Mechanism Of MiR-200c Deletion In Alleviating Cholestatic Hepatic Fibrosis

Objectives:Some studies have shown that miR-200c has abnormal expression in liver fibrosis,but its role in the occurrence and development of liver fibrosis is still unclear.Therefore,we performed bile duct ligation on wild type C57BL/6J mice and C57BL/6J mice with miR-200c knock out to induce cholestasis and simulate cholestatic liver fibrosis to explore the effect of miR-200c on BDL induced liver fibrosis.Methods:Six to eight week old healthy male wild-type mice and miR-200c KO C57BL/6J mice were selected for sham operation and BDL.They were divided into four groups:WT-sham,WT-BDL,miR-200c KO-sham and miR-200c KO-BDL.The mice in the sham operation opened the abdominal cavity to free the common bile duct without ligation,while the mice in the BDL opened the abdominal cavity to the bile duct ligation.The experimental period was two weeks.The weight of the four groups of mice was measured at 0,1,3,5,7,9,11,13 and 14 days respectively,and the amount of food and water intake was recorded.Observe the general state of mice in each group after operation.On the 14^th day after operation,take materials and weigh the weight of liver,kidney and spleen,calculate the organ index（viscera weight/body weight,%）,and measure the serum aspartate transferase（AST）,alanine aminotransferase（ALT）and total bile acid（TBA）.H&E staining and Masson staining were used to observe the pathological changes of liver.Western blot was used to quantitatively detect the expression of liver fibrosis,inflammation and apoptosis related proteins of mice in each group.Detection of liver fibrosis markers by Quantitative RT-PCR:α-Sma,Tgf-βand so on.LO2 cells were cultured,and the necrosis of cells treated with cholic acid（CA）,chenodeoxycholic acid（CDCA）,taurocholic acid（TCA）and taurochenodeoxycholic acid（TCDCA）was detected with the cell necrosis test kit.After liver tissue was taken,it was quickly frozen,and frozen sections were prepared for Mass Spectral Imaging detection to observe the spatial distribution of bile acids.Immunohistochemistry（IHC）was used to stain liver tissue sections to observe the expression and distribution of ZO-1.LO2 cells were transfected with miR-200c mimics to detect the m RNA expression of apoptosis related factors.Results:1.The activity of mice in the four groups decreased after operation.The activity of mice in WT-sham and miR-200c KO-sham basically recovered to the preoperative level 1-2 days after operation,and the rest had no significant change,while the activity of mice in WT-BDL and miR-200c KO-BDL basically did not recover,and the weight of mice in BDL group decreased significantly.Three days after operation,the ears,limbs and tail of mice in BDL group began to appear jaundice,the urine color gradually deepened to dark yellow,and the skin of mice in BDL group was rough and lusterless at the late stage of operation.On the 14^th day after operation,it can be seen that with the extension of common bile duct ligation time,mice in BDL group showed yellow staining of peritoneum,gallbladder and common bile duct gradually increased due to cholestasis,liver texture became hard,and granular nodules appeared on the uneven surface.2.The indexes of liver,kidney and spleen in WT-BDL were significantly higher than those in WT-sham（p<0.05）.The indexes of liver,kidney and spleen in miR-200c KO-BDL were significantly lower than those in WT-BDL（p<0.05）.ALT,AST and TBA in WT-BDL were significantly higher than those in WT-sham（p<0.05）.AST and ALT in miR-200c KO-BDL were significantly lower than those in WT-BDL（p<0.05）.3.Microscopic observation of liver H&E staining showed that:in WT-sham and miR-200c KO-sham,normal and complete structure of liver lobules,orderly arrangement of liver plates,no expansion of bile ducts,no inflammatory infiltration and necrosis of liver tissue were found.In WT-BDL,there were many inflammatory cells infiltrating into the liver tissue,hyperplasia of bile duct epithelioid cells,bile duct dilatation and proliferation of fibrous tissue around the bile duct in the portal area,and large necrotic hepatocytes and structural disorder of hepatic lobules were seen.Compared with WT-BDL,miR-200c KO-BDL was relieved in inflammatory cell infiltration,bile duct expansion,hepatocyte necrosis,etc.4.Masson staining of liver under microscope showed that the liver structure of mice in WT-sham and miR-200c KO-sham was normal,without collagen fibers,liver lobules were arranged orderly,cytoplasm was dyed dark red,and no obvious blue was seen around.Bile duct dilatation,tissue structure disorder and collagen fiber hyperplasia were observed in WT-BDL.Compared with WT-BDL,the degree of bile duct dilatation and collagen fiber formation in miR-200c KO-BDL were significantly reduced.5.The necrosis of LO2 cells was stained.It was found that the necrosis of LO2 cells was significantly increased after bile acid treatment.6.Mass spectrometry of frozen sections of liver tissue showed that TCA and TCDCA had no difference between WT-sham and miR-200c KO-sham.The content of TCA and TCDCA in miR-200c KO-BDL was significantly lower than that in WT-BDL,and the content of ATP,ADP and UDP was also significantly different.7.Apoptotic factors increased significantly after overexpression of miR-200c in LO2 cells.8.The expression of ZO-1 in the liver of BDL mice was decreased by immunohistochemical staining of liver sections,but increased in miR-200c KO-BDL.Conclusion:miR-200c KO has the effect of alleviating liver fibrosis caused by cholestasis in mice,and can inhibit the inflammatory reaction and hepatocyte apoptosis,regulate bile acid metabolism,and improve the morphology and function of the liver.