高糖条件下沉默LRP6通过Wnt/β-catenin途径抑制大鼠视网膜Müller细胞的自噬与凋亡

Silencing LRP6 inhibits the autophagy and apoptosis of rat retinal Müller cells through the Wnt/β-catenin pathway under high-glucose conditions

目的:探究在高糖作用下大鼠视网膜Müller细胞中低密度脂蛋白受体相关蛋白6(LRP6)的表达及其对高糖条件诱导的Müller细胞自噬与凋亡的作用和机制。方法:体外培养大鼠视网膜Müller细胞,采用RT-PCR与Western blotting检测高糖条件下Müller细胞中LRP6 mRNA和蛋白的表达水平;利用siRNA干扰技术沉默Müller细胞中的LRP6,并分别在葡萄糖浓度为5.6 mmol·L^(-1)(NG)与35 mmol·L^(-1)的DMEM培养液(HG)中进行培养,按处理方式的不同将Müller细胞分为葡萄糖浓度为5.6 mmol·L^(-1)的DMEM培养的正常葡萄糖组(NG组)、葡萄糖浓度为35 mmol·L^(-1)的DMEM培养的转染si-NC组(HG+si-NC组)和si-LRP6的Müller细胞组(HG+si-LRP6组);采用Western blotting检测各组Müller细胞中自噬相关蛋白P62的表达、LC3Ⅱ/LC3Ⅰ之值、Beclin1与Atg12-Atg5复合体的表达;共聚焦显微镜观察RFP-GFP-LC3串联质粒转染后各组Müller细胞中自噬通量的变化;TUNEL染色检测各组Müller细胞的凋亡率,Western blotting法检测细胞中抗凋亡蛋白Bcl-2、促凋亡蛋白Bax与cleaved Caspase-3及Wnt/β-catenin途径中β-catenin蛋白的表达。结果:与NG组相比,HG组Müller细胞中LRP6 mRNA和蛋白的表达水平明显增高(P<0.01);与NG组比较,HG+si-NC组、HG+si-LRP6组Müller细胞中除P62和Bcl-2蛋白表达显著降低外(P<0.05),LC3Ⅱ/LC3Ⅰ之值、Beclin1与Atg12-Atg5复合体、细胞中自噬通量、TUNEL阳性细胞率及细胞中Bad、cleaved Caspase-3蛋白表达水平均明显升高(P<0.05);而与HG+si-NC组比较,HG+si-LRP6组中除P62、Bcl-2蛋白明显升高外(P<0.05),其他检测指标均显著降低;此外,与NG组比较,HG+si-NC组中β-catenin蛋白表达显著升高(P<0.05),而HG+si-LRP6组明显降低(P<0.05);其中与HG+si-NC组相比,HG+si-LRP6组中β-catenin蛋白的降低趋势更为显著(P<0.01)。结论:高糖可促进Müller细胞中LRP6的表达,采用siRNA干扰技术沉默细胞中LRP6表达可能通过下调Wnt/β-catenin途径抑制高糖诱导的Müller细胞自噬,并减少细胞的凋亡。

Objective:To investigate the expression of low density lipoprotein receptor-associated protein 6(LRP6)in Müller cells of rat retina under high-glucose condition and its effect on autophagy and apoptosis induced by high glucose condition and related mechanisms.Methods:Müller cells were cultured in vitro and the expression levels of LRP6 mRNA and protein in Müller cells were detected by RT-PCR and Western blotting under high-glucose conditions.LRP6 in Müller cells was silenced by siRNA interference method,and cultured in DMEM medium with glucose concentration of 5.6 mmol·L^(-1)(NG)and 35 mmol·L^(-1)(HG),respectively.Müller cells were divided into 3 groups according to different treatment methods:normal glucose group cultured in DMEM with glucose concentration of 5.6 mmol·L^(-1)(NG group)and Müller cell group transfected with si-NC or si-LRP6 cultured in DMEM with glucose concentration of 35 mmol·L^(-1)(HG+si-NC group or HG+si-LRP6 group).Western blotting was used to detect the expression of autophagy related protein P62,LC3Ⅱ/LC3Ⅰratio,Beclin1 and Atg12-Atg5 complex in Müller cells.Confocal microscopy was used to observe the changes of autophagy flux in Müller cells after transfection with RFP-GFP-LC3 tandem plasmids.TUNEL staining was used to detect the apoptosis rate of Müller cells in each group.Western blotting was used to detect the expression of anti-apoptotic protein Bcl-2,pro-apoptotic protein Bax and cleaved caspase-3,andβ-catenin protein in Wnt/β-catenin pathway.Results:Compared with NG group,HG could significantly promote the expression of LRP6 mRNA and protein in Müller cells(P<0.01).Compared with NG group,the expression of P62 and Bcl-2 protein in Muller cells in HG+si-NC group and HG+si-LRP6 group was significantly decreased(P<0.05).LC3Ⅱ/LC3Ⅰratio,Beclin1 and Atg12-Atg5 complex,autophagy flux,TUNEL positive cell rate,and Bad and cleaved Caspase-3 protein expression levels in cells were significantly increased(P<0.05).Compared with HG+si-NC group,the protein levels of P62 and Bcl-2 in HG+si-LRP6 group were significantly increased(P<0.05),and other indicators were significantly decreased.In addition,compared with NG group,β-catenin protein expression in HG+si-NC group was significantly increased(P<0.05),while that in HG+si-LRP6 group was significantly decreased(P<0.05).The decrease trend ofβ-catenin protein in HG+si-LRP6 group was more significant than that in HG+si-NC group(P<0.01).Conclusion:High glucose can promote the expression of LRP6 in Müller cells,while silencing LRP6 expression by siRNA interference may inhibit the autophagy of Müller cells induced by high glucose and reduce cell apoptosis by down-regulating Wnt/β-catenin pathway.