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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 effect and mechanism of oxymatrine (OMT) on cardiomyocyte injury induced by aldosterone (ALD). Methods: Cardiomyocytes were isolated and purified by trypsin digestion and differential adherent method. Cardiomyocytes were injured by 1 × 10 ~ (-5) mol·L -1 ALD. The experiment was divided into blank group, model group (1 × 10 -5 mol·L -1 ALD), ALD + OMT high concentration (3.78 × 10 -4 mol·L -1) + OMT low concentration (1.89 × 10-4mol·L -1), ALD + JNK inhibitor (JNK I, 5 × 10 -6 mol·L -1), ALD + aspirin (Asp, 1 × 10 ~ (-5) mol·L ~ (-1)) groups, that is, OMT, JNK inhibitor and aspirin group were pretreated with the corresponding drugs for 2 h, then added to a final concentration of 1 × 10 ~ 5) mol·L -1 ALD for 24 h. Cell viability was assayed by MTT assay and morphological changes of cardiomyocytes were observed by Giemsa staining. The effects of OMT on ALD-induced JNK protein expression, JNK phosphorylation (p-JNK) and mRNA expression were analyzed by Western blot and Real-time PCR. Results: Compared with the blank group, ALD significantly decreased the survival rate of cardiomyocytes (P <0.01) and changed the morphology of cells. OMT significantly decreased the aldosterone - induced cardiomyocyte survival rate (P <0.01) and restored the cells to normal morphology. Western blot results showed that ALD induced an increase in phosphorylation of JNK (P <0.01), while OMT significantly inhibited ALD-induced increase of p-JNK expression (P <0.01). Real-time PCR showed that compared with the blank group , ALD induced JNK mRNA expression increased (P <0.01), the use of OMT for pre-protection, the gene expression in each group did not change significantly. CONCLUSION: OMT has a protective effect on ALD-induced cardiomyocyte injury and its mechanism is closely related to inhibition of JNK protein phosphorylation.