MSCs Regulate STAT3 To Influence The Autophagy Flux Of Intrahepatic Bile Duct Epithelial Cells In The Treatment Of Primary Biliary Cirrhosis

Background Primary biliary cirrhosis(PBC)is characterized as cholangitis of small bile ducts.There are few studies on the pathogenesis of bile duct loss in PBC.It has been suggested that abnormal autophagyof the intrahepatic biliary epithelial cells(IBECs)may be involved in the progressive loss of bile duct in the PBC.It has been reported that human mesenchymal stem cells(MSCs)could enhance the autophagy.Previously,we demonstrated that Umbilical cord-derived mesenchymal stem cells(UCMSCs)transplantation had certain effects on PBC mouse.This study aimed at the regulation of STAT3 on MSCs and the relationship between autophagy and BECs,provide the basis for the treatment of BECs damage in PBC patients with MSCs.Objective This study aimed at the regulation of STAT3 on MSCs and the relationship between autophagy and IBECs,provide the basis for the treatment of IBECs damage in PBC patients with MSCs.Methods To set up the PBC model,thirty female C57 BL / 6 mice(SPF grade)were randomly divided into experimental group(n = 18),control group(n = 6)and an untreated group(n = 6).2-octynyl acid(2-OA)-bovine serum albumin(BSA)+ Freund's Adjuvant/Incomplete Freund's adjuvant(CFA/IFA)by intraperitoneal injection was used in 18 mice in the experimental group.The control group were injected with the same amount of BSA+IFA,non-drug intervention was used in the untreated group.After induced PBC successfully,the PBC mice were randomly divided into the control group(PBC group,n=4),MSCs transplantation group(MSCs group,n=6)and STAT3 inhibitors treated group(Stattic group,n=6).Two weeks later,all the mice were sacrificed and the peripheral blood sera were collected.The liver pathology and immunohistochemical study were performed in for that randomly taken out from each group,and the intrahepatic biliary tree and intrahepatic biliary epithelial cells(IBECs)were obtained by perfusion and centrifugationin from the remaining mice.Serum liver function were determined by the automatic biochemical analyzerand the serum pyruvate dehydrogenase complex E2 subunit(PDC-E2)antibody titers were measured by ELISA.Hepatocytes were removed by the liver perfusion digestion and then the intrahepatic biliary trees were isolated.IBECs were purified after digestion extraction.cytokeratin 19(CK19),hepatocyte nuclear factor 4?(HNF-4?)immunohistochemistry were used to identify the target cell.IBECs from the control group,PBC model group,MSCs transplantation group and Stattic treatment group were collected and then STAT3/phosphorylated STAT3,p62,LC3-I/II,LAMP-1,PKR/phosphorylated PKR,Beclin1,e IF2 alpha/phosphorylated e IF2 alpha were determined by western blot.Also the m RNA of those proteins were detected by RT-PCR.The autophagy bubble formation in IBECs isolated from each group were investigated by electronmicroscope.Hi BECs were incubated in the presence of glycochenodeoxcholat(GCDC)for 0h,2h,6h,12 h,24h,36 h,48h,54 h and 60 h,then the GCDC-treated Hi BECs were cocultured with UCMSCs through transwell for 0h,6h,12 h,48h and 60 h.The expression of intracellular LC3-II/LC3-I,STAT3/p STAT3,PKR and e IF2?/pe IF2? of Hi BECs were detected by western blot.Electron microscopy(EM)analyses of m Cherry-GFP-LC3 were uesd to evaluate whether MSCs coculture had an effect on autophagyrelated structures of autophagosomes and autolysosomes.Reconstruction of STAT3 silencing Hi BECs(si STAT3)by si RNA.Western blot was employed to determine the effect of MSCs on the intracellular proteins above of Hi BECs.Results(1)Compared with the control group and the untreated group,after 22 weeks 2-OA-treatment,high-titer of the anti-PDE-2 antibody was detected in the serum from PBC mice,lymphocytic infiltration,bile duct injury surrounded by the infiltrated mononuclear cell and scattered formation of granulomas were also found in the livers of the mice,and small bile duct injury was similar to chronic non-suppurative cholangitis.Compared with the PBC mice,periportal inflammatory cell infiltration was less in both MSCs group and Stattic group,and no granuloma formation was found.Spleen weight was significantly higher in the model group than the control group(P<0.05).Compared with the control group,serum GGT,LDH,globulin and adenosine deaminase were higher in the model group(P<0.05)and the valley GGT levels decreased in the MSCs transplantation group(P<0.05).There is a reasing trend of albumin and white ball ratio(P>0.05).(2)Compared with the control group,IBECs autophagy protein Beclin-1 and STAT3,p STAT3 were higher in PBC group(P<0.05),those protein decreased in MSCs transplantation and Stattic treatment group(P<0.05).LC3-II/I and p62 expression showed no difference between the four groups.STAT3 m RNA,p62 m RNA of PBC group were lower than those of the control group(P<0.05),p62 m RNA of MSCs transplantation group was higher than that of the PBC group(P<0.05).(3)GCDC treatment lead to induction of autophagosome.The markedly increased expression of LC3-II/LC3-I was detected in 48 h.After STAT3 silencing,the inceased expression of LC3-II/LC3-I were observed in the Hi BECs,and the reverse result was detected in MSCs coculture group,while the decreased expression of pe IF2?,PKR were obseved in the Hi BECs with or without MSCs coculture.EM analyses revealed that MSCs upregulated autolysosome formation in the GCDC-treated Hi BECs.Conclusion MSCs enhanced the autophagy and decreased the expression of STAT3 in the primary biliary cirrhosis model and MSCs regulated STAT3/PKR/pe IF2? expression to enhance autophagy in vitro and in vivo.