Oxidized Low-Density Lipoprotein Induces Rat Hepatic Stellate Cell Autophagy by Wnt5a/PKCδ Signaling Pathway

This study was aimed to investigate the effect of ox-LDL (oxidized low-density lipoprotein) on autophagy of rat hepatic stellate cell HSC-T6, and explore the pathogenesis of non-alcoholic steatohepatitis. HSC-T6 cells were cultured in vitro and treated with different concentrations of ox-LDL (0, 10, 20, 40, 60 μg/mL) at different time points (0, 3, 6, 12, 24 h), and the expression levels of LC3 II, Beclin1 and p62 were detected by Western blot. HSC-T6 cells treated with different concentrations of ox-LDL (0, 10, 20, 40, 60 μg/mL) for 12 h. The expression levels of Wnt5a, p-PKCδ and p-STAT3 were detected by Western blot. HSC-T6 cells were divided into Control group, ox-LDL group, ox-LDL+si-NC group and ox-LDL+si-Wnt5a group. After treatment, the expression levles of LC3 II, Beclin1, p62, p-PKCδ and p-STAT3 in each group were detected by Western blot and qRT-PCR; the expression of LC3 II was detected by immunofluorescence assay; the lipid droplet of HSC-T6 was observed by oil red O staining; the content of Hyp (hydroxyproline) in cell culture supernatant was determined by colorimetric method and the contents of HA (hyaluronic acid) and LN (laminin) were determined by ELISA. HSC-T6 cells were pretreated with PKCδ inhibitor (Rottlerin) and were divided into Control group, ox-LDL group, ox-LDL+DMSO and ox-LDL+Rottlerin group. The detection method was consistent with Wnt5a knockdown. The levels of LC3 II and Beclin1 were significantly increased (P<0.05), while p62 content decreases (P<0.01) after HSC-T6 cells were treated with 20 μg/mL ox-LDL for 12 h. The levels of Wnt5a, p-PKCδ and p-STAT3 were significantly increased after HSC-T6 cells were treated with 20 μg/mL ox-LDL for 12 h (P<0.01). After Wnt5a knockdown, the mRNA and protein expression levels of Wnt5a in HSC-T6 cells were significantly reduced (P<0.001). Western blot showed that knockdown of Wnt5a reduced the expression levels of LC3 II, Beclin1, p-PKCδ, p-STAT3, and induced the expression of p62 (P<0.05). qRT-PCR showed that the expression levels of LC3 II and Beclin1 mRNA were significantly decreased in ox-LDL+si-Wnt5a group compared with ox-LDL+si-NC group (P<0.05). Knockdown of Wnt5a reduced the LC3 II fluorescent spots, inhibited cellular lipid accumulation, while decreased the levels of Hyp, HA, and LN (P<0.05). The results of PKCδ inhibitor pretreatment were consistent with knockdown Wnt5a. ox-LDL induces autophagy of HSC-T6 cells through the Wnt5a/PKCδ pathway.