Effects And Mechanism Of Clostridium Intestinal Metabolites PCS/pCG On Intrahepatic Inflammation And Biliary Epithelial Cells

Objective:Primary Biliary Cholangitis(PBC)is an autoimmune liver disease c haracterized by progressive chronic cholestasis and intrahepatic non-purulent infl animation of the small and medium bile ducts,and the pathogenesis is currentl y unknown.Targeted destruction of Biliary Epithelial Cells(BEC)is an import ant feature of PBC.BEC damage caused by bile composition disorder may bet he beginning of the pathogenesis of PBC.The bile secreted by the liver is exc reted into the intestine through the gallbladder,and part of it is reabsorbed at the end of the small intestine and returned to the liver,which is the hepatointe stinal circulation.Gut microb es play a role in maintaining the stability of the host environment.The disorder of intestinal bacteria in PBC has been confirme d,and the levels of tyrosine and phenylalanine in serum of patients with PBC have been changed.P-cresyl sulfate(pCS)/p-cresyl gluronide(pCG)is a metabol ite of tyrosine and phenylalanine,which has a variety of biological activities a nd toxicity.Persistent inflammation is a key link in the occurrence and develo pment of PBC,and disordered intestinal bacteria and metabolites are important factors in the persistence of inflammation.The mechanism of pCS/pCG,a me tabolite of Clostridium,on intrahepatic inflammation and BEC is unclear.There fore,to study the effect of pCS/pCG on intrahepatic inflammation and intrahep atic biliary epithelial cells in animals,and to culture HlBEpiC.with different c oncentrations of pCS/pCG,to study the effect of pCS/pCG on human intrahepa ticbile epithelial cells,and to clarify the effect of pCS/pCG on intrahepatic infl ammation and biliary epithelial cells,which may participate in the pathogenesis of PBC and provide a theoretical basis for clinical search for new therapeutic targets.Methods:Part ?:Study the effects of pCS/pCG on intrahepatic inflammation and int rahepatic biliary epithelial cells in animals?Intraperitoneal injection of pCS/pCG in SPF grade C57BL/6 female mice;?HE staining was taken from the liver of mice to observe the inflammation;?The liver of mice was taken to observe the changes of intrahepatic biliary e pithelial cells by immunohistochemistry;?The serum of mice was taken to detect the expression of TGF-? and IFN-?by ELISA;?The expression of Caspase-1 and Bcl-2 in mice liver was detected by qPCR;?The expression levels of Caspase-3,Bax and Bcl-2 proteins in mice liver wa sdetected by western blotting.Part ?:Study the effect of pCS/pCG on human intrahepatic biliary epithe lial cells in vitro?Cultured human intrahepatic biliary epithelial cell(HIBEpiC),and was added with different concentrations of pCS/pCG,observe changes in cell morphology;?The viability of cells was detected by CCK-8;?The cell cycle changes of HIBEpiC was detected by flow cytometry;?The apoptosis of HIBEpiC was detected by flow cytometry.Results:Part ?:PCS/pCG promotes intrahepatic inflammation and injures intrahep atic biliary epithelial cells in mice?In pCS group and pCG group,inflammatory cells infiltrated in the portal are a of liver and the severity of inflammation increased with the increase of admi nistration time;?Reactive bile duct hyperplasia occurred on the 4th week after administration in pCS group and pCG group;?The expression of serum inflammatory factors IFN-? and TGF-? increased wi th the administration time in pCS group and pCG group;?The expression of Caspase-1,Caspase-3 and Bax in liver of pCS group and pCG group increased,while the expression of Bcl-2 decreased gradually.Part ?:pCS/pCG injures human intrahepatic biliary epithelial cells by pro moting apoptosis in vitro?The addition of different concentrations of pCS and pCG has toxic effect on HIBEpiC;?The morphology of HIBEpiC was significantly changed by adding different c oncentrations of pCS and pCG;?PCS and pCG block S stage of HIBEpiC;?PCS and pCG promote the apoptosis of HIBEpiC.Conclusion:?The pCS and pCG can induce inflammatory cell infiltration in the intrahepati c portal area of mice and promote the occurrence and development of intrahep atic inflammation;?The pCS and pCG injure intrahepatic biliary epithelial cells by promoting ap optosis;?The pCS and pCG on intrahepatic inflammation in mice is related to the ex pression of inflammatory factors IFN-? and TGF-?;?The pCS and pCG have toxic effects on HIBEpiC;?The pCS and pCG block the S phase and may promoted the apoptosis of H IBEpiC.