The Mechanisms On The Prevention Of Gallstone Formation By Berberine In Mice

Background: Cholesterol gallstone disease is a common disease.The formation of cholesterol gallstone is caused by the interaction of both genetic and environment factors leading to supersaturation of biliary cholesterol,imbalance of pro/anti-nucleating factors and dysfunction of gallbladder.Among them,supersaturation of biliary cholesterol is the prerequisite for gallstone formation,which is often related with high cholesterol/fat diet.Berberine(BBR)is a traditional medicine that is prescribed to diarrhea caused by acute intestinal infection.In recent years,BBR has attracted more and more attention,because of its effects in anti-dyslipidimia,anti-diabetes,anti-cancer and immune regulations.In the first part of this study,we aimed to investigate the alternations in the composition of gut microbiota in mice fed with lithogenic diet.In the second part of this study,we aimed to investigate the effect of BBR on intestinal cholesterol absorption,regulation of gut microbiota and its involvement in the regulation of entero-hepaitc cholesterol and bile acid metabolism as well as it possible role in the prevention of gallstone formation.In the third part of this study,we aimed to investigate the role of prevention of cholesterol gallstone formation by ezetimibe(Eze)that is an inhibitor targeting intestinal cholesterol transporter,Niemann Pick C1 like 1,in mice fed with lithogenic diet.Material and manners: Adult male C57BL6 J mice were fed with lithogenic diet containing 1.25% cholesterol and 0.5% cholic acid for 8 weeks,to induce gallstone formation(LD group).In the BBR treatment group,mice were given by gavage each day with a dose of 50 and 100 mg/kg·d of BBR,respectively(BBR50 and BBR100 group).In the Eze treatment group,mice were given by gavage each day with a dose of 5 mg/kg·d of Eze.A group of mice were fed with chow diet as blank control(Chow group).On sacrifice,occurrence of gallstone was observed.Liver,gallbladder,intestines and feces were collected from each mice.Expression of genes involved in metabolism of cholesterol in enterohepatic circulation were measured by realtime PCR.Gut microbiota were measured by 16 sRNA sequencing.The lipids in liver and bile were quantified by enzymatic methods.Result:(1)Lithogenic diet could alternate the gut microbiota in mice.The abundance,diversity and composition of gut microbiota were changed significantly,but BBR show its capability to restore the abundance and diversity,and rebuild the composition of gut microbiota.(2)BBR could inhibit the absorption of cholesterol in intestine(Chow group 59.37±2.53,LD group 59.43±3.22,BBR50 group 36.96±5.31,BBR100 group 28.41±3.41),the degree of reduction were 38%~52% when comparing with LD group.(3)BBR could reduce the free cholesterol(Chow group 0.50±0.02,LD group 0.78±0.02,BBR50 group 0.76±0.02,BBR100 group 0.68±0.04)and cholersteryl ester(Chow group 3.24±0.11,LD group 40.37±2.30,BBR50 group 32.97±2.94,BBR100 group 19.11±2.82)in liver tissue.(4)BBR could reduce the production of secondary bile acid through inhibiting the bacteria belong to Clostridium that possess 7?-dehydroxylase activity,and then reduce the hydrophobic index of bile acid in gallbladder bile(Chow group-0.1945±0.0096,LD group 0.1211±0.0085,BBR50 group 0.0407±0.0130,BBR100 group 0.0090±0.0050).(5)BBR could improve the composition of bile and reduce the cholesterol saturation index(CSI)(Chow group 0.29±0.02,LD group 1.44±0.22,BBR50 group 1.20±0.05,BBR100 group 0.90±0.12),which can help to inhibite the precipitation of cholesterol.(6)Gallstone formed in mice fed with lithogenic diet(100%),but ezetimibe completely inhibited gallstone formation(0%).(7)The intestinal cholesterol absorption rate significantly reduced in eze group(9.29%±4.32%)compared with LD group(58.62%±3.10%),P<0.01.(8)Eze group had lower plasma cholesterol level(1.11±0.10 mmol/L)and hepatic cholesterol level(Eze: 2.70±0.07 mg/g tissue)compared with LD group(4.99±0.50 mmol/L and 22.92±2.39 mg/g tissue),P<0.05.(9)Biliary cholesterol content and cholesterol saturation index(CSI)decreased markedly in Eze group compared with LD group(cholesterol: Eze: 2.72±0.29 vs LD: 10.87±1.46 mmol/L;CSI: Eze: 0.57±0.07 vs LD: 1.42±0.19,P<0.01).Conclusions:(1)Berberine could inhibit intestinal cholesterol absorption and ameriolate hepatic cholesterol burden in mice fed with lithogenic diet.It aslo remodelled gut microbiota under such diet and decreased secondary bile acid contents.These regulatory effects by BBR collectively led to inhibition of gallstone formation in mice.Our results may provide novel experimental evidences for the mechanisms through which BBR regulates the metabolism of cholesterol and bile acid.(2)Ezetimibe could prevent gallstone formation in mice through inhibition of intestinal cholesterol absorption.