Curcumin Inhibits Hypoxia-induced Epithelial-mesenchymal Transition In Hepatocytes And Its Mechanism

Background and Aims:Hepatic fibrosis,characterized by abnormal accumulation of extracellular matrix(ECM),is a common pathological process of many chronic liver diseases.In this process,the activation and proliferation of the polytopic myofibroblasts(MFB)is a key link in the incidence of HF.Hepatic stellate cell(HSC)has long been recognized as the main source of MFB.A new study has found that hepatocytes undergo transdifferentiation to myofibroblasts through EMT pathway,and participate in the synthesis of collagen in liver fibrogenesis.Prophase research showed that curcumin plays a prominent role in protecting hepatocytes,inhibiting liver fibrogenesis and inhibiting the epithelial to mesenchymal transition(EMT)of renal tubular epithelial cell and tumor cells through regulate the protein expression of TGF-?/smad and Hedgehog signal pathway.So this project is to investigate:1)whether hepatocytes undergo EMT in the process of hepatic fibrosis;2)whether curcumin could inhibit the EMT of hepatocytes,and which mechanism involved?This project has great significance on study to further understand the pathogenesis of liver fibrosis and rich research foundation of curcumin.Methods:This research carried on the molecular mechanism of the effect of curcumin on hepatocytes EMT,combining the in vivo and in vitro study,and using molecular biological methods.1)The in vivo study:Intervention of curcumin(100,200,400 mg/kg)was proceeded in CCl4 induced rat liver fibrosis model.The degree of liver injury was evaluated biochemically by pathological examination.Immunofluorescence detected the expression of Vimentin and Collagen I in liver tissue;Furthermore,Western Blot detected Vimentin,Collagen I and a-SMA protein expression in liver tissue.2)The in vitro study:We used the oxygen inducer(CoCl2)to build the epithelial mesenchymal transition model of human liver cell L02 induced by hypoxia;Using cell wound healing assay and Trans well migration test detected the influence of curcumin on cell movement and migration ability of L02 induced by hypoxia;Western Blot and immunofluorescence detected the effect of curcumin in different concentrations(10,20,30 ?M)on protein expression of E-cadherin,N-cadherin,Vimentin,Collagen I and a-SMA in L02 cells induced by hypoxia;Microscope observed the influence of curcumin(30 ?M)on morphology of L02 induced by hypoxia in different times.3)Molecular mechanisms discussion:we used Western Blot detected the effect of curcumin on the protein expression of Wnt and TGF-P related to signaling pathways which is closely related to the regulation of epithelial mesenchymal transformation.Results:As shown in tissue sections with HE staining,compared with sections from livers in the control,CCl4 caused prominent hepatic steatosis,necrosis,and formation of regenerative nodules and fibrotic septa between the nodules.Immunofluorescence results show that the expression of Vimentin and Collagen I increased significantly in the model group.Curcumin can inhibit CCl4 pathology change of liver tissue induced by CC14,and can inhibit protein expression of Vimentin,Collagen I and a-SMA in the liver tissue with a dose-dependent manner(P<0.05).Hypoxia revulsant CoCl2 have no obvious effect on L02 proliferation activity under 100?M,at the same time,Eilsa results show that CoC12 have no influence on L02 function under the concentration of 400 ?M compared with the control group(P>0.05).L02 cells appeared obvious mesenchymal characteristics induced by CoCl2(100 ?M)for 72 h.Western Blot results showed that hypoxia could induced the reducing of the expression of epithelial protein E-cadherin,the increasing of the expression of mesenchymal protein N-cadherin and Vimentin,and enables the expression of fibrosis marker Collagen I and a-SMA in L02 cells.Curcumin has no effect on cell proliferation of L02 below 70?M.The concentration of curcumin with hypoxia inducers(CoCl2,100 ?M)presenting no inhibition to L02 cell proliferation curcumin is under 50 ?M.And cell movement experiment suggests that curcumin could inhibit horizontal and vertical migrationand movement of L02 cells induced by hypoxia.Moreover,curcumin could inhibit the expression of epithelial protein E-cadherin,and increase the expression of mesenchymal protein N-cadherin and Vimentin and fibrosis protein Collagen I and a-SMA dose-dependently(P<0.05).Curcumin(30 ?M)could inhibit hypoxia inducing mesenchymal transition of morphology of L02 cell.Immunofluorescence showed that curcumin can inhibit the expression of fibrosis protein a-SMA and the mesenchymal change of morphology of L02.Western blot results suggested that Wnt was not affected by curcumin,but curcumin could increase GSK-3? and lower ?-catenin protein expression.Moreover,TGF?-Smad signaling pathways and its downstream signaling molecules Smad 2,Smad 2/3 was obvious inhibited by curcumin,but we found the fact that the effect if curcumin inhibiting epithelial mesenchymal transition of hepatocytes induced by hypoxia is partly dependent on TGF-? signaling pathways through blocking the TGF-?R.Curcumin can raise the protein expression of HGF and c-met in hepatocytes induced by hypoxia.Conclusion and significance:Hepatocytes can transform into MFB through the epithelial mesenchymal transition in liver fibrosis,participating and increasing the synthesis and deposition of extracellular matrix.Curcumin could inhibit the process of epithelial mesenchymal transition of hepatocyte induced by hypoxia,and reduce the generation of extracellular matrix.This effect may be due to the inhibiting of the protein expression of TGF-?,Wnt and HGF/c-met signaling pathway.Our research provides a new perspective in the treatment of liver fibrosis and involved mechanisms.