达格列净对高糖诱导内皮细胞损伤保护作用及机制的研究

Protective effect and its mechanism of Dapagliflozin on high glucose induced endothelial cell injury

目的探讨达格列净(DAP)对高糖诱导内皮细胞损伤的保护作用。方法将人微血管内皮细胞HMEC-1分为正常对照组(Con)组、高糖(HG)组、HG+10 nmol/L DAP组(HG+10 DAP)、HG+100 nmol/L DAP组(HG+100 DAP)、HG+100 nmol/L DAP+anti-miR-Con组(HG+100 DAP+anti-miR-Con)、HG+100 nmol/L DAP+anti-miR-423-5p组(HG+100 DAP+anti-miR-423-5p)。构建荧光报告基因载体,将荧光报告基因与miR-Con、miR-423-5p、anti-miR-Con、anti-miR-423-5p共转染至HMEC-1细胞分别为miR-Con、miR-423-5p、anti-miR-Con、anti-miR-423-5p组。细胞计数试剂盒(CCK-8)法检测内皮细胞活力,试剂盒乳酸脱氢酶(LDH)水平,流式细胞仪活性氧(ROS),TUNEL试剂盒检测细胞凋亡情况,RT-PCR测定内皮细胞中miR-423-5p表达,双荧光素酶报告实验检测miR-423-5p与WNT3的结合力。结果与Con组比较,HG、HG+10 DAP、HG+100 DAP组细胞活性、miR-423-5p表达降低(P<0.05),LDH浓度、ROS活性、TUNEL阳性细胞升高(P<0.05)。与HG组比较,HG+100 DAP组细胞活性、miR-423-5p表达升高(P<0.05),LDH浓度、ROS活性、TUNEL阳性细胞降低(P<0.05)。与HG+10 DAP组比较,HG+100 DAP组ROS活性、TUNEL阳性细胞降低(P<0.05),细胞miR-423-5p表达升高(P<0.05)。与HG+100 DAP+anti-miR-Con组比较,HG+100 DAP+anti-miR-423-5p组miR-423-5p表达、细胞活性降低(P<0.05),LDH浓度、ROS活性和TUNEL阳性细胞率升高(P<0.05)。miR-423-5p组WNT3蛋白表达低于miR-Con组(P<0.05),anti-miR-423-5p组细胞中WNT3蛋白表达高于anti-miR-Con组(P<0.05)。双荧光素酶报告基因检测实验结果显示,WT-WNT3中miR-423-5p组荧光酶活性低于miR-Con组。结论DAP可抑制高糖诱导的内皮细胞损伤,作用机制与调控miR-423-5p/WNT3信号通路相关。

Objective To investigate the protective effect of Dapagliflozin(DAP)on endothelial cell injury induced by high glucose and its related mechanism.Methods Human microvascular endothelial cells(HMEC-1)were divided into normal control group(Con),high glucose group(HG),HG+10 nmol/L DAP group(HG+10 DAP),HG+100 nmol/L DAP group(HG+100 DAP),HG+100 nmol/L DAP+anti-miR-Con group(HG+100 DAP+anti-miR-Con),HG+100 nmol/L DAP+anti-mir-423-5p group(HG+100 DAP+anti-miR-423-5p).The fluorescent reporter gene vector was constructed,and the fluorescent reporter gene was co-transfected with miR-Con,miR-423-5p,anti-miR-Con and anti-miR-423-5p into HMEC-1 cells,which were miR-Con group,miR-423-5p group,anti-miR-Con group and anti-miR-423-5p group.Cell counting kit(CCK-8)was used to detect endothelial cell viability.Lactate dehydrogenase(LDH)level of kit,reactive oxygen species(ROS)of flow cytometry,TUNEL kit was used to detect cell apoptosis.RT-PCR was used to detect the expression of miR-423-5p in endothelial cells,and the binding force between miR-423-5p and WNT3 was detected by double luciferase report experiment.Results Compared with Con group,cell activity and miR-423-5p expression decreased(P<0.05),while LDH concentration,ROS activity and TUNEL positive cells increased(P<0.05)in HG,HG+10 DAP and HG+100 DAP groups.Compared with HG group,cell activity and miR-423-5p expression increased(P<0.05),while while LDH concentration,ROS activity and TUNEL positive cells decreased(P<0.05)in HG+100 DAP group.Compared with HG+10 DAP group,the activity of ROS and TUNEL positive cells decreased(P<0.05),and the expression of miR-423-5p increased(P<0.05)in HG+100 DAP group.Compared with HG+100 DAP+anti-miR-Con group,the expression and cell activity decreased(P<0.05),while LDH concentration,ROS activity and TUNEL positive cell rate increased(P<0.05)in miR-423-5p in HG+100 DAP+anti-miR-423-5p group.WNT3 protein expression was lower in miR-423-5p group than in miR-Con group(P<0.05),and WNT3 protein expression was higher in anti-miR-423-5p group than in anti-miR-Con group(P<0.05).The results of double luciferase reporter gene detection showed that in WT-WNT3,the luciferase activity was lower in miR-423-5p group than in miR-Con group.Conclusion DAP could inhibit endothelial cell injury induced by high glucose.The mechanism of this effect may be related to the regulation of miR-423-5p/WNT3 signaling pathway.