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本研究旨在观察缺锌对心肌细胞损伤作用并探讨其可能的机制。利用锌离子螯合剂四吡啶甲基乙二胺[N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine,TPEN]处理H9c2心肌细胞建立模拟缺锌模型,采用MTT法测定细胞存活率,用光学显微镜观察细胞形态学改变,用乳酸脱氢酶(lacate dehydrogenase,LDH)试剂盒检测心肌细胞上清液LDH水平,使用JC-1染色法检测线粒体膜电位(mitochondrial membrane potential,ΔΨm),采用活性氧(reactive oxygen species,ROS)试剂盒检测细胞内ROS水平,用Western blot检测ERK蛋白的磷酸化水平。结果显示,TPEN引起心肌细胞形态改变,LDH释放量明显增高,细胞ΔΨm降低,细胞内ROS水平明显增加。同时,TPEN下调心肌细胞ERK蛋白磷酸化水平,降低细胞存活率;MEK/ERK信号通路抑制剂PD98059预处理可进一步抑制ERK蛋白磷酸化水平,进一步降低细胞存活率;而S-亚硝基-N-乙酰-DL-青霉胺(SNAP,可激活MEK/ERK信号通路)和MPG(ROS清除剂)均可逆转TPEN的上述抑制作用。以上结果表明,缺锌通过促进细胞内ROS生成而抑制ERK通路,引起心肌细胞损伤。
The purpose of this study was to observe the effect of zinc deficiency on cardiomyocyte injury and to explore its possible mechanism. H9c2 cardiomyocytes were treated with zinc ion chelator tetrapyridylethylenediamine (N, N, N ’, N’-tetrakis (2-pyridylmethyl) ethylenediamine, TPEN] to establish simulated zinc deficient model and the cell viability The morphological changes of cells were observed with light microscope. LDH levels in cardiomyocyte supernatants were detected by lacde dehydrogenase (LDH) kit. The mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Intracellular ROS was detected by reactive oxygen species (ROS) kit, and phosphorylation of ERK protein was detected by Western blot. The results showed that TPEN induced morphological changes of cardiac myocytes, the release of LDH was significantly increased, the ΔΨm of cells was decreased and the level of ROS in cells was significantly increased. Meanwhile, TPEN down-regulated the phosphorylation of ERK in cardiomyocytes and decreased the cell survival rate. Pretreatment with MEK / ERK signal pathway inhibitor PD98059 further inhibited the phosphorylation of ERK and further decreased the cell survival rate; while S-nitroso-N -acetyl-DL-penicillamine (SNAP, activatable MEK / ERK signaling pathway) and MPG (ROS scavenger) reverses the above-mentioned inhibition of TPEN. The above results show that zinc deficiency can inhibit ERK pathway by promoting intracellular ROS production and cause cardiomyocyte injury.