摘要
目的研究丹参喷干粉对缺氧/复氧(H/R)所致的H9c2心肌细胞损伤的保护作用及其机制。方法将H9c2细胞置于无糖DMEM培养基中缺氧培养5 h,再复氧培养1 h,用于复制心肌细胞H/R模型,采用CCK8法检测细胞活力,并确定丹参喷干粉质量浓度,丹参喷干粉及ROS抑制剂(N-乙酰半胱氨酸)干预细胞后,Western blot、RT-qPCR法检测丹参喷干粉对GPX1蛋白及基因表达的影响,进一步通过流式细胞仪检测凋亡细胞和ROS水平,黄嘌呤氧化酶法和TBA法检测H9c2细胞SOD活性和MDA水平,Western blot、RT-qPCR法检测凋亡相关因子cleaved-caspase3、cleaved-RARP的表达,通过细胞转染方法过表达GPX1基因观察其联合丹参喷干粉对氧化应激及细胞凋亡的干预效应。结果与H/R模型组比较,丹参喷干粉及NAC预处理能增强GPX1表达(P<0.01),减少细胞凋亡(P<0.01),降低细胞氧化应激指标ROS、MDA水平(P<0.01),增加SOD活性(P<0.01)。过表达GPX1能够增强丹参喷干粉对氧化应激的效应,并减少凋亡相关蛋白cleaved-caspase3、cleaved-RARP的表达。结论丹参喷干粉可通过减轻氧化应激来抑制细胞凋亡,降低缺氧/复氧所致的心肌细胞损伤,其作用机制与GPX1信号通路相关。
AIM To study the protective effects and mechanism of Salvia miltiorrhiza powder on hypoxia/reoxygenation(H/R)-induced H9c2 myocardial cell injury.METHODS H9c2 cells cultured in sugar-free DMEM medium for 5 h with hypoxia were subsequently reoxygenated for 1 h to replicate the H/R model of cardiomyocytes for exposure to the different concentrations of Salvia miltiorrhiza powder,and had the cell viability detected by CCK8 assay.After the treatment of Salvia miltiorrhiza powder and ROS inhibitor(NAC),H/R-induced H9c2 myocardial cells had their expressions of GPX1 protein and mRNA detected by Western blot and RT-qPCR,their levels of cells apoptosis and ROS detected by flow cytometry,their SOD activity and MDA level detected by xanthine oxidase assay and TBA assay,their expressions of apoptosis-related factors cleaved-caspase3 and cleaved-RARP determined by Western blot and RT-qPCR,and finally their oxidative stress and apoptosis observed.based on the GPX 1 gene overexpressed by cell transfection method,and the combinative intervention of GPX1 gene and Salvia miltiorrhiza powder.RESULTS Compared with H/R model group,groups treated with Salvia miltiorrhiza powder and NAC shared enhanced GPX1 expression(P<0.01),reduced cell apoptosis(P<0.01),decreased levels of oxidative stress indices of ROS and MDA(P<0.01),and increased SOD level(P<0.01).Overexpression of GPX1 enhanced the effect of Salvia miltiorrhiza powder on oxidative stress and reduced the expressions of apoptosis related proteins cleaved-caspase3 and cleaved-RARP.CONCLUSION Salvia miltiorrhiza powder can inhibit cell apoptosis and reduce H/R-induced myocardial damage through regulation of GPX1 signaling pathway to alleviate the oxidative stress.
作者
谢君
芦波
李建华
桂明泰
姚磊
周训杰
符德玉
XIE Jun;LU Bo;LI Jian-hua;GUI Ming-tai;YAO Lei;ZHOU Xun-jie;FU De-yu(Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 200437,China)
出处
《中成药》
CAS
CSCD
北大核心
2021年第11期2989-2996,共8页
Chinese Traditional Patent Medicine
基金
国家自然科学基金(81774111)
上海市科学技术委员科研计划项目(19401970400)
上海中医药大学“研究生创新培养”专项科研项目(Y2021077)
上海雷允上药业有限公司科研项目(2019)。
