黄芪甲苷通过下调miR-327缓解缺氧对脑微血管内皮细胞凋亡及炎症反应的影响

AstragalosideⅣAlleviates the Effects of Hypoxia on Brain Microvascular Endothelial Cells Apoptosis and Inflammation by Down-Regulating miR-327

目的探讨黄芪甲苷对缺氧处理的脑微血管内皮细胞(BMECs)损伤的影响及分子机制。方法体外培养BMECs,将其分为对照组、模型组、低、中、高剂量黄芪甲苷组、高剂量黄芪甲苷+miR-NC、高剂量黄芪甲苷+miR-327;四甲基偶氮唑盐比色法(MTT)检测细胞活力;流式细胞术检测细胞凋亡;蛋白质印迹(western blot)法检测蛋白表达;试剂盒检测BMECs损伤标志物,包括乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px);酶联免疫吸附实验(ELISA)检测白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α);实时荧光定量PCR(RT-qPCR)检测miR-327的表达水平。结果与对照组比较,缺氧处理9 h与12 h处,BMECs存活率显著降低(P<0.05)。与对照组相比,模型组细胞凋亡率显著升高,Bcl-2表达水平显著降低,Bax表达水平显著升高(P<0.05);与模型组相比,低、中、高剂量黄芪甲苷组细胞凋亡率均显著降低,Bcl-2表达水平升高,Bax表达水平降低,且呈剂量依赖性(P<0.05)。与对照组相比,模型组LDH、MDA、IL-6、IL-1β和TNF-α表达水平显著升高,SOD和GSH-Px表达水平显著降低(P<0.05);与模型组相比,低、中、高剂量黄芪甲苷组LDH、MDA、IL-6、IL-1β和TNF-α表达水平显著降低,SOD和GSH-Px表达水平显著升高,且呈剂量依赖性(P<0.05)。与对照组相比,模型组miR-327表达水平显著升高(P<0.05);与模型组相比,低、中、高剂量黄芪甲苷组miR-327表达水平显著降低,且呈剂量依赖性(P<0.05)。过表达miR-327逆转了黄芪甲苷对缺氧处理BMECs凋亡和炎症反应的影响。结论黄芪甲苷可能通过下调miR-327表达缓解缺氧对BMECs凋亡及炎症反应的影响。

OBJECTIVE To explore the effect of astragalosideⅣon the damage of brain microvascular endothelial cells(BMECs)treated with hypoxia and its molecular mechanism.METHODS BMECs were cultured in vitro and divided into control group,model group,low,medium and high dose astragalosideⅣgroups,high dose astragalosideⅣ+miR-NC group,and high dose astragalosideⅣ+miR-327 group.The tetramethylazozolate colorimetric method(MTT)was used to detect cell activity.Flow cytometry was used to detect cell apoptosis.Western blot was used to detect protein expression.The kits detected damage markers of BMECs,including lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX).ELISA was used to evaluate the levels of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α).Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of circDONSON.RESULTS Compared with the control group,the survival rate of BMECs decreased significantly at 9 h and 12 h after hypoxia treatment(P<0.05).Compared with the control group,the apoptosis rate of the model group was significantly increased,Bcl-2 expression level was significantly decreased,and Bax expression level was significantly increased(P<0.05).Compared with the model group,the apoptosis rates of the low,medium and high dose astragalosideⅣgroups were significantly reduced,the expression level of Bcl-2 was increased,and the expression level of Bax was decreased in a dose-dependent manner(P<0.05).Compared with the control group,LDH,MDA,IL-6,IL-1 β and TNF-αexpression levels in the model group were significantly increased,while SOD and GSH-PX expression levels were significantly decreased(P<0.05).Compared with the model group,the expression levels of LDH,MDA,IL-6,IL-1β and TNF-αin the low,medium and high dose astragalosideⅣgroups were significantly decreased,and the expression levels of SOD and GSH-PX were significantly increased in a dose-dependent manner(P<0.05).Compared with the control group,miR-327 expression level in the model group was significantly increased(P<0.05).Compared with the model group,the expression level of miR-327 in the low,medium and high dose astragalosideⅣgroups was significantly reduced dose-dependently(P<0.05).The overexpression of miR-327 reversed the effects of astragalosideⅣon the apoptosis and inflammatory response of hypoxia treated BMECs.CONCLUSION AstragalosideⅣmay alleviate the effects of hypoxia on BMECs apoptosis and inflammation by down-regulating the expression of miR-327.