恩格列净通过miR-497-3p/GPLD1通路调控高糖诱人肾小球微血管内皮细胞增殖凋亡的机制研究

doi:10.3969/j.issn.1006-6233.2022.02.04

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摘要 目的: 探究恩格列净(EMPA)调控人肾小球微血管内皮细胞(HRGEC)增殖、凋亡的作用机制。方法: 30mmoL/L的葡萄糖诱导HRGEC建立高糖环境下的肾损伤细胞模型。恩格列净(250、500、1000nmoL/L)处理受损细胞。脂质体法将miR-NC组(转染miR-NC)、miR-497-3p组(转染miR-497-3p)、si-NC组(转染si-NC)、si-鱼糖基磷脂酰肌醇特异性磷脂酶D1(GPLD1)组(转染si-GPLD1)、EMPA+miR-497-3p+pcDNA组(共转染miR-497-3p和pcDNA)、EMPA+miR-497-3p+pcDNA-GPLD1组(共转染miR-497-3p和pcDNA-GPLD1)转染HRGEC细胞,并用30mmoL/L的葡萄糖或500nmoL/L恩格列净处理。细胞计数试剂盒(CCK-8)、流式细胞术检测细胞增殖率、凋亡率。蛋白免疫印迹(WB)实验检测细胞GPLD1、增殖细胞核抗原(PCNA)、细胞周期依赖性蛋白激酶抑制因子1A(P21)、半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3、Caspase-9蛋白表达。双荧光素酶报告实验、RNA 结合蛋白免疫沉淀(RNA binding protein immunoprecipitation assay,RIP)实验检测细胞中miR-497-3p与GPLD1的结合力。结果: 与NG组相比,HG组HRGEC细胞增殖率显著降低,凋亡率显著升高,miR-497-3p表达显著降低(P<0.05);与HG组相比,恩格列净(250、500、1000nmoL/L)组细胞增殖率显著升高,凋亡率显著降低,miR-497-3p表达显著升高(P<0.05),选出恩格列净的最适浓度500nmoL/L。miR-497-3p显著抑制野生型GPLD1细胞的荧光活性,促进Ago2-GPLD1的表达,负向调控GPLD1的蛋白表达。过表达miR-497-3p、敲减GPLD1具有相似的促进高糖诱导HRGEC细胞增殖,抑制凋亡的作用,并上调PCNA蛋白,下调P21、Caspase-3、Caspase-9蛋白。过表达GPLD1显著削弱恩格列净、过表达miR-497-3p对高糖诱导HRGEC细胞的增殖、凋亡调控作用。结论: 恩格列净促进高糖诱导HRGEC细胞增殖,抑制凋亡,其机制与调控miR-497-3p/GPLD1信号通路相关。

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关键词 ：恩格列净, 糖尿病, miR-497-3p, GPLD1

Abstract：Objective: To explore the mechanism of empagliflozin (EMPA) regulating the proliferation and apoptosis of human glomerular microvascular endothelial cells (HRGEC). Methods: HRGEC was induced by 30 mmoL/L glucose to establish the renal injury cell model in high glucose environment. The damaged cells were treated with englitazin (250, 500, 1000nmoL/L). HRGEC cells were transfected with miR-NC group (transfected miR-NC), miR-497-3p group (transfected miR-497-3p), si-NC group (transfected si-NC), si-fish glycosylphosphatidylinositol specific phospholipase D1 (GPLD1) group (transfected si-GPLD1), EMPA + miR-497-3p + pcDNA group (co-transfected miR-497-3p and pcDNA), EMPA + miR-497-3p + pcDNA-GPLD1 group (co-transfected miR-497-3p and pcDNA-GPLD1), were all treated with 30mmoL/L glucose or 500 nmol / L empagliflozin. The cell proliferation rate and apoptosis rate were detected by cell counting kit (CCK-8) method and flow cytometry. The expressions of GPLD1, proliferating cell nuclear antigen (PCNA), cell cycle dependent protein kinase inhibitor 1a (p21), Caspase-3 and Caspase-9 were detected by western blotting (WB) assay. The binding ability of miR-497-3p to GPLD1 was detected by dual luciferase assay and RNA binding protein immunoprecipitation assay (RIP). Results: Compared with NG group, the proliferation rate significantly decreased in HG group, the apoptosis rate significantly increased, and the expression of miR-497-3p significantly decreased (P<0.05). Compared with HG group, the cell proliferation rate, apoptosis rate and the expression of miR-497-3p were significantly increased in empagliflozin (250, 500 and 1000nmoL/L) group (P<0.05). The optimal concentration of empagliflozin was 500 nmol/L. MiR-497-3p significantly inhibited the fluorescence activity of wild-type GPLD1 cells, promoted the expression of Ago2-GPLD1, and negatively regulated the protein expression of GPLD1. Overexpression of miR-497-3p and knockdown of GPLD1 had similar effects on promoting the proliferation and inhibiting apoptosis, up-regulating PCNA protein and down-regulating p21, caspase-3 and caspase-9 protein of HRGEC cells induced by high glucose. Overexpression of GPLD1 significantly weakened the regulatory effects on the proliferation and apoptosis of empagliflozin and overexpression of miR-497-3p in HRGEC cells induced by high glucose. Conclusion: Empagliflozin could promote the proliferation and inhibit apoptosis of HRGEC cells induced by high glucose, and its mechanism is related to the regulation of miR-497-3p / GPLD1 signal pathway.

Key words： Empagliflozin Diabetes MiR-497-3p GPLD1

基金资助:河北省卫生和计划生育委员会科研基金项目,(编号:20211203)

通讯作者: 檀增桓

引用本文:

高瑞超, 李敏, 金朋然, 刘旋, 冯雪, 檀增桓. 恩格列净通过miR-497-3p/GPLD1通路调控高糖诱人肾小球微血管内皮细胞增殖凋亡的机制研究[J]. 河北医学, 2022, 28(2): 194-201.
GAO Ruichao, LI Min, JIN Pengran, et al. Mechanism of Empagliflozin Regulating Proliferation and Apoptosis of Human Glomerular Microvascular Endothelial Cells Induced by High Glucose Through MiR-497-3p/GPLD1 Pathway. HeBei Med, 2022, 28(2): 194-201.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2022.02.04 或 http://www.hbyxzzs.cn/CN/Y2022/V28/I2/194

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