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被引量：4

Mechanism of Empagliflozin Regulating Proliferation and Apoptosis of Human Glomerular Microvascular Endothelial Cells Induced by High Glucose Through MiR-497-3p/GPLD1 Pathway

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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。 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.