氧化低密度脂蛋白通过Wnt5a/PKCδ信号通路诱导大鼠肝星状细胞自噬

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

该文旨在研究氧化低密度脂蛋白(ox-LDL)对大鼠肝星状细胞(HSC-T6)自噬的影响及机制,探讨非酒精性脂肪肝炎的发病机理。体外培养的HSC-T6细胞以不同质量浓度(0、10、20、40、60μg/mL)的ox-LDL分别处理不同时间(0、3、6、12、24 h)后,用Western blot检测LC3 II、Beclin1、p62的含量。不同质量浓度(0、10、20、40、60μg/mL)的ox-LDL处理HSC-T6细胞12 h后,Western blot检测Wnt5a、p-PKCδ、p-STAT3的含量。将HSC-T6细胞分为对照组(Control)、ox-LDL组、ox-LDL+si-NC组和ox-LDL+si-Wnt5a组,经相应处理后用Western blot、qRT-PCR分别检测LC3 II、Beclin1、p62、p-PKCδ、p-STAT3的蛋白和mRNA含量变化;免疫荧光检测LC3 II的变化;油红O染色观察HSC-T6脂滴含量变化;比色法检测各组细胞培养上清中羟脯氨酸(Hyp)含量;ELISA检测细胞培养上清中透明质酸(HA)和层黏连蛋白(LN)含量。PKCδ抑制剂Rottlerin预处理细胞:将HSC-T6细胞分为对照组(Control)、ox-LDL组、ox-LDL+DMSO组和ox-LDL+Rottlerin组,检测方法与敲低Wnt5a一致。经ox-LDL处理后,HSC-T6细胞中LC3 II、Beclin1含量增加(P<0.05),p62含量减少(P<0.01),且在ox-LDL质量浓度为20μg/mL、作用12 h时达到峰值。ox-LDL质量浓度为20μg/mL、作用12 h时,HSC-T6细胞中Wnt5a、p-PKCδ、p-STAT3蛋白表达显著升高(P<0.01)。敲低Wnt5a后,HSC-T6细胞中Wnt5a、LC3 II、Beclin1 mRNA和蛋白表达水平显著降低(P<0.001),p62蛋白表达增多(P<0.01),p-PKCδ、p-STAT3蛋白表达减少(P<0.05),细胞内LC3 II点状聚集减少,脂滴含量减少,细胞培养上清中Hyp、HA、LN含量也减少(P<0.05)。抑制PKCδ后,结果与敲低Wnt5a一致。ox-LDL可通过增强Wnt5a/PKCδ通路诱导HSC-T6细胞自噬。

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(0,10,20,40,60μg/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(0,10,20,40,60μg/mL)for 12 h.The expression levels of Wnt5a,p-PKCδand 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-PKCδand 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 PKCδinhibitor(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 20μg/mL ox-LDL for 12 h.The levels of Wnt5a,p-PKCδand p-STAT3 were significantly increased after HSC-T6 cells were treated with 20μg/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 PKCδinhibitor pretreatment were consistent with knockdown Wnt5a.ox-LDL induces autophagy of HSC-T6 cells through the Wnt5a/PKCδpathway.