高糖条件下沉默LRP6通过...üller细胞的自噬与凋亡_周敏华.pdf

东南大学学报(医学版)J Southeast Univ(Med Sci Edi)2023，Feb;42(1):32-40收稿日期2022-08-08 修回日期2022-11-11基金项目广东省医学科研基金资助项目(B2021203)作者简介周敏华(1977 )，女，广东东莞人，副主任医师。E-mail:nanali1986163 com通信作者朱咏瑶E-mail:Zhuyy417163 com引文格式周敏华，吴颖，陈毅光，等 高糖条件下沉默 LP6 通过 Wnt/-catenin 途径抑制大鼠视网膜 Mller 细胞的自噬与凋亡J 东南大学学报(医学版)，2023，42(1):32-40 论著 高糖条件下沉默 LP6 通过 Wnt/-catenin途径抑制大鼠视网膜 Mller 细胞的自噬与凋亡周敏华，吴颖，陈毅光，陈庆隆，李文翀，刘枘岢，朱咏瑶(东莞市松山湖中心医院 内分泌科，广东 东莞523326)摘要目的:探究在高糖作用下大鼠视网膜 Mller 细胞中低密度脂蛋白受体相关蛋白6(LP6)的表达及其对高糖条件诱导的 Mller 细胞自噬与凋亡的作用和机制。方法:体外培养大鼠视网膜 Mller 细胞，采用 T-PC 与 Western blotting 检测高糖条件下 Mller 细胞中 LP6 mNA 和蛋白的表达水平;利用 siNA 干扰技术沉默 Mller 细胞中的 LP6，并分别在葡萄糖浓度为 5 6 mmolL1(NG)与 35 mmolL1的 DMEM 培养液(HG)中进行培养，按处理方式的不同将 Mller 细胞分为葡萄糖浓度为 5 6 mmolL1的 DMEM 培养的正常葡萄糖组(NG 组)、葡萄糖浓度为35 mmolL1的 DMEM 培养的转染 si-NC 组(HG+si-NC 组)和 si-LP6的 Mller 细胞组(HG+si-LP6 组);采用 Western blotting 检测各组 Mller 细胞中自噬相关蛋白 P62 的表达、LC3/LC3之值、Beclin1 与 Atg12-Atg5 复合体的表达;共聚焦显微镜观察 FP-GFP-LC3 串联质粒转染后各组 Mller 细胞中自噬通量的变化;TUNEL 染色检测各组 Mller 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/-catenin 途径中-catenin 蛋白的表达。结果:与 NG 组相比，HG 组 Mller 细胞中 LP6 mNA 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-LP6 组 Mller 细胞中除 P62 和 Bcl-2 蛋白表达显著降低外(P 0 05)，LC3/LC3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、cleavedCaspase-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-LP6 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-catenin 蛋白表达显著升高(P 0 05)，而 HG+si-LP6 组明显降低(P 0 05);其中与 HG+si-NC 组相比，HG+si-LP6组中-catenin 蛋白的降低趋势更为显著(P 0 01)。结论:高糖可促进 Mller 细胞中 LP6 的表达，采用siNA 干扰技术沉默细胞中 LP6 表达可能通过下调 Wnt/-catenin 途径抑制高糖诱导的 Mller 细胞自噬，并减少细胞的凋亡。关键词高糖;低密度脂蛋白受体相关蛋白 6;视网膜 Mller 细胞;自噬;凋亡;大鼠 中图分类号774 1 文献标志码A 文章编号1671-6264(2023)01-0032-09doi:10 3969/j issn 1671-6264 2023 01 00523Silencing LP6 inhibits the autophagy and apoptosis of rat retinalMller cells through the Wnt/-catenin pathway underhigh-glucose conditionsZHOU Minhua，WU Ying，CHEN Yiguang，CHEN Qinglong，LI Wenchong，LIU ongke，ZHU Yongyao(Department of Endocrinology，Dongguan Songshanhu Central Hospital，Dongguan 523326，China)Abstract Objective:To investigate the expression of low density lipoprotein receptor-associated protein 6(LP6)in Mller cells of rat retina under high-glucose condition and its effect on autophagy and apoptosis inducedby high glucose condition and related mechanisms Methods:Mller cells were cultured in vitro and the expressionlevels of LP6 mNA and protein in Mller cells were detected by T-PC and Western blotting under high-glu-cose conditions LP6 in Mller cells was silenced by siNA interference method，and cultured in DMEM mediumwith glucose concentration of 5 6 mmolL1(NG)and 35 mmolL1(HG)，respectively Mller cells were di-vided into 3 groups according to different treatment methods:normal glucose group cultured in DMEM with glucoseconcentration of 5 6 mmolL1(NG group)and Mller cell group transfected with si-NC or si-LP6 cultured inDMEM with glucose concentration of 35 mmolL1(HG+si-NC group or HG+si-LP6 group)Western blottingwas used to detect the expression of autophagy related protein P62，LC3/LC3 ratio，Beclin1 and Atg12-Atg5complex in Mller cells Confocal microscopy was used to observe the changes of autophagy flux in Mller cells aftertransfection with FP-GFP-LC3 tandem plasmids TUNEL staining was used to detect the apoptosis rate of Mllercells in each group Western blotting was used to detect the expression of anti-apoptotic protein Bcl-2，pro-apoptot-ic protein Bax and cleaved caspase-3，and-catenin protein in Wnt/-catenin pathway esults:Compared withNG group，HG could significantly promote the expression of LP6 mNA and protein in Mller 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-LP6 group was significantly decreased(P 0 05)LC3/LC3 ratio，Beclin1 and Atg12-Atg5 complex，au-tophagy flux，TUNEL positive cell rate，and Bad and cleaved Caspase-3 protein expression levels in cells were sig-nificantly increased(P 0 05)Compared with HG+si-NC group，the protein levels of P62 and Bcl-2 in HG+si-LP6 group were significantly increased(P 0 05)，and other indicators were significantly decreased In addition，compared with NG group，-catenin protein expression in HG+si-NC group was significantly increased(P 0.05)，while that in HG+si-LP6 group was significantly decreased(P 0 05)The decrease trend of-cateninprotein in HG+si-LP6 group was more significant than that in HG+si-NC group(P 0 01)Conclusion:Highglucose can promote the expression of LP6 in Mller cells，while silencing LP6 expression by siNA interferencemay inhibit the autophagy of Mller cells induced by high glucose and reduce cell apoptosis by down-regulatingWnt/-catenin pathway Key wordshigh glucose;low density lipoprotein receptor-associated protein 6;retinal Mller cells;autophagy;apoptosis;rats糖尿病视网膜病变(diabetic retinopathy，D)是造成全世界劳动年龄人群视力受损与失明的主要原因1。流行病学数据表明，截至 2019 年，我国糖尿病患者高达 1 16 亿，占全世界糖尿病人口总数的 1/4 以上，居世界首位2。但迄今为止，D 的确切发病机制尚不清楚，而目前治疗 D 的方法，如抗血管内皮生长因子和激光凝固术等，对 D 患者的疗效有限3。因此，积极探究 D 的发病机制具有重要意义。视网膜33周敏华，等 高糖条件下沉默 L