The Effects And Mechanisms Of Ischemia/Reperfusion On Epithelial Mesenchymal Transitions In Human Introhepatic Biliary Epithelial Cells

Objective1.To investigate the effects of ischemia/reperfusion on Epithelial Mesenchymal Transitions in human introhepatic biliary epithelial cells.2.To research the mechanisms of ischemia/reperfusion on the promotion of Epithelial Mesenchymal Transitions in human introhepatic biliary epithelial cells.Methods1.Human introhepatic biliary epithelial cells were divided into two groups,that is the normal group and the ischemia/reperfusion group,and each group was cultured in its corresponding conditions.Then the protein of the cells were harvested,including the normal group cells and the cells which reperfused for 6h,12 h,24h after ischemia.BCA assay was used to make sure equal amounts of proteins were loaded in the experiments of Western blot.The expression of proteins related to Epithelial Mesenchymal Transitions(E-cadherin,Vimentin,FSP-1)were detected.2.Human introhepatic biliary epithelial cells were preconditioned with cyclopamine,a kind of inhibitors of Hedgehog pathway,in different concentrations of 0,5,10,15,20,25,30,35,40?mol/L for 12 h.Then CCK-8 assay was used to detect the viability of the cells and the IC50 Curve was illustrated.We also used CCK-8 assay to investigate the proliferation of cells in normal group,ischemia/reperfusion group and ischemia/reperfusion with cyclopamine group and draft the growth curves.Western blot was used here to detect and compare the expressions of proteins related to Hedgehog pathway(SHH,SMO,Gli)and Epithelial Mesenchymal Transitions(E-cadherin,Vimentin)among the normal group,ischemia/reperfusion group and ischemia/reperfusion with cyclopamine group.Results1.The expression of E-cadherin in ischemia/reperfusion group was lower than that in the normal group,while on the opposite,the expressions of Vimentin and FSP-1 were higher in the ischemia/reperfusion group,and the phenomenon was especially apparent when the cells were reperfused for 6h.2.The IC50 curve was illustrated according to the results of cell viability measured by CCK-8,and 18.68?mol/L was calculated as the optimum concentration of cyclopamine,and during the experiments we chose a concentration of 20?mol/L to precondition human introhepatic biliary epithelial cells for 12 h in order to attenuate the complexity of the experiments.Among the growth curves of different groups of cells,the growth rates of of cells which went though ischemia/reperfusion were lower than cells cultured in normal conditions,and the outcomes are significant at 1h,6h and 12h(P<0.05).And the groups preconditioned with cyclopamine had lower rates than other groups,especially in the ischemia/reperfusion groups(P<0.001).The outcomes of Western blot showed the expression of SHH,SMO and Gli were up-regulated in ischemia/reperfusion groups,which indicated that the Hedgehog pathway was activated.After the preconditioning of cyclopamine,the expression of Gli was down-regulated significantly,indicating the inhibition of Hedgehog pathway.In the meantime,compared with the ischemia/reperfusion group cells,the expression of Vimentin was lower,while the expression of E-cadherin was higher,indicating the Epithelial Mesenchymal Transitions was down-regulated.Conclusions1.Ischemia/reperfusion can lead to the Epithelial Mesenchymal Transitions of human introhepatic biliary epithelial cells.2.Ischemia/reperfusion up-regulate the Epithelial-Mesenchymal Transitions of human introhepatic biliary epithelial cells by activating the Hedgehog pathway,and it can be alleviated by blocking the process.