Zhang Ying, Zhao Qihong, Li Xiaohong, Ji Fuhai
Department of Anesthesiology, The First Affiliated Hospital of Soochow University, No. 899 Pinghai Road, Suzhou, 215006, Jiangsu Province, China.
Department of Anesthesiology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, 233004, China.
Mol Cell Biochem. 2021 Jul;476(7):2803-2812. doi: 10.1007/s11010-021-04102-8. Epub 2021 Mar 16.
We aimed to investigate the protective role and mechanism of dexmedetomidine (DEX) on H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R) injury. Six experimental groups were designed as follows: normal control group (group C), H/R group, H/R + DEX group, H/R + gastrodin group, H/R + Ex527 (SIRT1 inhibitor) group, and H/R + DEX + Ex527 group. Lactate dehydrogenase (LDH) activity and the levels of oxidative stress-related enzymes such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were measured using corresponding commercial kits. Cell counting kit (CCK)-8 assay was used to detect cell survival rate while flow cytometry and caspase 3/7 activity were used to determine cell apoptosis, respectively. Western blot was used to detect the expression of silent information regulator 1 (SIRT1), C/EBP homologous protein (CHOP), cleaved-caspase-12/3 and pro-caspase-12/3 in each group. From our findings, when compared with H/R, H/R + Ex527 and H/R + DEX + Ex527 groups, DEX pretreatment of cells in H/R + DEX group significantly increased cell survival rate, and simultaneously reduced LDH activity, oxidative stress and the apoptosis rate of H9c2 cells with H/R injury. Moreover, DEX up-regulated SIRT1 expression level and down-regulated the levels of endoplasmic reticulum (ER) stress-related markers such as CHOP, cleaved-caspase-12 and cleaved-caspase-3, respectively. Ex527 could completely block DEX-induced upregulated expression of SIRT1, and partially blocked the DEX-induced downregulated expression levels of CHOP, cleaved-caspase-12 and cleaved-caspase-3. These results proved that DEX reversed H/R injury-induced oxidative stress and ER stress-dependent apoptosis of cardiomyocytes via SIRT1/CHOP signaling pathway.
我们旨在研究右美托咪定(DEX)对缺氧/复氧(H/R)损伤后H9c2心肌细胞的保护作用及机制。设计了六个实验组,如下:正常对照组(C组)、H/R组、H/R + DEX组、H/R + 天麻素组、H/R + Ex527(SIRT1抑制剂)组和H/R + DEX + Ex527组。使用相应的商业试剂盒测量乳酸脱氢酶(LDH)活性以及氧化应激相关酶如丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽(GSH)的水平。细胞计数试剂盒(CCK)-8法用于检测细胞存活率,而流式细胞术和半胱天冬酶3/7活性分别用于测定细胞凋亡。蛋白质免疫印迹法用于检测每组中沉默信息调节因子1(SIRT1)、C/EBP同源蛋白(CHOP)、裂解的半胱天冬酶-12/3和前半胱天冬酶-12/3的表达。根据我们的研究结果,与H/R组、H/R + Ex527组和H/R + DEX + Ex527组相比,H/R + DEX组中对细胞进行DEX预处理可显著提高细胞存活率,同时降低H/R损伤的H9c2细胞的LDH活性、氧化应激和凋亡率。此外,DEX分别上调SIRT1表达水平并下调内质网(ER)应激相关标志物如CHOP、裂解的半胱天冬酶-12和裂解的半胱天冬酶-3的水平。Ex527可完全阻断DEX诱导的SIRT1表达上调,并部分阻断DEX诱导的CHOP、裂解的半胱天冬酶-12和裂解的半胱天冬酶-3表达下调。这些结果证明,DEX通过SIRT1/CHOP信号通路逆转H/R损伤诱导的心肌细胞氧化应激和内质网应激依赖性凋亡。