醋酸铅诱导心肌细胞凋亡的机制研究

Mechanism of lead acetate-induced cardiomyocyte apoptosis

目的 明确铅暴露对心肌细胞的毒性损伤,进一步探索其诱导心肌细胞凋亡的具体机制。方法 将正常培养的AC16人源心肌细胞随机分为正常对照组和不同浓度的醋酸铅组;通过Cell Counting Kit-8和乳酸脱氢酶(LDH)细胞毒性检测试剂盒测定细胞存活率以及LDH的释放率;利用活性氧荧光探针(DCFH-DA)和线粒体膜电位检测试剂盒(JC-1)检测活性氧(ROS)水平及线粒体膜电位变化;Western blot法检测凋亡蛋白及内质网应激相关蛋白的表达;采用Image J对Western blot条带进行量化。结果 相较于正常对照组,当醋酸铅浓度≥25μg/mL时,AC16细胞存活率明显降低且具有浓度依赖性,差异具有统计学意义(P<0.05);同时当醋酸铅浓度≥25μg/mL时,AC16细胞LDH释放率显著升高且具有浓度依赖性,差异有统计学意义(P<0.05);最后发现醋酸铅暴露将引起AC16细胞ROS的产生以及线粒体膜电位的下降。此外,相较于正常对照组,醋酸铅暴露后凋亡相关蛋白Cleaved-caspase3、Cleaved-PARP和内质网应激相关蛋白Calpain 1、Bip的表达均明显增高,差异均具有统计学意义(P<0.05)。结论 醋酸铅存在明显心肌毒性,通过内质网应激和氧化应激诱导心肌细胞凋亡的发生。

Objective To clarify the toxic damage of lead exposure to cardio-myocytes, and further explore the specific mechanism of its induced cardiomyocytes apoptosis. Methods The normally cultured human cardiomyocytes(AC16) were randomly divided into a control group and a lead acetate group with different concentrations. Cell viability and lactate dehydrogenase(LDH) release rate were determined by Cell Counting Kit-8 and LDH Cytotoxicity Detection Kit. Reactive oxygen species fluorescent probe(DCFH-DA) and mitochondrial membrane potential detection kit(JC-1)were used to detect reactive oxygen species(ROS) levels and mitochondrial membrane potential changes. Western blot was applied to detect the expression of apoptosis proteins and endoplasmic reticulum stress-related proteins, and Image J was employed to quantify the Western blot bands. Results Compared with the control group, when the concentration of lead acetate was greater than or equal to 25 μg/mL, the survival rate of AC16 cells was significantly reduced and in a concentration-dependent manner(P<0.05). At the same time, when the concentration of lead acetate was greater than or equal to 25 μg/mL, the release rate of lactate dehydrogenase in AC16 cells was significantly increased in a concentration-dependent manner(P<0.05). Secondly, lead acetate exposure was found to induce ROS production and decrease mitochondrial membrane potential in AC16 cells. In addition, compared with the control group, the expression of apoptosis-related proteins Cleaved-caspase 3 and Cleaved-PARP, endoplasmic reticulum stress-related proteins Calpain 1 and Bip were significantly increased after lead acetate exposure(P<0.05). Conclusion Lead acetate has obvious cardiotoxicity, which may induce cardiomyocyte apoptosis through endoplasmic reticulum stress and oxidative stress.