Du Zhong-Jun, Cui Guan-Qun, Zhang Juan, Liu Xiao-Mei, Zhang Zhi-Hu, Jia Qiang, Ng Jack C, Peng Cheng, Bo Cun-Xiang, Shao Hua
Department of Toxicology, Shandong Academy of Occupational Health and Occupational Medicine, Shandong Academy of Medical Sciences.
Department of Respiratory Medicine, Qilu Children's Hospital of Shandong University, Jinan.
Int J Nanomedicine. 2017 Mar 20;12:2179-2188. doi: 10.2147/IJN.S127904. eCollection 2017.
Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological effects.
心肌细胞间的缝隙连接细胞间通讯（GJIC）对于心脏同步收缩至关重要，且依赖于含连接蛋白的通道。连接蛋白43（Cx43）是心脏组织中参与GJIC的主要成分，其异常表达与多种心脏疾病密切相关。已知二氧化硅纳米颗粒（SNPs）会诱发心血管毒性。然而，GJIC在SNPs诱导的心肌细胞凋亡中发挥作用的机制仍不清楚。本研究的目的是使用CCK-8试剂盒、刮擦加载染料转移技术、膜联蛋白V/碘化丙啶双染色检测以及蛋白质印迹分析，确定SNPs降低的GJIC是否通过线粒体途径促进大鼠心肌细胞系（H9c2细胞）凋亡。结果表明，SNPs以时间和浓度依赖性方式在H9c2细胞中引发细胞毒性。SNPs还通过下调Cx43和上调磷酸化Cx43（P-Cx43）以浓度依赖性方式降低H9c2细胞中的GJIC。缝隙连接阻滞剂甘珀酸钠抑制缝隙连接导致细胞存活率降低和凋亡增加，而视黄酸增强缝隙连接则导致细胞存活率提高但凋亡减少。此外，SNPs通过破坏功能性缝隙连接诱导的凋亡与线粒体途径相关凋亡所涉及的蛋白质如Bcl-2/Bax、细胞色素C、半胱天冬酶-9和半胱天冬酶-3的异常表达相关。综上所述，我们的结果首次证明SNPs降低的GJIC通过线粒体途径促进心肌细胞凋亡。此外，SNPs在心肌细胞中下调GJIC是通过下调Cx43和上调P-Cx43介导的。这些结果表明，在大鼠心肌细胞系中，GJIC在SNPs诱导的凋亡中起保护作用，并且GJIC可能是SNPs诱导生物学效应的靶点之一。
