Zhu Zhipeng, Ling Xiaoyan, Zhou Hongmei, Zhang Caijun
Department of Anesthesiology, The Second Affiliated Hospital of Jiaxing University, Jiaxing, Zhejiang 314000, P.R. China.
Outpatient Nursing Department, The Second Affiliated Hospital of Jiaxing University, Jiaxing, Zhejiang 314000, P.R. China.
Exp Ther Med. 2021 Feb;21(2):132. doi: 10.3892/etm.2020.9564. Epub 2020 Dec 10.
Myocardial ischemia-reperfusion injury (MIRI) has been confirmed to induce endoplasmic reticulum stress (ERS) during downstream cascade reactions after the sufficient deterioration of cardiomyocyte function. However, clinically outcomes have been inconsistent with experimental findings because the mechanism has not been entirely elucidated. Dexmedetomidine (DEX), an α adrenergic receptor agonist with anti-inflammatory and organ-protective activity, has been shown to attenuate IRI in the heart. The present study aimed to determine whether DEX is able to protect injured cardiomyocytes under hypoxia/reoxygenation (H/R) conditions and evaluate the conditions under which ERS is efficiently ameliorated. The cytotoxicity of DEX in H9c2 cells was evaluated 24 h after treatment with several different concentrations of DEX. The most appropriate H/R model parameters were determined by the assessment of cell viability and injury with Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays after incubation under hypoxic conditions for 3 h and reoxygenation conditions for 3, 6, 12 and 24 h. Additionally, the aforementioned methods were used to assess cardiomyocytes cultured with various concentrations of DEX under H/R conditions. Furthermore, the degree of apoptosis and the mRNA and protein expression levels of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12 were evaluated in all groups. The addition of 1, 5 and 10 µM DEX to the cell culture significantly increased the proliferation of H9c2 cells by >80% under normal culture conditions. In the H/R model assessment, following 3 h of anoxia exposure, H9c2 cell viability decreased to 62.67% with 3 h of reoxygenation and to 36% with 6 h of reoxygenation compared with the control. The viability of H9c2 cells subjected to hypoxia for 3 h and reoxygenation for 3 h increased by 61.3% when pretreated with 1 µM DEX, and the LDH concentration in the supernatant was effectively decreased by 13.7%. H/R significantly increased the percentage of apoptotic cells, as detected by flow cytometry, and increased the expression levels of GRP78, CHOP and caspase-12, while treatment with either DEX or 4-phenylbutyric acid (4-PBA) significantly attenuated these effects. Additionally, despite the protective effect of DEX against H/R injury, 4-PBA attenuated the changes induced by DEX and H/R. In conclusion, treatment with 1 µM DEX alleviated cell injury, apoptosis and the increases in GRP78, CHOP and caspase-12 expression levels in H9c2 cells induced by 3 h of hypoxia and 3 h of reoxygenation.
心肌缺血再灌注损伤（MIRI）已被证实在心肌细胞功能充分恶化后的下游级联反应中会诱导内质网应激（ERS）。然而，临床结果与实验结果并不一致，因为其机制尚未完全阐明。右美托咪定（DEX）是一种具有抗炎和器官保护活性的α肾上腺素能受体激动剂，已被证明可减轻心脏的缺血再灌注损伤（IRI）。本研究旨在确定DEX是否能够在缺氧/复氧（H/R）条件下保护受损的心肌细胞，并评估有效改善ERS的条件。在用几种不同浓度的DEX处理24小时后，评估DEX对H9c2细胞的细胞毒性。通过在缺氧条件下孵育3小时并在复氧条件下孵育3、6、12和24小时后，使用细胞计数试剂盒-8和乳酸脱氢酶（LDH）释放试验评估细胞活力和损伤，从而确定最合适的H/R模型参数。此外，上述方法用于评估在H/R条件下用不同浓度DEX培养的心肌细胞。此外，在所有组中评估凋亡程度以及葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP）和半胱天冬酶-12的mRNA和蛋白质表达水平。在正常培养条件下，向细胞培养物中添加1、5和10μM DEX可使H9c2细胞的增殖显著增加>80%。在H/R模型评估中，缺氧暴露3小时后，与对照组相比，复氧3小时时H9c2细胞活力降至62.67%，复氧6小时时降至36%。用1μM DEX预处理3小时缺氧和3小时复氧的H9c2细胞，其活力增加了61.3%，上清液中的LDH浓度有效降低了13.7%。通过流式细胞术检测，H/R显著增加了凋亡细胞的百分比，并增加了GRP78、CHOP和半胱天冬酶-12的表达水平，而用DEX或4-苯基丁酸（4-PBA）处理可显著减轻这些影响。此外，尽管DEX对H/R损伤有保护作用，但4-PBA减弱了DEX和H/R诱导的变化。总之，用1μM DEX处理可减轻3小时缺氧和3小时复氧诱导的H9c2细胞的细胞损伤、凋亡以及GRP78、CHOP和半胱天冬酶-12表达水平的增加。
