Mechanism Of Hif-1 Target Gene Bnip3 Regulating Autophagy And Activation Of Hepatic Stellate Cells

Objective:Activation of hepatic stellate cells（HSCs）is a key mechanism for the occurrence and development of hepatic fibrosis.Phenotypic changes in the process of activation have been recognized,but the precise mechanisms leading to these phenotypic changes are still poorly understood.It has been demonstrated that hypoxia inducible factor-1（Hif-1）affects the activation of hepatic stellate cells by regulating autophagy.The expression of autophagy related genes in hypoxia-induced hepatic stellate cells was analyzed by whole-genome expression microarray and a differentially expressed autophagy related gene,bnip3（Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3）,was screened out from hypoxia-stimulated LX-2hepatic stellate cells.The purpose of this study is to explore the possible mechanism of Hif-1 target gene Bnip3 regulating autophagy and activation in hepatic stellate cells,thus to reveal the mechanism of hepatic fibrosis and look for the target of reversing hepatic fibrosis.Methods:LX-2 cells were treated with CoCl₂ and the expression of Bnip3 in LX-2cells was detected by Real-time quantitative polymerase chain reaction and Western blot.The expression of Bnip3 in liver tissue of mice infected with Schistosoma japonicum was detected by immunohistochemistry.LX-2 cells were treated with hypoxia and the expression of P62,α-SMA and LC3B was detected with Western blot and the puncta of p62 was detected by confocal microscopy.The expression of Hif-1was inhibited by Hif-1 chemical inhibitor YC-1 and the expression of Bnip3 was detected by Western blot and confocal microscopy in LX-2 cells exposed to hypoxia or LPS treatment.The expression of Bnip3,α-SMA and LC3B in CoCl₂-treated or LPS-treated LX-2 cells was tested by Western blot,as Bnip3 expression was interfered by specific siRNA.Primary hepatic stellate cells were isolated from BALB/c mice with collagenase perfusion-percoll separation method and the expression of Hif-1,Bnip3,LC3B,P62 andα-SMA was detected with Western blot during the natural activation of primary hepatic stellate cells.The expression of GFAP,Bnip3 and formation of P62 puncta were detected with confocal microscopy.Hif-1chemical inhibitor YC-1 was added into culture medium of primary hepatic stellate cells and Western blot was used to detect the expression of Hif-1 and Bnip3.Bnip3expression was interfered by specific siRNA and the expression of Bnip3,α-SMA and LC3B was detected by Western blot in primary hepatic stellate cells during natural activation.Results:The expression of Bnip3 was increased in hypoxia-treated LX-2 cells and in liver tissue of Schistosoma japonicum infected mice.Hypoxia induced autophagy in LX-2 cells.Inhibition of Hif-1αexpression reduced the expression of Bnip3 in LX-2 cells.Silencing of Bnip3 expression inhibited autophagy and the activation of LX-2 cells.The expression of Bnip3 increased in the in-vitro naturally activated primary hepatic stellate cells.Occurrence of autophagy and increased expression of Hif-1 was observed in primary hepatic stellate cells.Inhibition of Hif-1αsuppressed expression of Bnip3 in primary hepatic stellate cells.Interfering Bnip3 expression with specific siRNA inhibited activation and autophagy of hepatic stellate cells.Conclusion:Hif-1 regulates activation and autophagy of hepatic stellate cells through the Hif-1 target molecule Bnip3.