Autophagy-related Protein,Bnip3 Interacts With Vimentin,an Intermediate Filament Protein,and Regulates Autophagy Of Hepatic Stellate Cells

Objective:Hepatic fibrosis is a pathological feature of many liver diseases.The essence of hepatic fibrosis is the imbalance of metabolism of extracellular matrix（ECM）secreted by hepatic stellate cells（HSCs）,which increases ECM synthesis and decreases ECM degradation.The activation of HSCs is the key cytological mechanism of liver fibrosis.We have reported a differentially expressed autophagy-related gene BNIP3 in activated hepatic stellate cells through whole-genome expression microarray.Bnip3（Bcl-2/adenovirus E1B-19k Da interacting protein 3）is a multifunctional molecule involved in regulating autophagy,apoptosis and gene transcription.We hypothesized that Bnip3 regulates the activation of hepatic stellate cells by regulating autophagy.This project aims to explore the mechanism of autophagy regulated by Bnip3 in hepatic stellate cells,to further reveal the possible mechanism of the pathophysiology and development of liver fibrosis,and provide a theoretical basis for intervention or reversal of the occurrence and development of hepatic fibrosis.Methods:The expression of Bnip3 was detected by immunohistochemical staining in the liver tissues of liver fibrosis patients and the normal liver tissues around the surgically resected hemangioma.Human hepatic stellate cell line,LX-2 cells,were stimulated by Co Cl₂ or LPS.Primary hepatic stellate cells of BALB/c mice were isolated by collagenase perfusion-Percoll density gradient centrifugation and cultured in vitro.The expression and the co-localization of Bnip3 and LC3B in hepatic stellate cells were observed by immunofluorescence staining under a confocal microscope.Cells were stained with Annexin V-FITC/PI apoptosis double staining kit,and the ratio of apoptosis cells was detected by flow cytometry.The expression of apoptosis-related proteins,caspase-3 and cytochrome C,were detected by Western blot.The expression of Bnip3 in mouse primary HSCs interfered with specific si RNA,and the autophagic flow was observed under a confocal microscope after transfecting GFP-RFP-LC3B plasmid into cells.The proteins that might bind to Bnip3 in HSCs were analyzed by immunoprecipitation and mass spectrometry.In the activated HSCs,the co-localization and interaction of Bnip3 and Vimentin were detected by immunofluorescence assay and immunoprecipitation.Phosphorylation of serine residues of Vimentin,including S56,S72,S82 and S38 was detected in the complex of Bnip3 and Vimentin with immunoprecipitation in the activated HSCs.The activity of Vimentin was inhibited by WFA（Withaferin A）and the expressions of Vimentin,Bnip3 and LC3B in hypoxia or LPS stimulated LX-2 cells were detected by Western blot and immunofluorescence assay.Results:The expression of Bnip3 was increased in fibrotic liver tissues from clinical patients and activated hepatic stellate cells.Autophagy occurred during the activation of hepatic stellate cells.Bnip3 was partially co-localized with autophagosome in activated hepatic stellate cells and inhibition of Bnip3 led to the blockage of autophagic flow.The interaction and co-localization of Bnip3 and Vimentin were observed in activated hepatic stellate cells.Inhibition of Vimentin activity inhibited Bnip3expression and autophagy in activated LX-2 cells.Conclusion:Autophagy associated protein Bnip3 is a key regulator of autophagy during the activation of hepatic stellate cells.Bnip3 regulates the autophagy of hepatic stellate cells by interacting with Vimentin,an intermediate filament protein.