曲美他嗪对缺氧/复氧诱导人心肌细胞凋亡的影响

Effects of trimetazidine on apoptosis in human cardiomyocytes induced by hypoxia and reoxygenation

目的研究曲美他嗪(trimetazidine)对缺氧/复氧致人心肌细胞凋亡及其信号改变的效应。方法通过采用胶原酶/胰酶联合消化法获取人心肌细胞,采用缺氧/复氧模拟缺血/再灌注诱导细胞凋亡,选取最佳凋亡时间为缺氧24h和复氧4h,并用免疫细胞化学法检测bcl-2、bax、细胞色素C和caspase-3蛋白的变化,用原位杂交法检测bcl-2mRNA和caspase-3mRNA表达的变化,用脂肪代谢试剂曲美他嗪不同剂量干预,观察对缺氧/复氧诱导人体外培养心肌细胞凋亡及其信号bcl-2、bax、细胞色素C和caspase-3蛋白及bcl-2mRNA和caspase-3mRNA表达的影响。凋亡信号表达用平均光密度表示。用SPSS10.0进行统计分析。结果①缺氧24h/复氧4h后细胞凋亡率明显升高(18.50%±1.00%);bcl-2(0.189±0.006)下降;bax(0.240±0.002)、caspase-3(0.230±0.002)和细胞色素C(0.225±0.003)升高;bcl-2mRNA(0.300±0.003)下降;caspase-3mRNA(0.307±0.004)升高;分别与对照组比较,差异均有显著性(P<0.01)。②曲美他嗪呈剂量依赖性明显降低细胞凋亡率,降低bax、caspase-3和cytochromeC蛋白阳性率和升高bcl-2蛋白阳性率,降低caspase3mRNA表达阳性率和升高bcl-2mRNA表达阳性率(均P<0.01)。结论曲美他嗪具有抗凋亡作用,可能与升高bcl-2蛋白和bcl-2mRNA表达与降低bax、caspase-3、cytochromeC蛋白和caspase-3mRNA表达水平有关。

Aim To investigate effects of trimetazidine on apoptosis and its signal regulation in human cardiomyocytes induced by hypoxia and reoxygenation. Methods Human cardiomyocytes were obtained from minced myocardium digested in trypsin and collagenase. The cultured cells apoptosis was induced by hypoxia and reoxygenation. Optional time of apoptosis was hypoxia 24 h and reoxygenation 4 h. The bcl-2, bax, caspase-3 and cytochrome C proteins were detected by using immunocytochemistry （ICC）. Bcl-2 mRNA and caspase-3 mRNA were detected by using in situ hybridization method （ISH）. Apoptosis signal was expressed by optical density. SPSS 10.0 was used for statistical analysis. Results （1）. Twenty-four hours of hypoxia followed by 4 h of reoxygenation significantly increased the rate of apoptosis [（18.50±1.00）%]. Results revealed that H/R significantly induced the down-regulation of bcl-2 proteins （0.189±0.006） and up-regulation of bax （0.240±0.002）, caspase-3 （0.230±0.002） and cytochrome C （0.225±0.003） proteins; down-regulation of bcl-2 mRNA （0.300±0.003） and up-regulation of caspase-3 mRNA （0.307±0.004） in cardiomyocytes comparing with control group respectively （all P 〈 0.01）. （2）. Trimetazidine significantly decreased the rate of apoptotic, up-regulated the expression level of bcl-2 and down-regulated expression levels of bax, caspase-3 and cytochrome C protein, respectively （all P 〈 0.01）. Trimetazidine also up-regulated the expression level of bcl-2 mRNA and down-regulated the expression level of caspase-3 mRNA. Conclusion Trimetazidine has anti-apoptosis effect and its mechanisms may be related to the increased expression levels of bcl-2 proteins and bcl-2 mRNA and decreased expression levels of bax, caspase-3, cytochrome C proteins and caspase-3 mRNA.