| Objective:Cholesterol gallstone(CGS)is a complex multifactorial disease affected by the interaction of genetic and environmental factors,and it is the expression of the steady-state imbalance of cholesterol in bile when the physical and chemical balance of cholesterol solubility in bile is disturbed.The main pathophysiological event is the persistent liver hypersecretion of biliary cholesterol.Most environmental factors may be related to western eating habits,including excessive intake of cholesterol.Laparoscopic cholecystectomy is one of the most common surgical operations,and it is the main method to treat gallstones at present.However,it is invasive and may lead to surgical complications.Not all patients with symptomatic gallstones are suitable for surgery.Ursodeoxycholic Acid(UDCA)has been used as a first-line drug,and has been suggested as a treatment option for patients with cholesterol-rich non-calcified gallstones<20mm in diameter.Taking UDCA for a long time can promote the dissolution of cholesterol gallstones.However,its long course of treatment may cause gastrointestinal dysfunction.Therefore,it is very important to develop new,effective and non-invasive treatment methods to prevent or treat CGS.In the treatment of cholesterol gallstones,Chinese herbal medicine not only has the advantages of safety,small side effects and obvious curative effect,but also has the characteristics of economy and practicality.Therefore,exploring the pathogenesis of cholesterol gallstones,devoting ourselves to the treasure of traditional Chinese medicine in China,developing safe and effective therapeutic drugs,and expounding the mechanism of action of drugs will bring good news to patients with cholesterol gallstones.Polygonum cuspidatum is a traditional Chinese herbal medicine in China,and Polydatin(PD)is a stilbene compound extracted from Polygonum cuspidatum root.Previous reports have shown that PD has many pharmacological effects,such as antiinflammatory,neuroprotective,antioxidant,immunomodulatory,cardioprotective and antiplatelet aggregation activities.However,the study of polydatin in cholesterol gallstones has not been reported,and the effect and mechanism of polydatin on cholesterol gallstones need to be revealed.In this study,firstly,the bile of patients with clinical cholelithiasis was analyzed by wide-target metabonomics.Then,a mouse model of cholesterol gallstone was established,and polydatin was used to intervene to verify the effect of polydatin on the differential metabolites in wide-target metabonomics.At the same time,the possible role of polydatin in cholesterol gallstone and its related mechanism were further studied at animal and cellular levels.Methods:Part I Analysis of bile metabolomics in patients with cholelithiasisGallbladder bile was collected from patients with non-calculi,cholesterol calculi,primary calculi and secondary calculi,and was detected by UPLC-MS chromatography and mass spectrometry for broad target metabolomics analysis to screen different bilirelated metabolites.Part Ⅱ The effect and mechanism of polygonin on cholesterol calculi were verified in vivoMale C57BL/6 mice aged 8 weeks were purchased and divided into 5 groups:Control,Model,Model+L-polydatin,Model+M-polydatin,Model+H-polydatin,with 6 mice in each group.The Control group was given ordinary feed.Model group was fed lithogenic diet(conventional diet supplemented with 1.25%cholesterol,15%total fat and 0.5%cholic acid)for 8 weeks,and each mouse was given the same amount of solvent(0.5%CMC-Na)once a day for 8 weeks.Model+polydatin group was given lithogenic diet for modeling,and each mouse was given polydatin(25,50,100 mg/kg)by intragastric administration once a day for 8 weeks.The gallbladder of mice was exposed,the lithiasis condition was evaluated by the 6-level judgment criteria,and the group with the best treatment effect of polygonin was selected for subsequent experimental detection.Liver and gallbladder tissues of mice in each group were collected,fixed and frozen for follow-up test.Experimental group:Control,Model,Model+polydatin.The group with the best treatment effect of polygonin was selected for follow-up experimental detection:1.Four different metabolites in broad-target metabolomics in bile of mice in each group were verified by commercial kit.2.After exposing the gallbladder of mice,the stones were taken and the stone formation of each group was analyzed.3.HE staining was used to detect the pathological changes of liver and gallbladder tissues,and to verify the effect of polygonin intervention on the pathological changes of liver and gallbladder tissues of mice with cholesterol calculus.4.Oil red O staining was used to verify the effect of polygonin intervention on lipid deposition in the liver of mice with cholesterol calculus.5.The serum triglycerides(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)of mice in each group were detected by the kit,and the effects of polygonin intervention on the changes of lipid levels of mice with cholesterol calculus were investigated.6.The kit was used to detect the levels of total bile acid and cholesterol in gallbladder bile,and to evaluate the effects of polydatin intervention on the changes of total bile acid and cholesterol levels in cholesterol calculus mice.7.ELISA kit was used to detect the levels of IL-1β,IL-6 and TNF-α in serum to verify the effect of polydatin intervention on inflammation induced by cholesterol stone mice8.The levels of ABCG5,ABCG8,Cyp7al and MUC5AC in liver tissues were detected by Real-time PCR and Western blot.Objective To investigate the effects of polygonin intervention on the expression of ABCG5,ABCG8,Cyp7a1,MUC5AC proteins related to cholesterol transport and stone formation in liver of mice with cholesterol calculus.9.The levels of PPAR-γ(cytoplasm)and PPAR-γ(nucleus)in liver tissues were detected by Real-time PCR and Western blot,and the effect of polygonin intervention on PPAR-γ signaling pathway in mice with cholesterol calculus was verified.Part Ⅲ:The effect of cuspidin on human intrahepatic bile duct epithelial cell inflammation and its mechanism1.Human intrahepatic bile duct epithelial cells were cultured and treated with 100 μg/ml LPS to construct the inflammation model of human intrahepatic bile duct epithelial cells.The cells were treated with different doses of polydatin(10 μ m,20 μ m,40 u m)for 24 h,and cell activity was detected by CCK-8.The optimal concentration of polydatin was screened for follow-up experiments.2.The levels of IL-1β,IL-6 and TNF-α in the supernatant of cells were determined to verify the effect of cuspidin on inflammatory factors in the inflammatory model of intrahepatic bile duct epithelial cells.3.Real-Time PCR and Western blo were used to detect the levels of ABCG5,ABCG8,Cyp7al and MUC5AC in cells,and to investigate the effects of cuspidin on the proteins related to cholesterol transport and stone formation in the inflammatory model of intrahepatic bile duct epithelial cells4.Western blot was used to detect the expression levels of PPAR-y(cytoplasm)and PPAR-y(nucleus)in cells,and to verify the regulatory effect of cuspidin on PPAR-y signaling pathway in the inflammatory model of intrahepatic bile duct epithelial cells5.Immunofluorescence(IF)was used to detect the expression of PPAR-y in cells and its entry into the nucleus,so as to further verify the regulatory role of PPAR-y signaling pathway in the inflammation model of intrahepatic bile duct epithelial cells with the intervention of polydatin.Results:Part I Analysis of bile metabolomics in patients with cholelithiasisA total of 105 kinds of bili-related differential metabolites were screened,among which 42 kinds were related to primary bile duct stones,34 kinds were related to secondary bile duct stones,and 29 kinds were related to cholesterol stones.Ultimately,Four kinds of bile differential metabolites related to cholesterol stones were screened out by broad-target metabolomics analysis,which were used for subsequent polydatin related experimentsPart Ⅱ the effect and mechanism of polygonin on cholesterol calculi were verified in vivo1.The expression level of the three differential metabolites in the cholesterol calculus model group was basically consistent with the expression level of heterometabolites in the broad target metabolomics.The above difference could be reversed in medium dose and high dose drug groups.2.The results showed that compared with the Control group,the grade of gallstones in the model group was significantly higher than that in the control group.After treatment with low,medium and high cuspidin,the gallstones in the model group showed a downward trend.Therefore,we selected the treatment group with high concentration of H-polydatin for the follow-up experimental study.3.HE staining results of liver showed that the liver cells in the control group were arranged neatly and tightly,while the liver cells in the cholesterol calculus model group were arranged loosely,with increased lipid vacuole deformation,hepatocyte injury and inflammatory cell infiltration,while the liver pathological symptoms in the polydatin intervention group were significantly reduced.The results of HE staining of gallbladder tissue showed that the gallbladder wall structure was normal in the normal group,the gallbladder wall connective tissue hyperplasia,gallbladder wall thickening and serous membrane structure and morphology were destroyed in the cholesterol stone model group,and the gallbladder histopathologic symptoms were significantly reduced in the polydatin intervention group.The results showed that polygonin had obvious therapeutic effect on cholesterol calculus in mice.4.The results of oil red O showed that the lipid deposition in the liver tissue of mice in the cholesterol calculus model group was significantly increased,while the lipid deposition in the cuspidate intervention group was gradually reduced,indicating that cuspidate could reverse the lipid deposition induced by cholesterol calculus.5.The blood lipid test results showed that compared with the control group,the serum levels of triglyceride(TG),total cholesterol(TC),high and low density lipoprotein cholesterol(LDL-C)in the cholesterol stone model group were significantly increased,and the levels of density lipoprotein cholesterol(HDL-C)were significantly decreased,and polydatin could significantly reverse the above changes.6.The test results of total bile acid and cholesterol showed that,compared with the control group,the level of total bile acid in the gallbladder bile of mice in the model group was significantly reduced,and the level of cholesterol was significantly increased,and polydatin could significantly recover the above changes.7.The results of inflammation-related indicators showed that the levels of IL-1β,IL-6 and TNF-α in serum of mice in the model group were significantly up-regulated,and the contents of IL-1β,IL-6 and TNF-α were significantly decreased after the intervention of polygonin,suggesting that polygonin could reverse the occurrence of cholesterol stone-induced inflammation.8.The gene and protein expressions of ABCG5,ABCG8,Cyp7al and MUC5AC related to cholesterol transport and stone formation showed that compared with the control group,the gene and protein expressions of ABCG5,ABCG8 and MUC5AC in the model group were significantly up-regulated,while the expression of Cyp7al was significantly down-regulated.The above changes could be reversed by intervention of polygonin.9.The detection results of PPAR-γ signaling pathway related genes and proteins showed that compared with the control group,the expression of PPAR-γ genes and proteins in the model group was significantly down-regulated,and the above changes could be restored by the intervention of polygonin.Part Ⅲ:The effect of cuspidin on human intrahepatic bile duct epithelial cell inflammation and its mechanism1.Inflammatory factor-related results showed that the levels of IL-1β,IL-6 and TNF-α in the supernatant of human intrahepatic bile duct epithelial cells in LPS group(LPS group)were significantly increased compared with the normal cell control group;Compared with LPS group,the levels of IL-1β,IL-6 and TNF-α in the cell supernatant of cuspidin intervention group were significantly decreased,suggesting that cuspidin could inhibit the expression of inflammatory cytokines in intrahepatic bile duct epithelial cells.2.The expression of ABCG5,ABCG8,Cyp7a1,MUC5AC genes and proteins related to cholesterol transport and stone formation showed that,compared with normal cells,the expressions of ABCG5,ABCG8,MUC5AC genes and proteins in LPS group were significantly up-regulated,while the expression of Cyp7a1 was significantly down-regulated.Compared with LPS group,the expression of genes and proteins of ABCG5,ABCG8 and MUC5AC in cuspine intervention group was significantly down-regulated,while the expression of Cyp7a1 was significantly up-regulated,suggesting that Cuspine intervention could reverse the LPS-induced up-regulation of ABCG5,ABCG8 and MUC5AC in human intrahepatic bile duct epithelial cells.Down-regulation of Cyp7a1 expression.3.Western blot analysis of PPAR-γ related proteins showed that the expression of PPARγ protein in nucleus and PPAR-γ protein in plasma of LPS group was significantly down-regulated and up-regulated compared with the control group.Compared with LPS group,the expression of PPAR-γ protein was significantly down-regulated in polygonin intervention group,suggesting that polygonin intervention could restore the above LPs-induced changes,suggesting that polygonin alleviated LPS-induced inflammation of human intrahepatic bile duct epithelial cells by promoting PPAR-γinto the nucleus and activating PPAR-γ signaling pathway.4.Immunofluorescence detection of the expression of PPAR-γ in vitro showed that compared with the control group,the expression of PPAR-γ in the nucleus of cells in LPS group was significantly reduced,and the expression of PPAR-γ in the nucleus was significantly increased after the intervention of polygonin.It was further revealed that sceptoside alleviated LPS-induced human intrahepatic bile duct epithelial cell injury by activating PPAR-γ signaling pathway.Conclusion:1.A total of 105 bili-related differential metabolites were screened by broad-target metabolomics in bile of patients with cholelithiasis,among which 29 kinds were related to cholesterol stones.After analysis and screening,four kinds of bile differential metabolites related to cholesterol stones were selected,and three kinds of differential metabolites were verified by intervention experiments related to polydatin,which were consistent with the results of metabolomics.2.Polydatin can inhibit the secretion of inflammatory factors through activating the PPAR-γ signaling pathway,regulate the expression of cholesterol transport and stone formation related genes ABCG5,ABCG8,Cyp7a1,MUC5AC and inhibit the formation of cholesterol stones in mice.3.Polydatin can inhibit the inflammatory factor of intrahepatic bile duct epithelial cells induced by LPS by activating PPAR-γ signaling pathway and inhibiting the secretion of inflammatory factors,regulate the expression of cholesterol transport and stone formation related genes including ABCG5,ABCG8,Cyp7al and MUC5AC.The effect and mechanism of polygonin on inflammatory changes of cholesterol calculi were further verified in vitro... |
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