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

作　　者：周敏华 吴颖 陈毅光 陈庆隆 李文翀 刘枘岢 朱咏瑶 ZHOU Minhua;WU Ying;CHEN Yiguang;CHEN Qinglong;LI Wenchong;LIU Rongke;ZHU Yongyao(Department of Endocrinology,Dongguan Songshanhu Central Hospital,Dongguan 523326,China)

机构地区：[1]东莞市松山湖中心医院内分泌科,广东东莞523326

出　　处：《东南大学学报（医学版）》2023年第1期32-40,共9页Journal of Southeast University(Medical Science Edition)

基　　金：广东省医学科研基金资助项目(B2021203)。

摘　　要：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

关 键 词：高糖 低密度脂蛋白受体相关蛋白6 视网膜MÜLLER细胞 自噬 凋亡 大鼠 

分 类 号：R774.1[医药卫生—眼科]