摘要
目的探讨苦参碱对H_(2)O_(2)诱导的大鼠心肌细胞H9c2氧化损伤的保护作用及可能的作用机制。方法大鼠心肌细胞H9c2分为空白组(正常培养)、苦参碱组(正常培养+50μmol/L苦参碱培养24 h)、H_(2)O_(2)模型组(200μmol/L H_(2)O_(2)预处理3 h)、H_(2)O_(2)+苦参碱组(200μmol/L H_(2)O_(2)预处理3 h后加入50μmol/L苦参碱培养24 h)、H_(2)O_(2)+苦参碱+AM组(200μmol/L H_(2)O_(2)预处理3 h后加入50μmol/L苦参碱和5μmol/L AM培养24 h)。采用CCK-8法、流式细胞术检测细胞活性和凋亡情况;荧光法检测细胞内活性氧簇(ROS)水平和线粒体膜电位变化;用试剂盒检测各组细胞培养上清中乳酸脱氢酶(LDH)和丙二醛(MDA)水平以及细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)活性。Western blot检测丝裂原活化蛋白激酶(MAPK)通路相关蛋白表达。结果与空白组相比,H_(2)O_(2)模型组细胞存活率降低,细胞凋亡率、ROS生成量、线粒体膜电位JC-1单体阳性细胞百分比及细胞培养上清中LDH和MDA水平增加,且细胞中SOD、CAT和GSH-Px活性降低,此外(p-P38 MAPK)/(P38MAPK)比值和(p-JNK)/(JNK)比值上调(P<0.05);然而苦参碱组上述指标与空白组相比无统计学差异(P>0.05)。与H_(2)O_(2)模型组相比,H_(2)O_(2)+苦参碱组细胞存活率升高,细胞凋亡率、ROS生成量、线粒体膜电位JC-1单体阳性细胞百分比以及细胞培养上清中LDH和MDA水平减少,且细胞中SOD、CAT和GSH-Px活性升高,此外(p-P38 MAPK)/(P38 MAPK)比值和(p-JNK)/(JNK)比值下调(P<0.05);然而与H_(2)O_(2)+苦参碱组相比,H_(2)O_(2)+苦参碱+AM组上述各种指标则被逆转(P<0.05)。结论苦参碱通过减少ROS的产生、增强抗氧化酶系统、维持线粒体功能等,对H9c2心肌细胞氧化应激损伤具有一定的保护作用,且作用机制与调控MAPK信号通路有关。
Objective To investigate the protective effect and possible mechanism of Matrine on H_(2)O_(2)induced oxidative damage in rat cardiomyocyte H9c2 cells.Methods Rat cardiomyocytes H9c2 were divided into blank group(normal culture),matrine group(normal culture+50μmol/L matrine culture for 24 h),H_(2)O_(2)model group(200μmol/L H_(2)O_(2)pretreatment for 3 h),H_(2)O_(2)+matrine group(200μmol/L H_(2)O_(2)pretreated for 3 h and then added 50μmol/L matrine for 24 h),H_(2)O_(2)+matrine+AM group(200μmol/L H_(2)O_(2)pretreated for 3 h and then added 50μmol/L matrine and 5μmol/L AM for 24 h).CCK-8 method and flow cytometry were used to detect cell viability and apoptosis;fluorescence method was used to detect intracellular reactive oxygen species(ROS)levels and mitochondrial membrane potential changes;kits were used to detect lactate dehydrogenation in cell culture supernatants of each group The levels of enzymes(LDH)and malondialdehyde(MDA)and the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT)in cells.Western blot was used to detect the expression of related proteins in the mitogen-activated protein kinase(MAPK)pathway.Results Compared with the blank group,the cell survival rate of the H_(2)O_(2)model group decreased,the apoptosis rate,the amount of ROS production,the percentage of mitochondrial membrane potential JC-1 monomer positive cells,and the LDH and MDA levels in the cell culture supernatant increased,and the cells The activities of SOD,CAT and GSH-Px decreased,and the ratios of(p-P38 MAPK)/(P38 MAPK)and(p-JNK)/(JNK)were increased(P<0.05);however,the above indicators in the matrine group were compared with the blank There was no statistical difference between the two groups(P>0.05).Compared with the H_(2)O_(2)model group,the cell survival rate of the H_(2)O_(2)+matrine group increased,the apoptosis rate,ROS production,the percentage of mitochondrial membrane potential JC-1 monomer-positive cells,and the LDH and MDA levels in the cell culture supernatant decreased.And the activity of SOD,CAT and GSH-Px in cells increased,in addition,the ratio of(p-P38 MAPK)/(P38 MAPK)and the ratio of(p-JNK)/(JNK)were down-regulated(P<0.05);however,compared with H_(2)O_(2)+Sophorae Compared with the alkali group,the above indicators in the H_(2)O_(2)+matrine+AM group were reversed(P<0.05).Conclusion Matrine can protect H9c2 cardiomyocytes from oxidative stress damage by reducing the production of ROS,enhancing the antioxidant enzyme system,and maintaining mitochondrial function,and one of the mechanisms of action is related to the regulation of the MAPK signaling pathway.
作者
赵希坤
闫鹏
赵飞龙
王可
Zhao Xikun;Yan Peng;Zhao Feilong;Wang Ke(Department of Cardiology,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang,Henan 471003;不详)
出处
《中国循证心血管医学杂志》
2023年第3期285-289,共5页
Chinese Journal of Evidence-Based Cardiovascular Medicine
基金
河南省医学科技攻关计划联合共建项目(2018020269)。
作者简介
通讯作者:王可,E-mail:hkdyfywk@163.com。
