天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (8): 791-795.doi: 10.11958/20210463

• 细胞与分子生物学 • 上一篇    下一篇

BMAL1对H2O2诱导的H9C2心肌细胞损伤的影响及机制探讨

易娜 1，袁李礼 2△   

  1. 1长沙市第四医院心内科（邮编410007）；2湖南省脑科医院心内科
  • 收稿日期:2021-02-25 修回日期:2021-04-09 出版日期:2021-08-15 发布日期:2021-08-19
  • 通讯作者: 袁李礼 E-mail:shushu622@qq.com
  • 作者简介:易娜（1984），女，硕士，主治医师，主要从事心肌损伤方面研究。E-mail：yina2028@163.com
  • 基金资助:
    湖南省自然科学基金资助项目（2018JJ3285）；湖南省卫生计生委科研计划课题项目（202103010510，B20180079）

Effects and mechanism of BMAL1 on H2O2-induced H9C2 cardiomyocyte injury

YI Na1, YUAN Li-li2△   

  1. 1 Department of Cardiology, the Fourth Hospital of Changsha, Changsha 410007, China; 2 Department of Cardiology,
    Brain Hospital of Hunan Province
    △Corresponding Author E-mail: shushu622@qq.com
  • Received:2021-02-25 Revised:2021-04-09 Published:2021-08-15 Online:2021-08-19

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摘要： 目的 探讨节律基因BMAL1对过氧化氢（H2O2）诱导的H9C2心肌细胞损伤的影响及机制。方法 构建 BMAL1稳定过表达的H9C2细胞系后，实验分为2个部分。（1）实验1。对照组、H2O2组（0.2 mmol/L的H2O2处理24 h）、 BMAL1 过表达（BMAL1-OE）组（BMAL1 稳定过表达 H9C2 细胞）、BMAL1 过表达+H2O2（BMAL1-OE+H2O2）组 （0.2 mmol/L的H2O2处理BMAL1稳定过表达H9C2细胞24 h）。（2）实验2。H2O2组、BMAL1-OE+H2O2组、BMAL1过表 达+抑制 NRF2+H2O2（BMAL1-OE+ML385+H2O2）组（NRF2 抑制剂 2 μmol/L 的 ML385 预处理 BMAL1 稳定过表达 H9C2 细胞 24 h，再用 0.2 mmol/L 的 H2O2处理 24 h）、BMAL1 过表达+抑制 HO-1+H2O2（BMAL1-OE+Znpp+H2O2）组 （HO-1抑制剂5 μmol/L的Znpp预处理BMAL1稳定过表达H9C2细胞24 h，再用0.2 mmol/L的H2O2处理24 h）。CCK-8 法检测细胞活力，羟胺法检测超氧化物歧化酶（SOD）活性、TBA 法检测丙二醛（MDA）含量，Western blot 法检测 BMAL1、核因子 E2 相关因子 2（NRF2）和血红素加氧酶-1（HO-1）蛋白表达水平。结果 （1）与 Control 组相比， BMAL1-OE组BMAL1 mRNA表达水平升高，H2O2组细胞活力和细胞上清液SOD活性下降、MDA含量增加，BMAL1、 NRF2和HO-1蛋白相对表达水平降低（P＜0.05）；与H2O2组相比，BMAL1-OE+H2O2组细胞活力和细胞上清液SOD活 性增加，MDA 含量减少，而 BMAL1、NRF2 和 HO-1 蛋白相对表达水平升高（P＜0.05）；（2）与 BMAL1-OE+H2O2组相 比，BMAL1-OE+ML385+H2O2组和BMAL1-OE+Znpp+H2O2组细胞活力和细胞上清液SOD活性降低，MDA含量增加 （P＜0.05）。结论 BMAL1可能通过上调NRF2/HO-1信号轴，减轻H2O2诱导的大鼠心肌细胞氧化应激损伤。

关键词: 肌细胞, 心脏, 过氧化氢, 氧化性应激, ARNTL转录因子类, NF-E2相关因子2, 血红素加氧酶-1, 节律基 因BMAL1

Abstract: Objective To investigate the effects and mechanism of BMAL1 on H2O2-induced H9C2 cardiomyocyte injury. Methods BMAL1 stable overexpression H9C2 cell lines were established. The experiments were divided into two parts. Part one：cells were divided into control group, H2O2 group (0.2 mmol/L H2O2 treatment for 24 h), BMAL1 overexpression group (BMAL1 stable overexpression cells, BMAL1-OE) and BMAL1 overexpression+H2O2 group (BMAL1 stable overexpression cells + 0.2 mmol/L H2O2 treatment for 24 h, BMAL1-OE+H2O2). Part two: there were H2O2 group, BMAL1-OE+H 2O2 group, BMAL1 overexpression+NRF2 inhibition+H2O2 group (NRF2 inhibitor 2 μmol/L ML385 pretreatment BMAL1 stable overexpression cells for 24 h, and 0.2 mmol/L H2O2 treatment for 24 h, BMAL1-OE+ML385+H2O2) and BMAL1-OE+HO-1 inhibition+H 2O2 group (HO-1 inhibitor 5 μmol/L Znpp pre-treatment BMAL1 stable overexpression cells for 24 h, and 0.2 mmol/L H2O2 treatment for 24 h, BMAL1-OE+Znpp+H2O2). CCK-8 was used to detect cell viability. Hydroxylamine method was used to detect SOD activity. TBA method was used to detect MDA content. Western blot assay was used to determine the protein expression of BMAL1, NRF2 and HO-1. Results (1) Experiment one. Compared with control group, the BMAL1 mRNA expression was increased in the BMAL1-OE group, the cell viability and the SOD activity of cell supernatant were decreased in H2O2 group, the MDA content was increased, and the expression levels of BMAL1, NRF2 and HO-1 protein were decreased (P＜0.05). Compared with H2O2 group, the cell viability and the SOD activity of cell supernatant were increased in BMAL1-OE+H2O2 group, the MDA content was decreased and the expression levels of BMAL1, NRF2 and HO-1 protein were increased (P＜0.05). (2) Experiment two. Compared with BMAL1-OE+H2O2 group, the cell viabilities and the SOD activity of cell supernatant were both decreased in BMAL1-OE+ML385+H2O2 group and BMAL1-OE+Znpp+H2O2 group, the MDA contents were increased (P＜0.05). Conclusion BMAL1 may up-regulate the NRF2/HO-1 signal axis, thereby reducing H2O2-induced cardiomyocyte oxidative stress injury in rat cardiomyocytes.

Key words: myocytes, cardiac, hydrogen peroxide, oxidative stress, ARNTL transcription factors, NF-E2-related factor 2, heme oxygenase-1, rhythm gene BMAL1

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