Oxidized Low-Density Lipoprotein Induced Rat Hepatic Stellate Cell Autophagy By Wnt5a/PKCδ Signaling Pathway

Objective:To investigate the effect of oxidized low-density lipoprotein(ox-LDL)on autophagy of rat hepatic stellate cell(HSC-T6),in order to explore the pathogenesis of non-alcoholic steatohepatitis.Method:(1)HSC-T6 cells were cultured in vitro,the dose-effect groups were treated with ox-LDL at the concentrations of 0,10,20,40,60 μg/m L for 24 hours.The time-effect groups were treated with 20 μg/m L ox-LDL for 0,3,6,12 and 24 hours,and the levels of autophagy-related protein LC3 II,Beclin1 and p62 were tested by Western blot.(2)HSC-T6 cells treated with different concentration of ox-LDL(0,10,20,40,60 μg/m L)for 12 hours.The expressions of pathway related proteins Wnt5 a,p-PKCδand p-STAT3 were detected by Western blot.(3)HSC-T6 cells were divided into Control group,ox-LDL group,ox-LDL+si-NC group and ox-LDL+ si-Wnt5 a group.The expressions of LC3 II,Beclin1,p62,p-PKCδ and p-STAT3 were tested by Western blot and q RT-PCR,respectively.The expression of LC3 II fluorescence spots were tested by immunofluorescence.The content of lipid droplets in HSC-T6 cells was observed by oil red O staining.The content of Hyp(hydroxyproline)in culture medium of HSC-T6 cells was tested by digestion method.The contents of HA(hyaluronic acid)and LN(laminin)in culture medium of HSC-T6 cells were tested by ELISA.(4)PKCδ inhibitor(Rottlerin)pretreatmen cells: HSC-T6 cells were divided into Control group,ox-LDL group,ox-LDL+DMSO group and ox-LDL+Rottlerin group.The expressions of LC3 II,Beclin1,p62 and p-STAT3 were detected by Western blot and q RT-PCR,respectively.The expression of LC3 II fluorescence spots were tested by immunofluorescence.The content of lipid droplets in HSC-T6 cells was observed by oil red O staining.The content of Hyp(hydroxyproline)in culture medium of HSC-T6 cells was tested by digestion method.The contents of HA(hyaluronic acid)and LN(laminin)in culture medium of HSC-T6 cells were tested by ELISA.Results:(1)After ox-LDL treatment,LC3 II and Beclin1 contents in HSC-T6 cells were increased,p62 degradation increased and reached the peak value at 20 μg/m L ox-LDL for 12 hours(P<0.05).(2)The levels of Wnt5 a,p-PKCδ and p-STAT3 were significantly increased after HSC-T6 cells were treated with ox-LDL at the concentraction of 20 μg/m L for 12hours(P<0.01).(3)After knocking down Wnt5 a,the autophagy-related proteins LC3 II,Beclin1 in HSC-T6 cells decreased and the p62 degradation decreased.The pathway related proteins p-PKCδ,p-STAT3 were also reduced.LC3 II fluorescence spots and content of lipid droplets in HSC-T6 cells were reduced,and the contents of Hyp,HA and LN was also reduced(P<0.05).(4)After inhibiting PKCδ,the LC3 II,Beclin1 in HSC-T6 cells decreased and the p62 degradation decreased.The expressions of pathway related proteins p-STAT3 were reduced.LC3 II fluorescence spots and content of lipid droplets in HSC-T6 cells were reduced.The contents of Hyp,HA and LN in the cell culture medium were also decreased(P<0.05).Conclusion:(1)ox-LDL could induce autophagy of HSC-T6 cells.(2)ox-LDL could promote the activation of Wnt5a/PKCδ signaling pathway in HSC-T6 cells.(3)ox-LDL may induce autophagy in HSC-T6 cells by enhancing the Wnt5a/PKCδ pathway...