Mechanism of BMAL1 attenuating H2O2-induced cardiomyocyte injury

doi:10.11958/20231027

Abstract

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

CLC Number:

R542.2

Figures/Tables 6

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^＊＊

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

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

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^＊＊

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

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

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Mechanism of BMAL1 attenuating H₂O₂-induced cardiomyocyte injury

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