CN114903905A - Application of glycodeoxycholic acid in preparation of medicine for treating intrahepatic cholestasis and medicine composition of glycodeoxycholic acid - Google Patents
Application of glycodeoxycholic acid in preparation of medicine for treating intrahepatic cholestasis and medicine composition of glycodeoxycholic acid Download PDFInfo
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- CN114903905A CN114903905A CN202110175232.0A CN202110175232A CN114903905A CN 114903905 A CN114903905 A CN 114903905A CN 202110175232 A CN202110175232 A CN 202110175232A CN 114903905 A CN114903905 A CN 114903905A
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Abstract
The invention discloses application of glycodeoxycholic acid or a stereoisomer or a derivative thereof in preparation of a medicament for preventing and/or treating intrahepatic cholestasis in humans or mammals. The invention also discloses a pharmaceutical composition used as FXR agonist. The invention has the beneficial effects that: the research discovers an action mechanism of intrahepatic cholestasis caused by the increase of the abundance of bacteroides enterobacter in intestinal tracts of ICP patients, and in vitro experiments and animal experiments prove that GDCA is used as a novel agonist of FXR and can play a role in preventing and/or relieving ICP by exciting FXR signals, which indicates that GDCA has the potential of being used for preparing the ICP treatment medicine.
Description
Technical Field
The invention belongs to the technical field of medical application of compounds, and particularly relates to application of glycodeoxycholic acid in the field of cholestasis diseases and a composition thereof.
Background
Intrahepatic Cholestasis of Pregnancy (ICP) is a specific complication in the middle and late gestation, is clinically characterized by skin pruritus and bile acid elevation, can obviously increase the risks of perinatal adverse events such as fetal distress, premature birth, sudden death of fetus and the like, and causes great harm to the reproductive health of women. Currently, the causes and mechanisms of ICP occurrence are still unclear. Because of this, treatment for ICP is more symptomatic and empirical. At present, the clinical first-line treatment drug is ursodeoxycholic acid (UDCA), and the UDCA is generated by metabolism of intestinal flora, is the most common drug for ICP treatment at present, can effectively relieve pruritus of pregnant women and protect liver dysfunction, but a large number of researches show that the protection effect of the UDCA on reducing serum bile acid and adverse events of fetus perinatal period is not obvious, so that the application of the UDCA to ICP treatment also presents a larger controversial debate.
Intestinal flora has become a hot spot of research in recent years, and plays an important role in various physiological and pathological regulation and control such as inflammation, metabolic syndrome, endocrine, reproduction and the like. The study of domestic scholars finds that the intestinal flora of a female polycystic ovary syndrome patient is obviously disordered, and the fecal bacteria of the polycystic ovary syndrome patient is transplanted to a mouse to cause the ovarian dysfunction of the mouse, so that the intestinal flora can promote the occurrence of the female polycystic ovary syndrome by regulating and controlling the metabolism of bile acid and the disturbance of immune function; the intestinal flora imbalance of the pregnant women with preeclampsia can increase the translocation of bacteria to placenta by disturbing the steady state of T cells and destroying intestinal barriers, so as to promote the occurrence of eclampsia, and the coprophilic transplantation of the pregnant women with preeclampsia can cause the phenotype of the mice with preeclampsia, so that the regulation and control function of the intestinal flora in the diseases of female reproductive systems is more and more concerned and more paid attention.
Various factors such as diet, genetic background of host and immune system are closely related to intestinal flora structure remodeling and change. In the gestation period of women, the body can be subject to serial changes such as hormone, metabolism, immunoregulation and the like, and great influence is generated on the steady state of the intestinal flora. The intestinal flora composition of normal pregnant women is obviously changed in different stages of pregnancy, and the intestinal flora composition and structure of the normal pregnant women and common women are also obviously different. It has been proved that if intestinal flora is disturbed during gestational period, pregnancy-related diseases such as gestational diabetes, gestational hypertension and preeclampsia can occur. The inventor selects normal pregnant women and ICP patients matched with age, gestation period and the like in the earlier stage, takes excrement and carries out 16S rRNA sequencing analysis respectively, and the result shows that the alpha diversity of intestinal flora of the ICP patients is obviously reduced, the composition of the intestinal flora of the two groups of people is also obviously different, and Bacteroides (Bacteroides) in the intestinal tracts of the ICP patients are obviously increased. Further intensive research finds that intestinal flora can participate in regulating and controlling the metabolism of specific bile acid to influence the function of Farnesoid X Receptor (FXR) so as to promote ICP, and research finds and proves that glycodeoxycholic acid (GDCA) is a novel FXR agonist, can target and excite FXR signals so as to effectively prevent and treat ICP, and provides a novel intervention means for the prevention and treatment of clinical ICP.
Disclosure of Invention
In view of the above, an object of the present invention is to provide the use of glycodeoxycholic acid.
The technical scheme is as follows:
use of glycodeoxycholic acid or a stereoisomer or a derivative thereof for the preparation of a medicament for the prevention and/or treatment of intrahepatic cholestasis in humans or mammals.
Preferably, the derivative of glycodeoxycholic acid is either a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable ester thereof.
Preferably, the intrahepatic cholestasis is caused at least in part by inhibition of Farnesoid X Receptor (FXR) downstream signaling in the human or mammal.
Preferably, the FXR downstream signal inhibition is caused by an increase in the abundance of Bacteroides in the intestinal tract of a human or mammal.
Preferably, the medicament is used for preventing and/or treating intrahepatic cholestasis in pregnancy of a human or a mammal.
It is another object of the present invention to provide another use of glycodeoxycholic acid. The technical scheme is as follows:
use of glycodeoxycholic acid or a stereoisomer or a derivative thereof for the preparation of a medicament intended to act as an FXR agonist in humans or mammals.
The invention also aims to provide a pharmaceutical composition. The technical scheme is as follows:
a pharmaceutical composition characterized by comprising glycodeoxycholic acid, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof, or a stereoisomer thereof.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, additive or excipient, and unavoidable impurities.
Preferably, the medicament is a tablet, a granule, a capsule or an oral liquid.
Drawings
Fig. 1 shows the diversity and composition differences of fecal flora in ICP patients (ICP) and normal pregnant women (Control), wherein: (A) alpha diversity; (B) beta diversity analysis; (C) the composition of flora; (D) LefSe analysis.
FIG. 2 shows the status of serum bile acid, hepatic transaminase and fetal damage in ICP patients and normal pregnant women by fecal bacteria transplantation, wherein: (A) serum bile acids; (B-D) biochemical indexes such as liver transaminase AST, ALT, ALP and the like; (E) fetal performance; (F) fetal mouse weight; the two groups are respectively a fecal bacteria transplanting pregnant mouse group (I-FMT) for ICP patients and a fecal bacteria transplanting pregnant mouse group (H-FMT) for normal pregnant women.
FIG. 3 is a KEGG analysis of metabolic pathways of differences in fecal bacteria in ICP patients and normal pregnant women;
fig. 4 is a graph of targeted metabolomics analysis of differences in bile acid composition between ICP patients (ICP) and normal Control pregnant women (Control), in which: (A) fecal bile acid levels; (B) serum bile acid levels; ** P<0.01。
FIG. 5 is a correlation analysis of the abundance of Bacteroides enteric in ICP patients with bile acid levels.
Figure 6 is a graph of changes in GDCA on mouse cholestasis and FXR signaling following bacteroid transplantation, in which: (A) serum bile acid levels; (B, C) biochemical index levels of hepatic transaminase ALT, AST, etc.; (D) fetal rat performance and fetal rat weight; (E-F) qPCR detecting the expression of FXR and SHP; the groups were respectively inactivated bacteroides transplantation pregnant rat group (Control), bacteroides transplantation pregnant rat group (B.fragilis), bacteroides transplantation and GDCA-administered gastric feeding pregnant rat group (B.fragilis + GDCA))； * P<0.05； ** P<0.01；ns,no significance。
FIG. 7 is a study of 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, qPCR is carried out to detect the expression condition result of downstream gene SHP, DMSO is used as a blank control, and chenodeoxycholic acid (CDCA) is used as a positive control agonist; ** P<0.01。
FIG. 8 shows that TR-FRET FXR coactivation analysis detects the agonism of several bile acids such as CDCA, GDCA, GUDCA and the like on FXR, and the fluorescence emission intensity ratio of 520nm to 495nm is used as a detection index.
FIG. 9 is a cell experiment to investigate the effect of GDCA on the mRNA expression of FXR downstream genes FGF19 and SHP, and to investigate the effect of FXR antagonists GUDCA and T- β MCA on the above effect.
FIG. 10 shows the expression of FXR downstream genes FGF15 and SHP in intestinal tissues of Bacteroides implantation pregnant mice and mice administered with GDCA gavage; the groups were respectively inactivated bacteroides transplantation pregnant rat group (Control), bacteroides transplantation pregnant rat group (b.fragiliss), bacteroides transplantation and GDCA gavage pregnant rat group (BF + GDCA).
Fig. 11 is a cholestasis symptom comparison of EE2 injection molded ICP mice and GDCA gavage mice given concurrently, wherein: (a) schematic diagram of experimental process; (b) bile acids, ALT, AST, ALP, GGT, liver index levels; (c) a liver tissue section HE staining picture; each group was a normal mouse group (Control), an EE2 injection molding ICP mouse group (EE2), and an EE2 injection molding and GDCA gavage mouse group (EE2+ GDCA).
FIG. 12 shows the expression of genes associated with bile acid metabolism in an ICP mouse injected with EE2 and a GDCA gavage mouse, wherein: (a) expression of FXR downstream genes FGF15 and SHP in intestinal tracts; (b) the expression conditions of FXR downstream gene SHP and NTCP genes in the liver; (c) expression of bile acid synthesis genes such as Cyp7a1, Cyp8b1, Cyp27a1, and bile acid excretion genes such as BSEP and MRP 2; the groups were normal mice (Control), EE2 injection molding ICP mice (EE2), EE2 injection molding and GDCA gavage mice (EE2+ GDCA), respectively.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings.
Example one
Characteristic analysis of intestinal flora of ICP patient and normal pregnant woman
1.1 metagenome analysis of differences in intestinal flora between ICP patients and control populations
Test materials and methods
(1) Collecting 100 ICP patients and 100 normal control pregnant woman feces and serum samples, enlarging sample amount, establishing a perfect sample library and a follow-up database, respectively taking two groups of population feces, dissolving in glycerol solution, and storing at-80 ℃;
(2) feces DNA extraction Kit (MinkaGene Stool DNA Kit) is utilized to extract feces according to the Kit operation steps: taking about 0.2g of excrement to a 2ml centrifugal tube, adding a lysis buffer solution for high-temperature lysis, centrifuging suspension, taking supernatant to a new centrifugal tube, adding proteinase K into the new centrifugal tube, digesting for 10 minutes at 70 ℃, adding an ethanol solvent, passing through a DNA binding column, repeatedly washing twice, adding an EB buffer solution into DNA precipitate for dissolution, and storing at-20 ℃ for later use;
(3) DNA is sent to Beijing Nuo cereal-originated biotechnology company to perform sequencing analysis based on Illumina HiSeq 2500 platform;
(4) after obtaining original sequencing data, firstly removing pollution and a linker sequence, and removing host genes according to a human genome reference sequence; further assembling the high-quality data of the decontamination and host genes through SPAdes; after assembly was complete, orf was predicted using MetaGene; clustering the predicted orf sequences by using CD-HI T software, wherein the longest orf sequence of each class is taken as a representative sequence to construct a non-redundant gene sequence set; after a non-redundant gene set is obtained, clear reads can be compared to the non-redundant gene set, and the abundance of each gene in each sample is calculated; after obtaining the gene abundance table, performing function comparison annotation on the genes by using software such as KEGG, GO, COG and the like; species abundance analysis using metaplan; respectively carrying out alpha and beta diversity analysis according to the abundance of the genes;
(5) according to the sequencing analysis result, the score of the abundance of each strain in each sample is further analyzed by using least square discriminant analysis (PLS-DA), and the variable importance is used for analyzing to obtain the bacteroides which has the most obvious difference between two groups of people.
Results of the experiment
16S rRNA sequencing analysis is carried out on fecal DNA of ICP patients and normal pregnant women respectively, and the result shows that the alpha diversity of intestinal flora of ICP patients is obviously lower than that of a control group (figure 1A). Beta diversity analysis suggests that the intestinal flora structure of ICP patients is significantly different from that of normal control group, and the two groups have significant difference in flora structure (FIG. 1B). Flora composition (fig. 1C) and LefSe analysis showed genera with significant differences between the two groups of people (fig. 1D), and Bacteroides (Bacteroides), lachnocrossdium, veillonella (Bacteroides), ross (Roseburia), Escherichia coli-shigella (Escherichia _ shigella) were found to be significantly enriched in the ICP patient group, while Blautia (Blautia) was significantly reduced. Among them, Bacteroides (Bacteroides) are significantly enriched in the intestinal tract of ICP patients.
1.2 bacterial population transplantation experiments
Test materials and methods
Respectively transplanting fecal bacteria from ICP patient and normal pregnant woman to sterile pregnant mouse, and detecting biochemical indexes such as bile acid and hepatic transaminase and fetal index. The method comprises the following specific steps:
(1) preparing a fecal bacteria suspension: respectively taking fresh feces from ICP patients and normal pregnant women, immediately suspending in PBS solution containing glycerol, subpackaging and storing at-80 deg.C for use;
(2) centrifuging the fecal suspension at low speed, removing excessive fecal residue, transplanting to sterile mice respectively, intragastrically administering 200 μ l (0.15g/ml) fecal suspension to each mouse, twice a week, and continuously intragastrically administering for 4 weeks;
(3) and (3) pressing the male mouse after the intragastric administration: the female mice are subjected to cage combination at a ratio of 1:2, the weight change of the female mice is observed and recorded, and the appearance of pessaries is observed and recorded as E0.5 d;
(4) pregnant female mice E18d, collecting excrement of the mice, anesthetizing, killing the mice, taking tissues such as blood, placenta and the like of the mice, observing and recording the number and weight of fetuses;
(5) detecting ALT, AST, ALP and total bile acid levels in serum by using a full-automatic biochemical analyzer;
(6) after the liver and the placenta are respectively fixed by formalin, embedding paraffin, slicing, and detecting pathological changes of the liver and the placenta tissue by HE (high intensity intrinsic tissue) staining;
results of the experiment
As shown in FIG. 2 (A-D), indexes of fecal strain transplanted mice (I-FMT) bile acid, liver transaminase (AST, ALT, ALP) and the like of ICP patients are obviously higher than those of normal pregnant woman fecal strain transplanted mice (H-FMT). As shown in FIG. 2(E, F), by observing fetal performance and comparing fetal mouse weights, it was found that ICP patients derived fecal bacteria transplanted mice (I-FMT) had significantly higher fetal damage than normal pregnant fecal bacteria transplanted mice (H-FMT).
This study showed that changes in gut flora structure did lead to the development of ICP-related symptoms in pregnant mice.
1.3 Metabolic differences between ICP patients and normal pregnant women
1.3.1KEGG analysis of differential metabolic pathways
Test materials and methods
The composition of intestinal flora of normal pregnant women and ICP patients is analyzed through metagenome sequencing, and KEGG metabolic pathway analysis is carried out through bioinformatics software.
Results of the experiment
As shown in figure 3, in the signal paths of the ICP patient and the normal pregnant woman in which the differentially enriched flora participates in regulation, the most obvious difference is the bile acid metabolism path, and the basis is provided for the follow-up study of bile acid composition and regulation.
1.3.2 Targeted metabonomics detection and analysis of bile acid composition and level in feces and serum of ICP patients and normal pregnant women
Test materials and methods
(1) Respectively taking feces and serum samples of ICP patients and normal pregnant women, and storing in an ultra-low temperature refrigerator for later use;
(2) the bile acid metabolism detection is finished by Shanghai Maite special drawing spectrum biotechnology company;
(3) preparing a test detection article: bile acid standards were purchased from Steralodids (Newport, RI, USA) and TRC Chemicals (Toronto, ON, Canada) and the laboratory at the inventor's premises, 10 isotopically labeled bile acid internal standards were purchased from C/D/N Isotopes (Quebec, Canada) and Steralodids (Newport, RI, USA). Bile acids and isotope internal standards were weighed accurately and dissolved in methanol to give a stock mother liquor of 5mM concentration. In application, the bile acid mother liquor is mixed and diluted in serum matrix without bile acid to 2500, 500, 250, 50, 10, 2.5 and 1nM concentration points. And preparing 1500, 150 and 5nM bile acid standard solutions as quality control samples with three concentrations of high, medium and low by using the bile acid-free serum matrix. The concentrations of the internal standards in the concentration point marking solution and the quality control sample are consistent, namely (GCA-d4, TCA-d4, TCDCA-d9, CA-d4, GCDCA-d4, CDCA-d4, LCA-d4 and aMCA-d5 are both 150 nM);
(4) sample pretreatment: stool or serum samples were added to a centrifuge tube, approximately 25mg of pre-cooled grinding beads was added, and 200 μ L of acetonitrile/methanol (v/v 8:2) mixed solvent containing 10 μ L of internal standard was added. After homogenization, the mixture was centrifuged at 13,500rpm for 20min at 4 ℃. Taking 10 mu L of supernatant, diluting with 90 mu L of mixed solvent of acetonitrile/methanol (80/20) and ultrapure water at a ratio of 1:1, oscillating, centrifuging, and waiting for sample injection analysis, wherein the sample injection volume is 5 mu L;
(5) ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS, ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) was used for quantitative bile acid detection.
Results of the experiment
In ICP patients, there was a significant average reduction in the concentration of three bile acids, glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA) and glycodeoxycholic acid (GDCA), in stool (fig. 4A) and serum (fig. 4B), respectively, compared to normal pregnant women.
1.4 Bacteroides correlation analysis with bile acid levels
Experimental materials and methods
(1) Obtaining the relative abundance value of bacteroides in each fecal sample of the ICP patient through metagenomics analysis;
(2) the content of bile acid in the excrement of the ICP patient is determined through targeted metabonomics, and the relevance of the bacteroid abundance and the content of the bile acid of the ICP patient is analyzed through relevance analysis.
Results of the experiment
Correlation analysis of bacteroides abundance and bile acid level of ICP patients shows that bacteroides abundance and GDCA level are obviously and negatively correlated as shown in figure 5, which indicates that bacteroides may participate in GDCA metabolic regulation.
Example two
Effect of GDCA in the Regulation of ICP Generation by Bacteroides
FXR is distributed in the intestine and liver and is involved in bile acid metabolism. FXR in the liver has been found to inhibit bile acid synthesis by inducing downstream signaling Small Heterodimer Partners (SHPs). Human fibroblast growth factor 19(FGF19) and mouse fibroblast growth factor 15(FGF15) are homologous bile acid metabolism regulators, which are FXR downstream signals. In the gut, FXR inhibits bile acid synthesis by inducing the signaling pathway of FGF19/FGF 15.
This section studies the effect of GDCA on mouse ICP and the mechanism of action.
2.1 Effect of GDCA on mouse ICP symptoms caused by Bacteroides transplantation
Experimental materials and methods
(1) Preparing strains: uniformly coating an anaerobic culture medium plate by a bacteroid plate marking method, culturing in an anaerobic glove box at 37 ℃, selecting a single colony to perform anaerobic culture in a liquid culture medium after the colony appears for 2-3 days, measuring the OD value of the culture medium and calculating the cfu value of bacteria;
(2) bacteroides (2X 10) 8 cfu) is dissolved in 200 mul of sterile water solution, and is respectively irrigated to a sterile female mouse, inactivated bacteroid species with the same concentration is irrigated in a contrast way, GDCA (30mg/Kg/d) is also irrigated to the stomach respectively, the stomach is irrigated twice a week for 4 weeks continuously, a male mouse and a female mouse are combined in a cage according to the proportion of 1:2, and the weight of the mouse and the appearance time of vaginal suppository are observed and recorded;
(3) the female mouse is pregnant E18d, the excrement of the mouse is collected before sacrifice, the mouse is anesthetized and sacrificed, the tissues of the blood, the intestinal tract, the liver, the placenta and the like of the mouse are taken, and the number and the weight of the fetus are observed and recorded;
(4) detecting ALT, AST and total bile acid levels in serum by using a full-automatic biochemical analyzer;
(5) respectively detecting and analyzing the excrement, the serum, the liver and the intestinal tissues by using an ultra-high performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS), wherein the sample processing and detecting and analyzing steps are the same as 1.3.2;
(6) taking small intestine and liver tissues of a mouse, and separating intestinal epithelial cells and liver cells, wherein the method comprises the following specific steps: transferring the small intestine to a culture dish, repeatedly cleaning DPBS, cleaning for several times by using a serum-free culture medium containing penicillin, cutting the intestinal tissue into fragments, cleaning the serum-free culture medium, centrifuging, removing the supernatant, and taking the precipitate for later use. Adding collagenase XI and neutral protease I into the precipitate, digesting for 20 minutes at 37 ℃, repeatedly blowing by a pipette gun, repeatedly cleaning the centrifugal precipitate, and then re-suspending for later use. Taking fresh liver tissue, cutting the liver tissue into small tissue blocks, repeatedly flushing and blowing by using a serum-free culture medium, and adding collagenase for digestion to obtain a liver cell suspension for later use. After the cell suspension is taken, RNA is respectively extracted by using a Trizol kit, and the expression of molecules such as FXR, SHP and the like is detected by qPCR.
Results of the experiment
As shown in 6 (A-D), bacteroides transplantation can obviously cause the increase of mouse bile acid level, ALT and AST liver transaminase level, and the appearance of fetal mice shows damage, and the weight of fetal mice shows reduction, namely shows the occurrence of cholestasis and the damage of fetal mice. The said performance caused by Bacteroides can be relieved obviously after GDCA is supplemented.
As shown in FIG. 6(E, F), in vivo experimental tests show that Bacteroides implantation does not affect the expression of FXR molecules, but can obviously inhibit the expression of FXR downstream signal SHP gene, and can recover FXR signal after GDCA supplementation, which suggests that Bacteroides can regulate and control the change of FXR signal through the change of bile acid such as GDCA.
2.2 in vitro experiments to investigate the relationship between GDCA and FXR signals
On the basis of the research, how GDCA influences the bile acid metabolism of the body and relieves ICP-related symptoms is further researched from the perspective of a signal path for controlling bile acid metabolism.
Experimental materials and methods
(1) Docking analysis predicted the binding conformation relationship of each of the bile acids described above to FXR: downloading the crystal structure (PDB ID:3dct, https:// www.rcsb.org /) of FXR receptor in RCSB Protein Data Bank, performing docking analysis of combination of GDCA and FXR by using Surflex-Dock GeomX (SFXC) of SYBYL-X2.0, and analyzing the combination of bile acid and FXR by using PyMOL and ligplot;
(2) effect of each bile acid on FXR signal activation was detected by TR-FRET FXR coactivation assay using LanthaScreen TM The detection kit of TR-FRET Farnesoid X Receptor activator Assay (Thermo Fisher, Cat # A15140) is carried out, and the specific brief steps are as follows: 1) preparing a Coregator buffer G according to the instruction, and adding DTT with the final concentration of 10 mM; 2) diluting a bile acid sample to be detected by using 100X sample diluent in a multiple ratio; 3) adding 10 mu L of diluted sample diluent into a measuring hole plate, and setting a measuring compound hole; 4) preparing 4X FXR-LBD diluent by using Coregulator buffer G buffer solution, and adding 5 mu L of diluent to each corresponding pore plate; 5) preparing a working solution with a final concentration of 2.0 mu M fluoroescein-SRC 2-2(4X) and 20nM Tb anti-GST antibody (4X) by using a Coregator buffer G buffer solution, and taking 5 mu L of the working solution to the reaction system; 6) gently shaking and uniformly mixing the reaction mixed solution, incubating at room temperature in a dark place, and reading the plate at the wavelengths of 520nm and 495nm respectively; 7) and (3) calculating a TR-FRET ratio (520nm/495nm), drawing a binding curve and calculating EC50, and analyzing the binding and functional effects of each bile acid and FXR in comparison with a standard agonist CDCA and an antagonist GUDCA.
(3) The dual-luciferase reporter gene detects the effect of each bile acid on FXR transcription activity: 1) HEK293 cells are cultured conventionally, after growth and confluence reach 85%, 6-hole plates are inoculated, and the cells are cultured at 37 ℃ until confluence reaches 85%; 2) the Kjeldahl gene company was entrusted to construct a human FXR expression plasmid, a PGL4-Shp-TK firefly luciferase reporter gene vector, and a Renilla luciferase control vector (pRL-luciferase; promega, Madison, WI), co-transfecting HEK293 cells with the transfection reagent Lipofectamine 3000, and culturing the transfected cells for 24 hours; 3) adding the bile acid with final concentration of 50-200 μ M into the cell culture solution, and using CDCA as positive control; 4) the activity of the luciferin reporter gene is respectively detected by using a dual-luciferase detection kit (Promega), and the specific operation is carried out according to the kit instruction.
(4) After incubation of each bile acid with primary intestinal epithelial cells or Caco2 cells, FXR signals and downstream gene expression were detected: 1) taking out small intestine of mouse from clean bench, repeatedly washing the intestinal contents with precooled PBS until the contents are clear, washing with penicillin and streptomycin-containing serum-free culture medium for several times, and shearing intestinal tissue to less than 1mm 2 Transferring the fragments to a centrifuge tube, repeatedly cleaning and blowing the serum-free culture medium, centrifuging to remove supernatant, adding collagenase and neutral protease to digest tissue blocks, repeatedly blowing and digesting, and completely resuspending and culturing the culture medium after centrifuging; 2) conventionally culturing Caco2 cells; 3) after the cells grow and converge to 85%, starving and culturing for 4h, respectively adding GDCA with the final concentration of 50-200 mu M, and incubating for 16 h; the contrast group is respectively added with GUDCA or T-beta MCA with the same final concentration; 3) after the incubation is finished, RNA and protein are respectively extracted, and qPCR is used for detecting the expression of molecules such as SHP, FGF19 and the like.
Results of the experiment
As shown in fig. 7(A, B), Docking analysis predicted that GDCA could bind to FXR, and in vitro experiments preliminarily found that GDCA could directly promote FXR downstream SHP gene expression, as shown in fig. 7(C), suggesting that GDCA may exert a regulatory role as an FXR agonist.
As shown in fig. 8, TR-FRET FXR co-activation analysis found that GDCA may act as an agonist of FXR to agonize FXR.
As shown in FIG. 9, the results of cell experiments show that GDCA can obviously promote the mRNA expression of FXR downstream genes FGF19 and SHP, and the promotion effect can be inhibited by FXR antagonists GUDCA and T-beta MCA.
2.3 in vivo experiments to investigate the relationship between GDCA and FXR signals
On the basis of in vitro experiments, the relation between GDCA and FXR signals is further researched through in vivo experiments.
Experimental materials and methods
(1) Preparing Bacteroides by the same method as in section 2.1;
(2) bacteroides sp(2×10 8 cfu) is dissolved in 200 mul sterile water solution, and is subjected to intragastric administration on a female mouse of a sterile female mouse, inactivated bacteroid species with the same concentration is subjected to intragastric administration on a control, GDCA (30mg/Kg/d) is respectively administered for intragastric administration twice a week for 4 weeks continuously, male mice and female mice are combined according to a ratio of 1:2, and the weight of the mice and the appearance time of vaginal suppository are observed and recorded;
(3) pregnant female mouse E18d, anaesthetizing and killing the mouse, taking the intestinal tract and liver tissue of the mouse, separating the intestinal epithelial cell and the liver cell according to the method of part 2.1, extracting RNA, and respectively detecting the expression of FXR, SHP and other downstream genes of the intestine and the liver by qPCR.
Results of the experiment
As shown in FIG. 10, after Bacteroides implantation into mice, the expression of FXR downstream genes in intestinal tissues is detected, and the Bacteroides implantation can obviously inhibit the expression of FGF15 and SHP downstream genes of FXR, while GDCA can obviously restore the FXR inhibition effect caused by Bacteroides.
This part of in vitro studies showed that GDCA indeed functions as an FXR agonist and is able to promote FXR downstream gene expression.
EXAMPLE III
Therapeutic Effect of GDCA on mouse ICP
Test materials and methods
(1) Standard ICP mouse model making, namely, establishing an ICP mouse model after subcutaneous injection of EE2 estrogen;
(2) gavage with GDCA (30mg/Kg/d) was performed before and after EE2 administration, twice a week for 4 consecutive weeks, male mice: the female mice are caged according to the proportion of 1:2, and the weight of the mice and the appearance time of the pessary are observed and recorded;
(3) and (3) when 18 days of pregnancy is needed, taking the intestinal tract and liver tissues of the mouse, detecting the phenotype change of the ICP mouse, separating intestinal epithelial cells according to the method of the 2.2 part, detecting the expressions of FGF15, SHP and the like by qPCR, separating liver cells, and respectively detecting the expressions of SHP, NTCP, CYP7A1, CYP8B1, BSEP, MRP2 and the like by qPCR.
Results of the experiment
The standard EE2 modeling ICP mouse is obtained by adopting the process shown in figure 11a, and the biochemical indexes such as bile acid level, ALT, AST, ALP, GGT, Liver Index (Liver Index) and the like related to ICP are found to be remarkably increased in a modeling ICP mouse group (EE2) compared with a normal mouse group (Control), which indicates that the modeling is successful, and the biochemical indexes of a GDCA gastric perfusion ICP mouse group (EE2+ GDCA) are remarkably inhibited (figures 11b and 11c), and the Liver pathological injury caused by EE2 can be relieved (figure 11 d).
As shown in fig. 12, the expression of FGF15, SHP and NTCP in FXR downstream genes of the EE2 modeled ICP mouse group (EE2) was significantly suppressed compared to the normal mouse group (Control), while GDCA returned FXR signals of ICP mice (fig. 12a and 12b), while GDCA significantly suppressed the expression of bile acid synthesis genes such as Cyp7a1, Cyp8b1 and Cyp27a1, and promoted the expression of bile acid excretion genes such as BSEP and MRP2 (fig. 12 c).
The results of the in vitro and in vivo experiments on the relationship between the GDCA and the FXR signals show that the ICP-related index and symptom can be relieved by supplementing the GDCA to the ICP mice, and the GDCA plays a role in preventing and treating ICP by enhancing the expression of liver and intestinal FXR downstream signal genes in the mice.
Compared with the prior art, the invention has the following beneficial effects: the research discovers an action mechanism of intrahepatic cholestasis caused by the increase of the abundance of bacteroides enterobacter in intestinal tracts of ICP patients, and in vitro experiments and animal experiments prove that GDCA, as a novel agonist of FXR, can play a role in preventing or/and relieving ICP by exciting FXR signals, and shows that GDCA has the potential of being used for preparing ICP treatment medicines.
Finally, it should be noted that the above-mentioned description is only a preferred embodiment of the present invention, and those skilled in the art can make various similar representations without departing from the spirit and scope of the present invention.
Claims (9)
1. Use of glycodeoxycholic acid or a stereoisomer or a derivative thereof for the preparation of a medicament for the prevention and/or treatment of intrahepatic cholestasis in humans or mammals.
2. 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. Use according to claim 1 or 2, characterized in that: the intrahepatic cholestasis is caused at least in part by inhibition of Farnesoid X Receptor (FXR) downstream signaling in the human or mammal.
4. Use according to claim 3, characterized in that: the FXR downstream signal inhibition is caused by the increase of the abundance of Bacteroides in intestinal tracts of humans or mammals.
5. Use according to claim 1 or 2, characterized in that: the medicament is used for preventing and/or treating intrahepatic cholestasis in pregnancy of a human or a mammal.
6. Use of glycodeoxycholic acid or a stereoisomer or a derivative thereof for the preparation of a medicament intended to act as an FXR agonist in humans or mammals.
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 a pharmaceutically acceptable carrier, additive or excipient, and unavoidable impurities.
9. The pharmaceutical composition of claim 8, wherein the drug is a tablet, a granule, a capsule, or an oral liquid.
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