香青兰总黄酮对阿霉素心肌毒性的保护机制研究

Protective effect and mechanism of total flavonoids of Dracocephalum moldevica on doxorubicin myocardial toxicity

目的 探讨香青兰总黄酮(TFDM)对阿霉素心脏毒性的保护作用及相关机制。方法 大鼠心肌细胞H9c2细胞随机分为对照组、模型组和TFDM治疗组(5、25、50、100μg/m L)。药物干预后,除对照组外,其余各组细胞采用阿霉素1μmol/L作用24 h制备心脏毒性模型。采用CCK-8法测定TFDM干预后阿霉素损伤对H9c2细胞活力的影响;采用试剂盒法测定各组细胞乳酸脱氢酶(LDH)释放情况及细胞内超氧化物歧化酶(SOD)、丙二醛(MDA)水平;采用Annexin-V FITC/PI双染法通过流式细胞仪检测各组细胞凋亡率;采用DCFH-DA、JC-1探针检测细胞内活性氧(ROS)和线粒体膜电位;采用Westernblotting检测各组细胞中p38丝裂原活化蛋白激酶(p38MAPK)、细胞外调节蛋白激酶1/2(ERK1/2)、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路相关蛋白表达。结果 与对照组比较,阿霉素诱导的模型组细胞活力下降,LDH、MDA水平升高,SOD活性降低,细胞凋亡率升高,ROS释放显著增多,线粒体膜电位显著下降,而TFDM则剂量依赖性地使细胞活力升高,LDH、MDA水平下降,SOD活性升高,细胞凋亡率降低,ROS释放显著减少,线粒体膜电位显著升高;Western blotting结果显示,与对照组比较,模型组细胞p-PI3K、p-Akt、p-ERK、Bcl-2蛋白表达水平降低,p-p38MAPK、Bax、Caspase-3蛋白表达水平显著升高;与模型组比较,TFDM使细胞中p-PI3K、p-Akt、p-ERK、Bcl-2蛋白表达水平升高,p-p38MAPK、Bax、Caspase-3蛋白表达水平降低。结论 TFDM能够保护心肌细胞,可能通过抗氧化应激,保护心肌细胞线粒体,调节MAPK、PI3K/Akt凋亡通路及其下游凋亡分子的表达抑制细胞凋亡。

Objective To explore the protective effect and mechanism of total flavonoids of Dracocephalum moldevica(TFDM)on doxorubicin-induced cardiotoxicity.Methods H9c2 cells were induced with 1μmol/L doxorubicin for 24 h to establish a cardiotoxicity model.H9c2 cells were randomly divided into control group,model group,and drug intervention group(four subgroups of 5,25,50,and 100(xg/mL).After the intervention of TFDM,the doxorubicin cardiotoxicity model was established in the other groups except the control group.The cell counting Kit-8 method was used to determine the viability of H9c2 cells induced by doxorubicin injury after the intervention of TFDM.The effects of lactate dehydrogenase release,intracellular superoxide dismutase and malondialdehyde in each group were determined by kit method.The apoptosis rate of each group was detected by flow cytometry using Annexin-V FITC/PI double staining method.Reactive oxygen species(ROS)and mitochondrial membrane potential in each group were detected by DCFH-DA and JC-1 probes.The expressions of p38MAPK,ERX1/2,and PI3K/Akt pathway-related proteins were detected by Western blotting.Results Compared with the control group,the cell viability of the model group induced by doxorubicin was decreased,the release of lactate dehydrogenase and the content of malondialdehyde were increased,the activity of superoxide dismutase was decreased,the apoptosis rate was increased,the release of reactive oxygen species was increased significantly,and the mitochondrial membrane potential was decreased significantly.However,TFDM increased H9c2 cell viability,decreased LDH and MDA levels,increased SOD activity,decreased apoptosis rate,significantly decreased ROS release,and significantly increased MMP in a dose-dependent manner.The difference was statistically significant.The results of Western blot showed that the expression levels of p-PI3K,p-Akt,p-ERKl/2,and Bcl-2 were decreased,and the expression levels of p-p38MAPK,Bax and Caspase-3 were significantly increased compared with the control group.However,in the TFDM-treated group,the expression of p-PI3K,p-Akt,p-ERKl/2,and Bcl-2 protein was increased,and the protein expression of p-p38MAPK,Bax,and Caspase-3 protein was decreased.Conclusion TFDM can protect cardiomyocytes,and its protective mechanism may be related to the resistance to oxidative stress,protection of cardiomyocyte mitochondria,and regulating MAPK enzyme family proteins,and PI3K/Akt signaling pathway and subsequent release of apoptotic cytokines to inhibit apoptosis.