氯离子通道-3在黄芪甲苷降低高糖诱导人脐静脉内皮细胞损伤中的作用

ROLES OF CHLORIDE CHANNEL-3 ON ASTRAGALOSIDE IV REDUCING INJURY OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS INDUCED BY HIGH GLUCOSE

目的探究氯离子通道-3(CIC-3)在黄芪甲苷(ASI)降低高糖诱导人脐静脉内皮细胞(HUVECs)损伤中的作用。方法体外培养HUVECs,使用MTT实验检测不同剂量ASI对HUVECs的影响;并将HUVECs分为正常对照组、高糖损伤组、低剂量黄芪甲苷组、中剂量黄芪甲苷组和高剂量黄芪甲苷组;通过高糖诱导制成HUVECs损伤模型;以高糖损伤模型为基础,低、中、高剂量黄芪甲苷组再分别加入1、2、4μmol/ml的ASI处理;采用流式细胞仪检测HUVEC的凋亡率;采用蛋白质印迹实验(WB)检测凋亡相关蛋白Bcl-2和Bax的表达情况;采用WB和实时聚合酶链反应(RT-PCR)检测黄芪甲苷处理24 h和48 h后氯离子通道(CIC3)蛋白和mRNA表达情况。结果随ASI使用剂量增加,HUVECs的存活率升高(P<0.05),但培养24h和培养48h存活率差异无统计学意义(P>0.05)。与正常对照组比较,高糖损伤组细胞凋亡率和Bcl-2蛋白相对表达水平、24h和48hCIC3蛋白和mRNA相对表达水平均显著升高,Bax蛋白相对表达水平显著降低(均P<0.05);与高糖损伤组比较,低、中、高剂量黄芪甲苷组细胞凋亡率和Bcl-2蛋白相对表达水平、24h和48hCIC3蛋白和mRNA相对表达水平均升高,且呈浓度依赖性(均P<0.05);与高糖损伤组比较,低、中、高剂量黄芪甲苷组Bax蛋白相对表达水平均降低,且随着剂量增加显著降低(均P<0.05);正常对照组、高糖损伤组、低剂量黄芪甲苷组培养24h和培养48hCIC3蛋白和mRNA相对表达水平均差异均无统计学意义(均P>0.05);中剂量黄芪甲苷组和高剂量黄芪甲苷组培养48hCIC3蛋白和mRNA相对表达水平均高于培养24h(均P<0.05)。结论 ASI可以通过抑制CIC-3的表达,调节HUVECs的凋亡并缓解其损伤。

Objective To explore the roles of chloride channel-3(CIC-3) on astragaloside IV(ASI) reducing injury of human umbilical vein endothelial cells(HUVECs) induced by high glucose.Methods HUVECs were cultured in vitro.The effects of different doses of ASI on HUVECs were detected by MTT.HUVECs were divided into normal control group,high glucose injury group,low-dose,medium-dose and high-dose ASI groups.HUVECs injury models were prepared by high glucose in high glucose injury group.On basis of high glucose injury model,low-dose,medium-dose and high-dose ASI groups were treated with 1,2 and 4 μmol/ml ASI,respectively.The apoptosis rate of HUVECs was detected by flow cytometry.The expression of apoptosis-related proteins Bcl-2 and Bax was detected by Western blot(WB).The expression of CIC3 protein and mRNA was detected by WB and real-time polymerase chain reaction(RT-PCR) after 24 h and 48 h of ASI treatment.Results With the increase of ASI dose,survival rate of HUVECs was significantly increased(P<0.05).However,the difference was not significant in survival rate after 24 h and 48 h of culture(P>0.05).Compared with normal control group,apoptosis rate and relative expression level of Bcl-2 protein,relative expression levels of CIC3 protein and mRNA at 24 h and 48 h were significantly increased,while relative expression level of Bax protein was significantly decreased in high glucose injury group(P<0.05).Compared with high glucose injury group,apoptosis rate and relative expression level of Bcl-2 protein,relative expression levels of CIC3 protein and mRNA at 24 h and 48 h were significantly increased,showing concentration-dependence(P<0.05),while relative expression level of Bax protein was significantly decreased,which was signifi-cantly decreased with the increase of ASI dose(P<0.05).The difference was not significant in the relative expression levels of CIC3 protein and mRNA among normal control group,high glucose injury group and low-dose ASI group after 24 h and 48 h of culture(P>0.05).In medium-dose and high-dose ASI groups,relative expression levels of CIC3 protein and mRNA after 48 h of culture were significantly higher than those after 24 h of culture(P<0.05).Conclusion ASI can regulate apoptosis of HUVECs and alleviate their injury by inhibiting expression of CIC3.