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缺锌对心肌细胞的损伤作用及机制研究

龚莹, 徐菁蔓, 习瑾昆, 徐哲龙^*

天津医科大学基础医学院，天津 300070

摘要

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。

关键词： 缺锌; H9c2心肌细胞; 细胞损伤; ERK信号通路

分类号：R363

[Injurious effect of zinc deficiency on cardiomyocytes.] [Article in Chinese]

GONG Ying, XU Jing-Man, XI Jin-Kun, XU Zhe-Long^*

Basic Medical College, Tianjin Medical University, Tianjin 300070, China

Abstract

The aim of the present study was to investigate the effect of zinc deficiency on cardiomyocyte survival and the underlying mechanisms. Simulated zinc deficiency model was developed in H9c2 cardiac cells with zinc chelator N, N, N’, N’-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). MTT assay was used to evaluate cell viability. Morphological changes of the cells were observed by optical microscope. Lacate dehydrogenase (LDH) levels of the cells were determined with LDH assay kit. Mitochondrial membrane potential (ΔΨ) was measured with confocal microscope using JC-1 dye. Intracellular reactive oxygen species (ROS) levels were determined by DCFH-DA staining. PD98059 (an inhibitor of ERK), SNAP, which can activate ERK, and the ROS scavenger, MPG, were respectively used to investigate mechanism of signal transduction. The phosphorylation of ERK was detected by Western blot. The results showed that TPEN significantly induced the cell morphological damage and the loss of ΔΨ, increased LDH leakage, and promoted ROS generation. In the H9c2 cells, TPEN significantly inhibited ERK phosphorylation and decreased cell viability, which was potentiated by PD98059, whereas both SNAP and MPG reversed the inhibitory effects of TPEN. These data suggest that zinc deficiency leads to the injury in H9c2 cardiac cells through down-regulating ERK pathway. Increased intracellular ROS may account for the effect of zinc deficiency.

Key words: Zinc deficiency; H9c2 cardiac cells; cell injury; ERK pathway

收稿日期：2016-02-07　　录用日期：2016-07-01

通讯作者：徐哲龙　　E-mail: jinkunxi@126.com

引用本文：

龚莹, 徐菁蔓, 习瑾昆, 徐哲龙. 缺锌对心肌细胞的损伤作用及机制研究[J]. 生理学报 2016; 68 (5): 677-683.

GONG Ying, XU Jing-Man, XI Jin-Kun, XU Zhe-Long. [Injurious effect of zinc deficiency on cardiomyocytes.] [Article in Chinese]. Acta Physiol Sin 2016; 68 (5): 677-683 (in Chinese with English abstract).