Based On LRH-1 Related Signal Pathways, We Explored The Effect And Mechanism Of Piperine On Inhibiting The Formation Of Cholesterol Stones In Mice

Purpose:The aim of this study is to establish an animal model of cholesterol gallstone in C57BL/6 mice induced by high-fat diet.By observing the effect of piperine on the expression of lipid in serum and bile,we further regulate the signal pathway of cholesterol transporter in liver based on LRH-1,and partly reveal the biological mechanism of piperine inhibiting cholesterol gallstone formation,so as to prevent cholesterol gallstone formation in clinic.The research and development of stone disease drugs provide experimental basisMaterial and method:1.Laboratory Animals Ninety SPF male C57BL/6 mice,aged 10-14 weeks and weighing 20±2 g,were fed in cages for 7 days.They were randomly divided into six groups:blank group,model group,ursodeoxycholic acid group?90mg kg₁?,low-dose piperine group?20 mg kg-1?,medium-dose piperine group?40 mg kg-1?and high-dose piperine group?60 mg kg-1?,with 15 mice in each group 2.Modeling and Medication Intervention At the same time,the drug dose was converted according to the equivalent dose of body weight of mice and adults.Except for the blank group,the other groups were fed with high-fat diet containing 2%cholesterol?basic diet+2%cholesterol+0.5%cholic acid+15%butter?The blank group and model group were given the same amount of 0.5%CMC-Na solution at 10:00 a.m.every day.The ursodeoxycholic acid group was given ursodeoxycholic acid solution at 10:00 a.m.And the piperine groups were given the same amount of piperine dissolved in 0.5%CMC-Na at 10:00 a.m.every day.Feeding for 4 weeks 3.Draw Materials Before the experiment,mice were fasting for 12 hours,weighed and recorded.Blood was collected by eyeball extraction;abdominal cavity was opened to observe the gallbladder,intracystic bile and gallstones,and calculating the incidence of gallstones?referred to as stone rate?;gallbladder length and width were measured by vernier caliper,gallbladder volume was calculated,and gallbladder bile was collected;all liver tissues were frozen and stored in the refrigerator at-80? for reserve.4.Detection Indicators ?1?Observation of the general state of mice:Observe the general state of weight,diet,fur,activity and reaction ability of mice in each group. ?2?Calculate the gallbladder stone rate of each group:gallbladder stone rate?%?=the number of mice producing gallbladder stone in each group/the number of mice in each group*100%;?3?Calculate the gallbladder volume of mice in each group:Calculate the gallbladder volume according to the elliptic formula:Volume?UL?=length?mm?*width?mm?*depth?mm?*pi/6?approximate to think that the gallbladder width is equal to the depth diameter?;?4?The lipid levels in bile of mice in each group were measured by automatic biochemical analyzer,CSI was calculated,and the lithogenicity of bile was evaluated by Carey scale.?5?The expression of TC,TG,LDL-C and HDL-C in serum of mice in each group was detected by automatic biochemical analyzer.?6?Western-blot technique was used to detect the expression of LRH-1,SRBI,ABCG5 and ABCG8 in liver tissue of mice in each group.?7?The expression of LRH-1,SRBI,ABCG5 and ABCG8 in liver tissues of mice in each group was detected by fluorescence quantitative PCR.Results:1.Effect of Piperine on Serum Lipid Level in C57BL/6 Mouse Cholesterol Gallstone Model 1.1 General state of mice in each group The mice in each group were active,with bright fur,good mental state and appetite,and no significant change in body weight.There was no death in all groups. 1.2 Lithogemc rate of mice in each group The lithiasis rates of mice in each group were blank group?O%?,model group?100%?and ursodeoxycholic acid group.The lithiasis rates of low,medium and high piperine group were 20%?3/15?,80%?12/15?,46.7%?7/15?and 13.3%?2/15?,respectively.Compared with the model group,there was no significant change in piperine low dose group?P>0.05?,and the other groups had statistical significance?P<0.05?. 1.3 Changes of gallbladder volume in mice of each group Compared with the blank group,the gallbladder volume of model group,ursodeoxycholic acid group and piperine group increased significantly?P<0.05?;compared with model group,the gallbladder volume of mice in ursodeoxycholic acid group,piperine medium dose group and piperine high dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the gallbladder volume of mice in low dose piperine group increased significantly?P<0.05?,and the gallbladder volume of mice in piperine low dose group increased significantly?P<0.05?,The gallbladder volume of mice in high dose group of capsaicin decreased significantly?P<0.05?.1.4 Comparison of CSI in gallbladder bile of mice in each group Compared with the blank group,CSI values of model group,ursodeoxycholic acid group and piperine groups were significantly higher?P<0.05?,and CSI values of model group and low-dose piperine group were higher than 1,suggesting cholesterol supersaturated bile;compared with model group,CSI values of ursodeoxycholic acid group and piperine group were significantly lower?P<0.05?;compared with ursodeoxycholic acid group,CSI values of low-dose piperine group were significantly lower?P<0.05?.The CSI value increased significantly?P<0.05?and decreased significantly in piperine medium and high dose groups?P<0.05?. 1.5 Comparison of TC and TG in Serum of Mice in Different Groups Compared with blank group,TC content in model group,ursodeoxycholic acid group and piperine group increased significantly?P<0.05?;compared with model group,TC content in ursodeoxycholic acid group,middle and high dosage piperine group decreased significantly?P<0.05?;TC content in low and middle dosage piperine group increased significantly compared with ursodeoxycholic acid group?P<0.05? Compared with blank group,TG content in model group increased significantly?P<0.05?;compared with model group,TG content in ursodeoxycholic acid group,middle and high dosage piperine group decreased significantly?P<0.05?;The content of TG in piperine low dose group was significantly higher than that in piperine high dose group?P<0.05? 1.6 Comparison of LDL-C and HDL-C in Serum of Mice in Different Groups Compared with the blank group,LDL-C content in model group,ursodeoxycholic acid group and piperine group increased significantly?P<0.05?;compared with model group,LDL-C content in ursodeoxycholic acid group,middle-dose piperine group and high-dose piperine group decreased significantly?P<0.05?.Compared with ursodeoxycholic acid group,the content of LDL-C in low and middle dose piperine group increased significantly?P<0.05?Compared with the blank group,HDL-C content in model group and low-dose piperine group decreased significantly?P<0.05?;compared with model group,HDL-C content in ursodeoxycholic acid group and high-dose piperine group decreased significantly?P<0.05?;Compared with ursodeoxycholic acid group,the content of HDL-C in low dose piperine group decreased significantly?P<0.05? 2.Effects of Piperine on LRH-l/SRBI and LRH-1/ABCG5/ABCG8 Signaling Pathway in Liver of Cholesterolithiasis Mice2.1 Comparison of LRH-1 Protein Expression in Liver Tissues of Mice in Different Groups Compared with the blank group,the expression of LRH-1 protein in model group,ursodeoxycholic acid group and piperine low and medium dose group increased significantly?P<0.05?;compared with model group,the expression of LRH-1 protein in ursodeoxycholic acid group,piperine medium and high dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of LRH-1 protein in low and medium dose group increased significantly?P<0.05?.The expression of LRH-1 protein in piperine high dose group decreased significantly?P<0.05? 2.2 Comparison of SRBI protein expression in liver tissue of mice in each groupCompared with blank group,SRBI protein expression in model group,ursodeoxycholic acid group and piperine low and medium dose group increased significantly?P<0.05?;compared with model group,SRBI protein expression in ursodeoxycholic acid group,piperine medium and high dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,SRBI protein expression in low dose group of piperine increased significantly?P<0.05?,and piperine increased significantly?P<0.05?.The protein expression of SRBI in the high dose group of capsaicin decreased significantly?P<0.05?. 2.3 Comparison of ABCG5 Protein Expression in Liver Tissues of Mice in Different Groups Compared with the blank group,the expression of ABCG5 protein in model group,ursodeoxycholic acid group and piperine low and medium dose group increased significantly?P<0.05?;compared with model group,the expression of ABCG5 protein in ursodeoxycholic acid group,piperine medium and high dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of ABCG5 protein in piperine low dose group increased significantly?P<0.05?.The expression of ABCG5 protein in piperine high dose group decreased significantly?P<0.05?.2.4 Comparison of ABCG8 Protein Expression in Liver Tissues of Mice in Different Groups Compared with blank group,the expression of ABCG8 protein in model group,ursodeoxycholic acid group and piperine low and medium dose group increased significantly?P<0.05?;compared with model group,the expression of ABCG8 protein in ursodeoxycholic acid group,piperine medium and high dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of ABCG8 protein in piperine low dose group increased significantly?P<0.05?.2.5 Comparison of LRH-1 expression in liver tissues of mice in each group Compared with the blank group,the expression of LRH-1 in model group,ursodeoxycholic acid group,piperine groups increased significantly?P<0.05?;compared with model group,the expression of LRH-1 in ursodeoxycholic acid group,piperine medium-dose group and high-dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of LRH-1 in piperine low-dose group and middle-dose group increased significantly?P<0.05?.?2.6 Comparison of SRBI mRNA Expression in Liver Tissues of Mice in Different Groups Compared with the blank group,the expression of SRBI in model group,piperine low-dose group and medium-dose group increased significantly?P<0.05?;compared with model group,the expression of SRBI in ursodeoxycholic acid group,piperine medium-dose group and high-dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of SRBI in piperine low-dose group and middle-dose group increased significantly?P<0.05?2.7 Comparison of the expression of ABCG5 and ABCG8 in liver tissue of mice in each group Compared with the blank group,the expression of ABCG5 in model group,piperine low-dose group and medium-dose group increased significantly?P<0.05?;compared with model group,the expression of ABCG5 in ursodeoxycholic acid group,piperine medium-dose group and high-dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of ABCG5 in low-dose piperine group increased significantly?P<0.05?2.8 Comparison of ABCG 8 mRNA expression in liver tissues of mice in groups Compared with the blank group,the expression of ABCG8 in model group,piperine groups increased significantly?P<0.05?;compared with model group,the expression of ABCG8 in ursodeoxycholic acid group,piperine medium-dose group and high-dose group decreased significantly?P<0.05?;compared with ursodeoxycholic acid group,the expression of ABCG8 in piperine low-dose group and middle-dose group increased significantly?P<0.05?Conclusion:In this study,C57BL/6 mice fed with high-fat diet were successfully established an efficient and stable cholesterol gallstone model.The moderate and high doses of piperine?40 mg kg-1,60 mg kg-1?may improve the abnormal lipid metabolism in serum by inhibiting the expression of LRH-l/SRBI and LRH-1/ABCG5/ABCG8 signaling pathways in liver tissue,thus inhibiting the formation of cholesterol gallstones in C57BL/6 mice induced by high-fat diet,and the high doses of piperine group is better than the medium doses of piperine group.