BMAL1减轻H2O2诱导的心肌细胞损伤机制研究

doi:10.11958/20231027

天津医药 ›› 2024, Vol. 52 ›› Issue (2): 119-123.doi: 10.11958/20231027

BMAL1减轻H₂O₂诱导的心肌细胞损伤机制研究

易娜¹(), 肖雯², 田源³, 袁李礼³^,^△()

1.长沙市第四医院心内科（邮编410007）
2.湖南师范大学生命科学学院
3.湖南省脑科医院心内科

收稿日期:2023-07-20 修回日期:2023-09-01 出版日期:2024-02-15 发布日期:2024-01-26
通讯作者: ^△ E-mail：shushu622@qq.com
作者简介:易娜（1983），女，副主任医师，主要从事心肌损伤方面研究。E-mail：yina2028@163.com
基金资助:
湖南省教育厅科研项目(21B0088);湖南省卫健委科研项目(202203012960);湖南省卫健委科研项目(202103010510);湖南省自然科学基金项目(2022JJ80062);湖南省自然科学基金项目(2022JJ80101);湖南省中医药科技项目(D2022024)

Mechanism of BMAL1 attenuating H₂O₂-induced cardiomyocyte injury

YI Na¹(), XIAO Wen², TIAN Yuan³, YUAN Lili³^,^△()

1. Department of Cardiology, the Fourth Hospital of Changsha, Changsha 410007, China
2. College of Life Science, Hunan Normal University
3. Department of Cardiology, Brain Hospital of Hunan Province

Received:2023-07-20 Revised:2023-09-01 Published:2024-02-15 Online:2024-01-26
Contact: ^△ E-mail: shushu622@qq.com

易娜, 肖雯, 田源, 袁李礼. BMAL1减轻H₂O₂诱导的心肌细胞损伤机制研究[J]. 天津医药, 2024, 52(2): 119-123.

YI Na, XIAO Wen, TIAN Yuan, YUAN Lili. Mechanism of BMAL1 attenuating H₂O₂-induced cardiomyocyte injury[J]. Tianjin Medical Journal, 2024, 52(2): 119-123.

摘要/Abstract

摘要：

目的探讨脑和肌肉组织芳香烃受体核转运蛋白的类似蛋白1（BMAL1）通过核因子E2相关因子2（NRF2）调节活性氧（ROS）/NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎症小体通路对过氧化氢（H₂O₂）诱导的心肌细胞损伤的影响。方法体外培养H9c2细胞和BMAL1稳定过表达的H9c2细胞，建立H₂O₂诱导的H9c2细胞损伤模型，并将细胞分为对照（Control）组、H₂O₂组、BMAL1过表达（BMAL1-OE）组、BMAL1过表达+H₂O₂（BMAL1-OE+H₂O₂）组、BMAL1过表达+NRF2抑制剂（BMAL1-OE+ML385）组、BMAL1过表达+NRF2抑制剂+H₂O₂（BMAL1-OE+ML385+H₂O₂）组。采用CCK-8法检测细胞活力，荧光探针2'，7'-二氯荧光素二乙酸酯检测ROS生成，Western blot检测BMAL1、NRF2和NLRP3蛋白表达，酶联免疫吸附试验法检测白细胞介素（IL）-1β释放。结果与Control组相比，H₂O₂组H9c2心肌细胞活力减弱，ROS生成增多，BMAL1和NRF2蛋白表达水平降低，NLRP3蛋白表达水平升高，IL-1β释放增多（P<0.05）；与H₂O₂组相比，BMAL1-OE+H₂O₂组H9c2心肌细胞活力升高，ROS生成减少，BMAL1和NRF2蛋白表达水平升高，NLRP3蛋白表达水平降低，IL-1β释放减少（P<0.05）。与BMAL1-OE+H₂O₂组相比，BMAL1-OE+ML385+H₂O₂组H9c2心肌细胞活力减弱，ROS生成增多，NLRP3蛋白表达水平升高，IL-1β释放增多（P<0.05）。结论 BMAL1可减轻H₂O₂诱导的H9c2心肌细胞损伤，其机制可能与NRF2调节ROS/NLRP3炎症小体通路有关。

关键词: ARNTL转录因子类, NF-E2相关因子2, 活性氧, NLR家族, 热蛋白结构域包含蛋白3, 脑和肌肉组织芳香烃受体核转运蛋白的类似蛋白1, 炎症小体

Abstract:

Objective To investigate the effect of BMAL1 on H₂O₂-induced cardiomyocyte injury through NRF2-regulated ROS/NLRP3 inflammasome pathway. Methods H9c2 cells and H9c2 cells with stable over-expressed BMAL1 were cultured and divided into the control group, the H₂O₂ group, the BMAL1-OE group, the BMAL1-OE+H₂O₂ group, the BMAL1-OE+ML385 group and the BMAL1-OE+ML385+H₂O₂ group. All groups were pre-intervened with corresponding inhibitors, and then treated with 0.2 mmol/L H₂O_2,except for the control group and the BMAL1-OE group. After the intervention, CCK-8 assay was used to measure cell viability, fluorescent probe DCFH-DA was used to measure ROS generation and Western blot assay was used to detect BMAL1, NRF2 and NLRP3 protein expressions. ELISA was used to determine IL-1β release. Results Compared with the control group, the cell viability was decreased, ROS generation was increased, BMAL1 and NRF2 protein expressions were decreased, NLRP3 expression and IL-1β release were increased in the H₂O₂ group (P<0.05). Compared with the H₂O₂ group, the cell viability was increased, ROS generation was decreased, BMAL1-OE and NRF2 protein expressions were increased, NLRP3 expression and IL-1β release were decreased in the BMAL1-OE+H₂O₂ group (P<0.05). Compared with the BMAL1-OE+H₂O₂ group, the cell viability was decreased, ROS generation was increased, NLRP3 expression and IL-1β release were increased in the BMAL1-OE+ML385+H₂O₂ group (P<0.05). Conclusion BMAL1 attenuates H₂O₂-induced H9c2 cardiomyocyte injury, and its mechanism may be related to the regulation of ROS/NLRP3 inflammasome pathway through NRF2.

Key words: ARNTL transcription factors, NF-E2-related factor 2, reactive oxygen species, NLR family, pyrin domain-containing 3 protein, BMAL1, inflammasome

中图分类号:

R542.2

图/表 6

表1 实验1各组H9c2心肌细胞活力和ROS水平比较 ($\bar{x}±s$)

Tab.1 Comparison of cell viability and ROS production of H9c2 cardiomyocytes between the four groups of part one

组别	n	细胞活力	ROS/%
Control组	6	0.81±0.07	100.90±2.20
H₂O₂组	6	0.38±0.04^a	195.50±12.37^a
BMAL1-OE组	6	0.83±0.06	99.17±1.89
BMAL1-OE+H₂O₂组	6	0.64±0.05^b	143.50±6.12^b
F		79.290^＊＊	248.700^＊＊

图1 实验1各组H9c2心肌细胞NRF2和NLRP3蛋白表达 A：Control组；B：H2O2组；C：BMAL1-OE组；D：BMAL1-OE+H2O2组；a与Control组比较，b与H2O2组比较，P<0.05。

Fig.1 NRF2 and NLRP3 protein expression of H9c2 cardiomyocytes in each group of part one

图2 实验1各组H9c2心肌细胞IL-1β释放情况 A：Control组；B：H2O2组；C：BMAL1-OE组；D：BMAL1-OE+H2O2组；a与Control组比较，b与H2O2组比较，P<0.05。

Fig.2 IL-1β release of H9c2 cardiomyocytes in each group of part one

表2 实验2各组H9c2心肌细胞活力和ROS水平比较

Tab.2 Comparison of cell viability and ROS production of H9c2 cardiomyocytes between the four groups of part two

组别	n	细胞活力	ROS/%
BMAL1-OE组	6	0.91±0.06	101.80±4.98
BMAL1-OE+H₂O₂组	6	0.65±0.04^a	142.30±10.34^a
BMAL1-OE+ML385组	6	0.87±0.07	100.70±2.42
BMAL1-OE+ML385+H₂O₂组	6	0.53±0.07^b	168.90±8.64^b
F		56.170^＊＊	124.900^＊＊

图3 实验2各组H9c2心肌细胞NLRP3蛋白表达 A：BMAL1-OE组；B：BMAL1-OE+H2O2组；C：BMAL1-OE+ML385组；D：BMAL1-OE+ML385+H2O2组；a与BMAL1-OE组比较，b与BMAL1-OE+H2O2组比较，P<0.05。

Fig.3 NLRP3 protein expression of H9c2 cardiomyocytes in each group of part two

图4 实验2各组H9c2心肌细胞IL-1β释放情况 A：BMAL1-OE组；B：BMAL1-OE+H2O2组；C：BMAL1-OE+ML385组；D：BMAL1-OE+ML385+H2O2组；a与BMAL1-OE组比较，b与BMAL1-OE+H2O2组比较，P<0.05。

Fig.4 IL-1β release of H9c2 cardiomyocytes in each group of part two

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BMAL1减轻H₂O₂诱导的心肌细胞损伤机制研究

Mechanism of BMAL1 attenuating H₂O₂-induced cardiomyocyte injury

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