficial effects of the present invention: This study found out the mechanism of action of the increased abundance of intestinal Bacteroides in ICP patients leading to intrahepatic cholestasis, and confirmed through in vitro experiments and animal experiments that GDCA, as a novel agonist of FXR, can stimulate FXR signaling plays a role in preventing and/or alleviating ICP, suggesting that GDCA has the potential to be used in the preparation of drugs for the treatment of ICP.

Description

Application of glycodeoxycholic acid in preparing medicine for treating intrahepatic cholestasis and its pharmaceutical composition

technical field

The invention belongs to the technical field of medicinal uses of compounds, in particular to the use of glycodeoxycholic acid in the field of cholestasis diseases and a composition thereof.

Background technique

Intrahepatic cholestasis of pregnancy (ICP) is a unique complication in the second and third trimesters of pregnancy. It is clinically characterized by skin pruritus and elevated bile acids, which can significantly increase perinatal adverse events such as fetal distress. , premature birth, sudden fetal death and other risks, causing great harm to women's reproductive health. At present, the etiology and mechanism of ICP is still unclear. Because of this, the treatment of ICP is more symptomatic and empirical. At present, the first-line clinical treatment drug is ursodeoxycholic acid (UDCA). UDCA is metabolized by intestinal flora and is the most commonly used drug for the treatment of ICP. It can effectively relieve itching in pregnant women and protect liver dysfunction. However, there are large samples. Population studies have shown that the protective effect of UDCA in reducing serum bile acids and reducing fetal perinatal adverse events is not obvious. Therefore, the application of UDCA in the treatment of ICP has also been controversial.

Intestinal flora has become a hot research topic in recent years, and plays an important role in various physiological and pathological regulation of inflammation, metabolic syndrome, endocrine and reproduction. Studies by domestic scholars have found that the intestinal flora of female polycystic ovary syndrome patients is significantly dysregulated. Transplanting fecal bacteria from polycystic ovary patients into mice can lead to ovarian dysfunction in mice. It has further been found that intestinal flora can regulate bile by regulating bile. The disorder of acid metabolism and immune function promotes the occurrence of polycystic ovary syndrome in women; the imbalance of intestinal flora in pregnant women with preeclampsia can increase the translocation of bacteria to the placenta by disturbing the homeostasis of T cells and destroying the intestinal barrier. The occurrence of eclampsia, and the fecal bacterial transplantation of pregnant women with preeclampsia can lead to the phenotype of preeclampsia in mice, suggesting that the regulatory role of gut microbiota in female reproductive system diseases has received more and more attention and attention.

A variety of factors, such as diet, the genetic background of the host, and the immune system, are closely related to the remodeling and alteration of the gut microbiota structure. During pregnancy, the body undergoes a series of changes in hormones, metabolism, and immune regulation, which have a huge impact on the homeostasis of the intestinal flora. The composition of intestinal flora of normal pregnant women has significant changes at different stages of pregnancy, and there are also significant differences in the composition and structure of intestinal flora between normal pregnant women and ordinary women. Studies have confirmed that if the intestinal flora is disturbed during pregnancy, it can lead to the occurrence of pregnancy-related diseases such as gestational diabetes, gestational hypertension and preeclampsia. The inventors of the present application selected normal pregnant women and ICP patients who were matched in age and gestational age in the early stage, and took feces for 16S rRNA sequencing analysis. The results found that the α diversity of intestinal flora in ICP patients was significantly reduced, and the intestinal flora of the two groups of people was significantly reduced. There were also significant differences in the composition of intestinal flora, and Bacteroides were significantly increased in the intestinal tract of patients with ICP. Further in-depth studies have found that intestinal flora can participate in the regulation of the metabolism of specific bile acids and thus affect the function of Farnesoid X Receptor (FXR), thereby promoting the occurrence of ICP. Deoxycholic acid (GDCA) is a new type of FXR agonist, which can target and stimulate FXR signaling to effectively prevent and treat ICP, providing a new intervention method for the prevention and treatment of clinical ICP.

SUMMARY OF THE INVENTION

In view of this, one of the objects of the present invention is to provide the use of glycodeoxycholic acid.

Its technical solutions are as follows:

Use of glycodeoxycholic acid or a stereoisomer or derivative thereof in the preparation of a medicament for preventing and/or treating intrahepatic cholestasis in humans or mammals.

Preferably, the derivatives of glycodeoxycholic acid are either pharmaceutically acceptable salts or pharmaceutically acceptable esters thereof.

Preferably, the above-mentioned intrahepatic cholestasis is caused, at least in part, by inhibition of downstream signaling of the farnesoid X receptor (FXR) in a human or mammal.

Preferably, the above-mentioned inhibition of downstream signaling of FXR is caused by an increase in the abundance of Bacteroides in the human or mammalian gut.

Preferably, the above-mentioned medicament is used for the prevention and/or treatment of intrahepatic cholestasis during pregnancy in humans or mammals.

Another purpose of the present invention is to provide another application of Glycodeoxycholic acid. Its technical solutions are:

Use of glycodeoxycholic acid or a stereoisomer or derivative thereof in the preparation of a medicament as an FXR agonist in humans or mammals.

The third object of the present invention is to provide a pharmaceutical composition. Its technical solutions are:

A pharmaceutical composition mainly comprises glycodeoxycholic acid, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof, or a stereoisomer thereof.

Preferably, the above-mentioned pharmaceutical composition also includes pharmaceutically acceptable carriers, additives or excipients, as well as inevitable impurities.

Preferably, the above-mentioned drugs are tablets, granules, capsules or oral liquids.

Description of drawings

Figure 1 shows the differences in fecal flora diversity and composition between ICP patients (ICP) and normal pregnant women (Control), in which: (A) α diversity; (B) β diversity analysis; (C) flora composition; (D) ) LefSe analysis.

Figure 2 shows the serum bile acid, liver aminotransferase and fetal damage in ICP patients and normal pregnant women after fecal bacteria transplantation, among which: (A) serum bile acid; (B ~ D) biochemical indicators such as liver aminotransferase AST, ALT, ALP; (E) Fetal manifestations; (F) Fetal body weight; the two groups were ICP patients with fecal bacteria transplanted pregnant mice (I-FMT) and normal pregnant women with fecal bacteria transplanted pregnant mice (H-FMT).

Figure 3 shows the metabolic pathways of KEGG analysis of differences in fecal bacteria between ICP patients and normal pregnant women;

Figure 4 shows the differences in bile acid composition between ICP patients (ICP) and normal control pregnant women (Control) by targeted metabolomics, in which: (A) fecal bile acid level; (B) serum bile acid level; ^** P<0.01.

Figure 5 shows the correlation analysis between the abundance of intestinal Bacteroides and bile acid levels in ICP patients.

Figure 6 shows the changes of GDCA on cholestasis and FXR signal in mice after Bacteroides transplantation, in which: (A) serum bile acid level; (B, C) biochemical index levels such as liver transaminases ALT and AST; (D) fetal mouse performance and fetal weight; (E-F) qPCR detection of the expression of FXR and SHP; each group was inactivated Bacteroides-transplanted pregnant mice (Control), Bacteroides-transplanted pregnant mice (B.fragilis), Bacteroides-transplanted and Pregnant mice were given GDCA by gavage (B.fragilis+GDCA); ^* P<0.05; ^** P<0.01; ns, no significance.

Figure 7 is a study on the regulation of GDCA and FXR signals, in which: (A, B) Docking analysis predicts the structural relationship and binding effect of GDCA and FXR; (C) After GDCA and Caco2 cells are incubated, the expression results of the downstream gene SHP detected by qPCR , DMSO as blank control, chenodeoxycholic acid (CDCA) as positive control agonist; ^** P<0.01.

Figure 8 is the TR-FRET FXR co-activation assay to detect the agonistic effect of several bile acids such as CDCA, GDCA, GUDCA on FXR, and the ratio of the fluorescence emission intensity at 520 nm and 495 nm was used as the detection index.

Figure 9 is a cell experiment to study the effect of GDCA on the mRNA expression of FXR downstream genes FGF19 and SHP, and to study the effects of FXR antagonists GUDCA and T-βMCA on the above effects.

Figure 10 shows the expression of FXR downstream genes FGF15 and SHP in intestinal tissue of Bacteroides-transplanted pregnant mice and pregnant mice given GDCA gavage at the same time; In the mouse group (B.fragilis), Bacteroidetes were transplanted and GDCA was administered to the pregnant mouse group (BF+GDCA).

Figure 11 is a comparison of cholestasis in EE2 injection modeling ICP mice and GDCA gavage mice, wherein: (a) schematic diagram of the experimental process; (b) levels of bile acid, ALT, AST, ALP, GGT, and liver index ; (c) HE staining photos of liver tissue sections; each group was a normal mouse group (Control), an EE2 injection modeling ICP mouse group (EE2), and an EE2 injection modeling and GDCA gavage mice group (EE2+). GDCA).

Figure 12 shows the expression of genes related to bile acid metabolism and bile acid metabolism in EE2 injection model ICP mice and GDCA gavage mice at the same time, in which: (a) FXR downstream gene FGF15 and SHP gene expression in the intestine; (b) FXR in the liver The expression of the downstream genes SHP and NTCP; (c) the expression of bile acid synthesis genes such as Cyp7a1, Cyp8b1, Cyp27a1, and the expression of bile acid excretion genes such as BSEP and MRP2; each group was a normal mouse group (Control), and the mice were injected with EE2. Model ICP mouse group (EE2), EE2 injection model and GDCA gavage mouse group (EE2+GDCA).

Detailed ways

The present invention will be further described below with reference to the embodiments and the accompanying drawings.

Example 1

1. Analysis of the characteristics of intestinal flora in ICP patients and normal pregnant women

1.1 Metagenomic analysis of gut microbiota differences between ICP patients and controls

Experimental materials and methods

(1) Collect feces and serum samples from 100 ICP patients and 100 normal control pregnant women, expand the sample size and establish a complete sample bank and follow-up database, respectively take the feces of the two groups of people, dissolve them in glycerol solution, and store at -80°C;

(2) Extract feces using the MinkaGene Stool DNA Kit (MinkaGene Stool DNA Kit) according to the kit operation steps: take about 0.2 g of feces into a 2 ml centrifuge tube, add lysis buffer for high temperature lysis, centrifuge the suspension, and take the supernatant to Add proteinase K to a new centrifuge tube and digest at 70°C for 10 minutes, add ethanol solvent, and then pass through the DNA binding column. After repeated washing twice, the DNA precipitate is dissolved in EB buffer, and stored at -20°C for later use;

(3) DNA was sent to Beijing Nuohezhiyuan Biotechnology Co., Ltd. for sequencing analysis based on the Illumina HiSeq 2500 platform;

(4) After obtaining the original sequencing data, first remove the contamination and linker sequences, and remove the host gene according to the reference sequence of the human genome; further assemble the high-quality data of the decontamination and host gene through SPAdes; after the assembly is completed, use MetaGene to predict orf; The predicted orf sequences are clustered using CD-HI T software, and the longest orf sequence of each class is taken as the representative sequence to construct a non-redundant gene sequence set; after obtaining the non-redundant gene set, cleanreads can be Align to the non-redundant gene set, and calculate the abundance of each gene in each sample; after obtaining the gene abundance table, use KEGG, GO, COG and other software to perform functional comparison and annotation of genes; use metaphlan for species abundance. degree analysis; α and β diversity analysis were performed according to gene abundance;

(5) According to the sequencing analysis results, the least squares discriminant analysis (PLS-DA) was used to further analyze the scores of the abundance of each bacterial species in each sample, and the variable importance analysis was used to obtain which Bacteroidetes were different in the two groups of populations. most notably.

Experimental results

The 16S rRNA sequencing analysis of fecal DNA from ICP patients and normal pregnant women showed that the α diversity of intestinal flora in ICP patients was significantly lower than that in the control group (Fig. 1A). β-diversity analysis showed that there were significant differences in the gut microbiota structure between ICP patients and normal controls, and the composition of the microbiota between the two groups was significantly different (Fig. 1B). The composition of the flora (Fig. 1C) and LefSe analysis showed that the two groups of populations had significantly different genera (Fig. 1D). It was found that Bacteroides, Lachnoclostridium, Bacteroides, Rosella ( Roseburia) and Escherichia_shigella were significantly enriched in the ICP patient group, while Blautia was significantly decreased. Among them, Bacteroides were significantly enriched in the intestinal tract of ICP patients.

1.2 Bacteria transplantation experiment

Experimental materials and methods

The fecal bacteria from ICP patients and normal pregnant women were transplanted into sterile pregnant mice, and biochemical indicators such as bile acids, liver transaminases, and fetal indicators were detected. The specific steps are:

(1) Preparation of fecal bacteria suspension: Take fresh feces from ICP patients and normal pregnant women respectively, resuspend them in glycerol-containing PBS solution immediately, and store them in aliquots at -80°C for later use;

(2) Centrifuge the above-mentioned fecal suspension at low speed, remove excess fecal residue, and transplant them to germ-free mice respectively. Each mouse is gavaged with 200 μl (0.15g/ml) of fecal suspension, twice a week, for 4 consecutive weeks. ;

(3) After the gavage is finished, press the male mouse: the female mouse in a ratio of 1:2 to close the cage, observe and record the weight change of the female mouse, observe the appearance of vaginal plug and record it as E 0.5d;

(4) The female mice were pregnant at E18d, the mouse stool was collected, the mice were anesthetized and killed, the mouse blood, placenta and other tissues were collected, and the number and weight of the fetuses were observed and recorded;

(5) The levels of ALT, AST, ALP and total bile acid in serum were detected by automatic biochemical analyzer;

(6) After the liver and placenta were fixed in formalin, they were embedded in paraffin, sectioned, and stained with HE to detect the pathological changes of the liver and placenta;

Experimental results

As shown in Figure 2 (A-D), the indexes of bile acid, liver transaminases (AST, ALT, ALP) in fecal bacteria transplanted mice derived from ICP patients (I-FMT) were significantly higher than those in normal pregnant women fecal bacteria transplanted mice (H-FMT). ). As shown in Figure 2 (E, F), observing the fetal performance and comparing the fetal weight, it was found that the fetal damage of the ICP patient-derived fecal bacteria transplanted mice (I-FMT) was also significantly higher than that of the normal pregnant women's fecal bacterial transplanted mice (H-FMT). ).

This study showed that changes in gut microbiota did lead to the occurrence of ICP-related symptoms in pregnant mice.

1.3 Metabolic differences between ICP patients and normal pregnant women

1.3.1 KEGG analysis of differential metabolic pathways

Experimental Materials and Methods

The gut microbiota composition of normal pregnant women and ICP patients was analyzed by metagenomic sequencing, and the KEGG metabolic pathway was analyzed by bioinformatics software.

Experimental results

The results showed that, as shown in Figure 3, among the signal pathways involved in the regulation of differentially enriched flora in the intestines of ICP patients and normal pregnant women, the most obvious difference was the bile acid metabolism pathway, which provided a basis for the follow-up study of bile acid composition and regulation.

1.3.2 Targeted metabolomics detection and analysis of bile acid composition and level in feces and serum of ICP patients and normal pregnant women

Experimental Materials and Methods

(1) Take feces and serum samples of ICP patients and normal pregnant women respectively, and store them in an ultra-low temperature refrigerator for future use;

(2) The bile acid metabolism test was entrusted to Shanghai Maitexipu Biotechnology Co., Ltd.;

(3) Preparation of test supplies: bile acid standards were purchased from Steraloids (Newport, RI, USA) and TRC Chemicals (Toronto, ON, Canada) and synthesized in the laboratory of the inventor, and 10 isotope-labeled bile acid internal standards were used. Available from C/D/N Isotopes Corporation (Quebec, Canada) and Steraloids Corporation (Newport, RI, USA). Bile acids and isotopic internal standards were accurately weighed and dissolved in methanol to obtain a stock solution of 5 mM concentration. The bile acid stock solutions were mixed at the time of use and serially diluted to 2500, 500, 250, 50, 10, 2.5 and 1 nM concentration points in bile acid free serum matrix. In addition, 1500, 150 and 5 nM bile acid standard solutions were prepared with bile acid-free serum matrix as quality control samples of three concentrations of high, medium and low. The concentrations of the internal standard in all concentration standard solutions and quality control samples are the same, namely (GCA-d4, TCA-d4, TCDCA-d9, CA-d4, GCDCA-d4, CDCA-d4, LCA-d4 and aMCA-d5 are both 150nM);

(4) Sample pretreatment: Take stool or serum samples into a centrifuge tube, add about 25 mg of pre-cooled grinding beads, and then add 200 μL of acetonitrile/methanol (v/v=8:2) mixed solvent containing 10 μL of internal standard. After homogenization, centrifuge at 13,500 rpm for 20 min at 4°C. Take 10 μL of supernatant, dilute it with 90 μL of 1:1 acetonitrile/methanol (80/20) and ultrapure water mixed solvent, and wait for sample injection analysis after shaking and centrifugation. The injection volume is 5 μL;

(5) Ultra-high performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS, ACQUITY UPLC-Xevo TQ-S, WatersCorp., Milford, MA, USA) was used for quantitative bile acid detection.

Experimental results

Compared with normal pregnant women, patients with ICP had significantly lower levels of three bile acids, glycocholic acid (GCA) and glycochenodeoxycholic acid, in both stool (Fig. 4A) and serum (Fig. 4B). (GCDCA) and glycodeoxycholic acid (GDCA).

1.4 Correlation analysis of Bacteroidetes and bile acid levels

Experimental Materials and Methods

(1) The relative abundance of Bacteroides in the stool samples of each ICP patient was obtained by metagenomics analysis;

(2) Targeted metabolomics was used to determine the content of bile acids in feces of ICP patients, and the correlation between the abundance of Bacteroides and the content of bile acids in ICP patients was analyzed by correlation analysis.

Experimental results

The correlation analysis between the abundance of Bacteroides and the level of bile acids in ICP patients showed that, as shown in Figure 5, the abundance of Bacteroides was significantly negatively correlated with the level of GDCA, suggesting that Bacteroides may be involved in the metabolic regulation of GDCA.

Embodiment 2

2. The role of GDCA in the regulation of ICP by Bacteroides

FXR is distributed in the intestine and liver and is involved in bile acid metabolism. It has been found that FXR in the liver inhibits bile acid synthesis by inducing a downstream signaling small heterodimeric partner (SHP). Human fibroblast growth factor 19 (FGF19) and mouse fibroblast growth factor 15 (FGF15) are homologous regulators of bile acid metabolism and are downstream signals of FXR. In the gut, FXR inhibits bile acid synthesis by inducing the FGF19/FGF15 signaling pathway.

This part studies the effect and mechanism of GDCA on ICP in mice.

2.1 The effect of GDCA on the symptoms of ICP in mice induced by Bacteroides transplantation

Experimental Materials and Methods

(1) Strain preparation: Bacteroidetes plate streaking method evenly spreads anaerobic medium plate, cultured in anaerobic glove box at 37°C, after 2-3 days of colony appearance, pick a single colony in liquid medium anaerobic Cultivate, measure the OD value of the medium to calculate the bacterial cfu value;

(2) Bacteroidetes (2×10 ⁸ cfu) were dissolved in 200 μl sterile aqueous solution, respectively gavaged sterile female mice, control gavage with the same concentration of inactivated Bacteroides species, and simultaneously administered GDCA (30mg/Kg/d) Gavage, twice a week, for 4 consecutive weeks, male and female mice were caged in a ratio of 1:2, and the weight of mice and the appearance time of vaginal plugs were observed and recorded;

(3) The female mice were pregnant at E18d, collected mouse feces before execution, anesthetized and killed the mice, collected mouse blood, intestine, liver, placenta and other tissues, and observed and recorded the number and weight of fetuses;

(4) The levels of ALT, AST and total bile acid in serum were detected by automatic biochemical analyzer;

(5) Feces, serum, liver and intestinal tissue were detected and analyzed by ultra-high performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) respectively, and the sample processing and detection and analysis steps were the same as 1.3.2;

(6) Take the mouse small intestine and liver tissue to separate intestinal epithelial cells and hepatocytes. Specific steps: move the small intestine to a petri dish, wash repeatedly with DPBS, wash with penicillin-containing serum-free medium for several times, and cut the intestinal tissue into The debris was washed with serum-free medium, and the supernatant was discarded by centrifugation to obtain the pellet for later use. Collagenase XI and neutral proteinase I were added to the pellet, digested at 37°C for 20 minutes, pipetted repeatedly with a pipette, and the pellet was washed repeatedly by centrifugation and then resuspended for use. Take fresh liver tissue, cut the liver tissue into small tissue pieces, rinse and pipet repeatedly with serum-free medium, add collagenase for digestion, and obtain a hepatocyte suspension for later use. After taking the above cell suspension, RNA was extracted by Trizol kit, and the expression of FXR, SHP and other molecules was detected by qPCR.

Experimental results

As shown in 6(A-D), Bacteroides transplantation can significantly increase the level of bile acid, ALT and AST in mice, and the appearance of fetal mice is damaged and body weight is reduced, that is, the occurrence of cholestasis and fetal damage. However, supplementation of GDCA can significantly alleviate the above-mentioned manifestations caused by Bacteroides.

As shown in Figure 6 (E, F), in vivo experiments showed that Bacteroides transplantation did not affect the expression of FXR molecules, but could significantly inhibit the expression of the FXR downstream signal SHP gene, while supplementation of GDCA could restore the FXR signal, suggesting that Bacteroides may Changes in FXR signaling are regulated by changes in bile acids such as GDCA.

2.2 In vitro experiments to study the relationship between GDCA and FXR signaling

On the basis of the above research, we further studied how GDCA affects the body's bile acid metabolism and alleviates ICP-related symptoms from the perspective of the signaling pathway of bile acid metabolism regulation.

Experimental Materials and Methods

(1) Docking analysis to predict the binding conformational relationship between the above-mentioned bile acids and FXR: Download the crystal structure of FXR receptor from RCSB Protein DataBank (PDB ID: 3dct, https://www.rcsb.org/), use SYBYL-X The Surflex-Dock GeomX (SFXC) of 2.0 performs docking analysis of the binding of GDCA to FXR, and the binding of bile acids to FXR is analyzed using PyMOL and ligplot;

(2) The effect of each bile acid and FXR signal activation was detected by TR-FRET FXR co-activation assay. The experiment was performed using LanthaScreen ^™ TR-FRET Farnesoid X Receptor Coactivator Assay Detection Kit (Thermo Fisher, Cat#A15140). The steps are as follows: 1) Prepare Coregulatorbuffer G according to the instructions, and add DTT with a final concentration of 10 mM; 2) Dilute the bile acid sample to be measured with 100X sample diluent; 3) Add 10 μL of the diluted sample diluent to the measurement well plate , set up measurement duplicate wells; 4) Prepare 4X FXR-LBD dilution with Coregulator buffer G buffer, add 5 μL to the above-mentioned corresponding well plates; 5) Use Coregulator buffer G buffer to prepare final concentration containing 2.0 μM fluorescein-SRC2-2 (4X) and 20nM Tb anti-GST antibody (4X) working solution, take 5 μL working solution to the above reaction system; 6) Gently shake and mix the reaction mixture, incubate at room temperature in the dark, and read the plate at wavelengths of 520nm and 495nm, respectively 7) Calculate TR-FRET ratio (520nm/495nm), draw binding curve and calculate EC50, compare with standard agonist CDCA and antagonist GUDCA, and analyze the binding and functional effects of each bile acid to FXR.

(3) Detection of the effect of bile acids on the transcriptional activity of FXR by dual luciferase reporter genes: 1) HEK293 cells were routinely cultured, and after the growth reached 85% confluence, they were seeded in 6-well plates, and cultured at 37°C to 85% confluence; 2) Entrusted Genkai Gene Company to construct human FXR expression plasmid, PGL4-Shp-TK firefly luciferase reporter gene vector and Renilla luciferase control vector (pRL-luciferase; Promega, Madison, WI), and co-transfected with transfection reagent Lipofectamine 3000 HEK293 cells were cultured for 24 hours after transfection; 3) The above bile acids were added to the cell culture medium with a final concentration of 50-200 μM, and CDCA was used as a positive control; 4) Fluorescence was detected by dual luciferase detection kit (Promega). The activity of the reporter gene was measured, and the specific operation was carried out according to the kit instructions.

(4) After incubation of each bile acid with primary intestinal epithelial cells or Caco2 cells, the FXR signal and downstream gene expression were detected: 1) The mouse small intestine was taken out from the ultra-clean workbench, and the intestinal contents were repeatedly washed with pre-cooled PBS until clear , washed several times in serum-free medium containing penicillin and streptomycin, cut the intestinal tissue into pieces less than 1 mm ² , transferred to a centrifuge tube, repeatedly washed with serum-free medium, pipetted, centrifuged to remove the supernatant, and added collagenase The tissue blocks were digested with neutral protease, repeatedly pipetted and digested, centrifuged, and resuspended and cultured in complete medium; 2) Routinely cultured Caco2 cells; 3) After the cells grew to 85% confluence, starvation and culture for 4 hours were added to the final concentration of 50 -200μM GDCA, incubated for 16h; control group was added with the same final concentration of GUDCA or T-βMCA; 3) After incubation, RNA and protein were extracted respectively, and the expression of SHP, FGF19 and other molecules was detected by qPCR.

Experimental results

As shown in Figure 7(A, B), Docking analysis predicts that GDCA can bind to FXR, and in vitro experiments initially found that GDCA can directly promote the expression of SHP downstream of FXR, as shown in Figure 7(C), suggesting that GDCA may function as an FXR agonist Regulation.

As shown in Figure 8, the TR-FRET FXR co-activation analysis found that GDCA could act as an agonist of FXR to stimulate FXR.

As shown in Figure 9, the cell experiment results found that GDCA can significantly promote the mRNA expression of FXR downstream genes FGF19 and SHP, and this promotion can be inhibited by the FXR antagonists GUDCA and T-βMCA.

2.3 In vivo experiments to study the relationship between GDCA and FXR signaling

On the basis of in vitro experiments, the relationship between GDCA and FXR signal was further studied through in vivo experiments.

Experimental Materials and Methods

(1) Preparation of Bacteroides species, the method is the same as that in Section 2.1;

(2) Bacteroides (2×10 ⁸ cfu) was dissolved in 200 μl of sterile aqueous solution, gavaged with sterile female mice, and the control was given the same concentration of inactivated Bacteroides species, and GDCA (30 mg/Kg/d) ) gavage, twice a week, continuous gavage for 4 weeks, male mice and female mice were caged at a ratio of 1:2, and the weight of mice and the appearance time of vaginal plugs were observed and recorded;

(3) The female mice were pregnant at E18d. The mice were anesthetized and sacrificed. The intestinal and liver tissues of the mice were collected. The intestinal epithelial cells and hepatocytes were isolated according to the method described in Section 2.1. RNA was extracted, and qPCR was used to detect FXR, SHP, etc. expression of downstream genes.

Experimental results

As shown in Figure 10, the expression of FXR downstream genes in intestinal tissue was detected after Bacteroides transplantation in mice, and it was found that Bacteroides transplantation could significantly inhibit the expression of FXR downstream genes FGF15 and SHP, while GDCA could significantly restore the FXR inhibitory effect caused by Bacteroides .

The in vitro and in vivo studies in this part show that GDCA indeed acts as an FXR agonist and can promote the expression of FXR downstream genes.

Embodiment 3

3. The therapeutic effect of GDCA on ICP in mice

Experimental Materials and Methods

(1) Standard ICP mouse model, that is, after subcutaneous injection of EE2 estrogen, the ICP mouse model was constructed;

(2) GDCA (30 mg/Kg/d) was administered by gavage before and after administration of EE2, twice a week, for 4 consecutive weeks. Male mice: female mice were caged at a ratio of 1:2, and the mice were observed and recorded. Weight and time of vaginal suppository appearance;

(3) At the 18th day of pregnancy, the intestinal and liver tissues of mice were collected to detect the phenotypic changes of ICP mice, and the intestinal epithelial cells were separated according to the method described in Section 2.2. The expression of FGF15, SHP, etc. was detected by qPCR, and the liver cells were separated. The expressions of SHP, NTCP, CYP7A1, CYP8B1, BSEP and MRP2 in liver were detected by qPCR.

Experimental results

The standard EE2 modeled ICP mice were obtained by the process shown in Figure 11a, and it was found that biochemical indicators such as bile acid levels, ALT, AST, ALP, GGT, and Liver Index related to ICP were significantly higher in the modeled ICP mice group (EE2 ) was significantly higher than that of the normal mouse group (Control), indicating that the modeling was successful, while the above biochemical indicators of the GDCA gavage ICP mouse group (EE2+GDCA) were significantly inhibited (Fig. 11b, 11c), and could alleviate the EE2 of liver pathological damage (Fig. 11d).

As shown in Figure 12, the expression of FXR downstream genes FGF15, SHP, and NTCP in the EE2 model ICP mouse group (EE2) was significantly inhibited compared with the normal mouse group (Control), while GDCA could restore the FXR signal of ICP mice (Fig. 12a, 12b), while GDCA can significantly inhibit the expression of bile acid synthesis genes such as Cyp7a1, Cyp8b1, Cyp27a1, and promote the expression of bile acid excretion genes such as BSEP and MRP2 (Fig. 12c).

Combined with the results of the above in vitro and in vivo experiments to study the relationship between GDCA and FXR signaling, it can be seen that supplementing GDCA in ICP mice can alleviate ICP-related indicators and symptoms, and GDCA works by enhancing the expression of FXR downstream signaling genes in the liver and intestine of mice. to the prevention and treatment of ICP.

Compared with the prior art, the beneficial effects of the present invention: this study found that the increased abundance of intestinal Bacteroides in patients with ICP leads to the mechanism of action of intrahepatic cholestasis, and through in vitro experiments and animal experiments, it was confirmed that GDCA is a new type of FXR. Agonists can prevent or/and alleviate ICP by activating FXR signaling, indicating that GDCA has the potential to be used to prepare drugs for the treatment of ICP.

Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and those of ordinary skill in the art can make a variety of similar It is indicated that such transformations fall within the protection scope of the present invention.

Claims (9)

1. Use of glycodeoxycholic acid or a stereoisomer or derivative thereof in the preparation of a medicament for preventing and/or treating intrahepatic cholestasis in humans or mammals. 2. The use according to claim 1, characterized in that: a derivative of glycodeoxycholic acid or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof. 3. The use according to claim 1 or 2, wherein the intrahepatic cholestasis is at least partly caused by the inhibition of downstream signaling of farnesoid X receptor (FXR) in humans or mammals. 4. The use according to claim 3, wherein the inhibition of the downstream signal of FXR is caused by the increased abundance of Bacteroides in the intestinal tract of humans or mammals. 5. The use according to claim 1 or 2, wherein the medicament is used for preventing and/or treating intrahepatic cholestasis during pregnancy in humans or mammals. 6. Use of glycodeoxycholic acid or a stereoisomer or derivative thereof in the preparation of a medicament as an FXR agonist in a human or mammal. 7. A pharmaceutical composition, characterized by comprising glycodeoxycholic acid, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof, or a stereoisomer thereof. 8. The pharmaceutical composition according to claim 7, further comprising pharmaceutically acceptable carriers, additives or excipients, and inevitable impurities. 9. The pharmaceutical composition according to claim 8, wherein the medicine is a tablet, a granule, a capsule or an oral liquid.

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