氧化苦参碱通过调控MAPK信息通路改善醛固酮诱导的心肌细胞损伤

Protective Effect of Oxymatrine Against Cardiac Myocytes Injury Induced by Aldosterone via MAPK Signaling Pathway

目的:研究氧化苦参碱(OMT)对醛固酮(ALD)诱导心肌细胞损伤的改善作用及其作用机制。方法:采用胰酶消化法和差速贴壁法分离和纯化新生大鼠心肌细胞进行培养,采用1×10^(-5)mol·L^(-1)ALD建立心肌细胞损伤模型。实验分为空白组,模型组(1×10^(-5)mol·L^(-1)ALD),ALD+OMT高浓度(3.78×10-4mol·L^(-1))组,ALD+OMT低浓度(1.89×10-4mol·L^(-1))组,ALD+JNK抑制剂(JNK I,5×10^(-6)mol·L^(-1))组,ALD+阿司匹林(Asp,1×10^(-5)mol·L^(-1))组,即OMT,JNK抑制剂和阿司匹林组先以相应药物预处理2 h后加入终浓度为1×10^(-5)mol·L^(-1)的ALD共同孵育24 h。二甲基噻唑蓝(MTT)法检测细胞存活率,Giemsa染色观察心肌细胞形态学变化情况。蛋白免疫印迹法(Western blot)及实时荧光定量PCR(Real-time PCR)分析OMT对ALD诱导JNK蛋白表达水平,JNK磷酸化(p-JNK)水平及mRNA表达的影响。结果:与空白组比较,ALD可显著降低心肌细胞存活率(P<0.01)并改变细胞形态,OMT可显著改善醛固酮诱导的心肌细胞存活率降低(P<0.01)并使细胞恢复至正常形态;Western blot结果显示ALD可诱导JNK蛋白磷酸化水平升高(P<0.01),而OMT可显著抑制ALD诱导的p-JNK表达增加(P<0.01);Real-time PCR结果显示,与空白组比较,ALD可诱导JNK mRNA表达增加(P<0.01),使用OMT进行预保护后,各组基因表达均没有显著改变。结论:OMT对ALD诱导的心肌细胞损伤具有保护作用,其作用机制与抑制JNK蛋白磷酸化密切相关。

Objective：To investigate the protective effect of oxymatrine（OMT） on cardiac myocytes injure induced by aldosterone and explore its mechanism.Method：The cardiomyocytes of neonatal rats were isolated and purified by trypsin digestion and differential adherence method for culture,and then 1×10-5mol·L-1ALD was used to establish myocardial injury models.The experiment was divided into blank group,model group（1×10-5mol·L-1ALD）,ALD ＋ OMT high dose group（3.78×10-4mol·L-1）,ALD＋OMT low dose group（1.89×10-4mol·L-1）,ALD ＋ JNK inhibitor group（JNK I,5×10-6mol·L-1）,ALD ＋ Aspirin group（Asp,1 ×10-5mol·L-1）.That is to say,in OMT,JNK inhibitor and Aspirin groups,after pre-treatment with corresponding medicines for 2 h,ALD with the final concentration of 1×10-5mol·L-1was added for co-incubation for 24 h.Then MTT assay was used to detect the cell survival rate;the morphological changes of cardiac myocytes were observed by Giemsa staining;Western blot and Real-time PCR were used to analyze the effect of OMT on the protein and mRNA expression of JNK and p-JNK.Result：As compared with the blank group,ALD significantly reduced the survival rate of cardiac myocytes（P〈0.01） and changed the cell morphology;OMT could reverse the decrease in survival rate of cardiac myocytes（P〈0.01） and restore the cells to normal form;Western blot showed that ALD could increase the phosphorylation of JNK（P〈0.01）,while the expression of p-JNK was significantly downregulated after pre-incubated with OMT as compared with ALD group（P〈0.01）.Real-time PCR results showed that the expression of JNK mRNA in ALD group was higher than that in normal control group（P〈0.01）,however no significant change of gene expression was observed in OMT group.Conclusion：OMT had protective effect on ALD-induced cardiac myocytes injury,and its mechanism may be closely related to inhibiting JNK protein phosphorylation.