Influence of resveratrol on H2O2-induced ferroptosis in alveolar epithelial cells by regulating the Nrf2-GPX4 pathway

doi:10.11958/20221584

Abstract

Abstract:

Objective To explore the influence of resveratrol (RES) on H₂O₂-induced ferroptosis in A549 cells by regulating nuclear factor E2-related factor 2 (Nrf2)-glutathione peroxidase 4 (GPX4) pathway. Methods Human alveolar epithelial cells A549 were randomly grouped into the control group, the H₂O₂ group, the RES low (RES-L, 50 μmol/L), the RES medium (RES-M, 100 μmol/L) and the RES high (RES-H, 150 μmol/L) concentration groups, the ML385 (Nrf2 inhibitor, 2 μmol/L ML385) group and the RES-H+ML385 (150 μmol/L RES+2 μmol/L ML385) group. Except for the control group, the other groups were treated with H₂O₂, and then intervened with corresponding doses of drugs and inhibitors. After the intervention, MTT method was performed to determine the viability of A549 cells in each group. The kit was applied to measure intracellular iron content. ELISA method was performed to measure levels of oxidative stress in A549 cells, including malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH). Transmission electron microscopy was applied to observe mitochondrial structure. Western blot assay was performed to measure the protein expression levels of Nrf2, GPX4 and heme oxygenase 1 (HO-1) in A549 cells. Results Compared with the control group, mitochondrial morphology was seriously damaged in the H₂O₂ group, and cell viability, SOD and GSH contents, and the Nrf2, GPX4 and HO-1 protein expression were significantly decreased, and the iron ion and MDA content were significantly increased (P<0.05). Compared with the H₂O₂ group, with the increase of the RES concentration, the mitochondrial morphological damage of A549 cells was improved, the cell viability, SOD and GSH contents, and the Nrf2, GPX4 and HO-1 protein expression gradually increased, while the iron ion and MDA content gradually decreased (P<0.05). However, the mitochondrial damage of A549 cells was aggravated in the ML385 group, and cell viability, SOD and GSH contents, and the Nrf2, GPX4 and HO-1 protein expression were significantly decreased, and iron ion and MDA content were significantly increased (P<0.05). ML385 was able to reverse the protective effect of RES on H₂O₂-induced A549 cells. Conclusion RES can reduce the level of oxidative stress in A549 cells induced by H₂O₂ and inhibit ferroptosis, which may be related to the activation of Nrf2-GPX4 signaling pathway.

Key words: resveratrol, A549 cells, ferroptosis, oxidative stress, NF-E2-related factor 2, glutathione peroxidase, mitochondria

CLC Number:

R349.5

Figures/Tables 5

References 20

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组别	细胞存活率（%）	铁离子含量（μg/g）
对照组	100.00±0.00	95.12±9.51
H₂O₂组	36.27±1.88^a	238.61±23.84^a
RES-L组	51.15±2.34^b	182.36±18.23^b
RES-M组	66.29±2.61^bc	142.31±14.23^bc
RES-H组	95.82±2.87^bcd	95.72±9.57^bcd
ML385组	21.35±1.25^b	279.64±27.94^b
RES-H+ML385组	37.01±1.69^ef	236.12±23.61^ef
F	1 368.036^＊＊	86.010^＊＊

组别	细胞存活率（%）	铁离子含量（μg/g）
对照组	100.00±0.00	95.12±9.51
H₂O₂组	36.27±1.88^a	238.61±23.84^a
RES-L组	51.15±2.34^b	182.36±18.23^b
RES-M组	66.29±2.61^bc	142.31±14.23^bc
RES-H组	95.82±2.87^bcd	95.72±9.57^bcd
ML385组	21.35±1.25^b	279.64±27.94^b
RES-H+ML385组	37.01±1.69^ef	236.12±23.61^ef
F	1 368.036^＊＊	86.010^＊＊

组别	SOD（U/mg）	GSH（mmol/g）	MDA（μmol/g）
对照组	35.16±3.56	239.68±23.96	4.68±0.46
H₂O₂组	16.25±1.62^a	126.26±12.52^a	14.52±1.45^a
RES-L组	22.36±2.24^b	153.65±15.36^b	8.34±0.83^b
RES-M组	28.68±2.87^bc	182.35±18.24^bc	6.65±0.62^bc
RES-H组	35.18±3.51^bcd	233.64±23.36^bcd	4.72±0.47^bcd
ML385组	10.59±1.05^b	93.36±8.53^b	21.21±2.12^b
RES-H+ML385组	16.35±1.63^ef	127.18±12.72^ef	14.48±1.45^ef
F	89.533^＊＊	63.216^＊＊	160.027^＊＊

组别	SOD（U/mg）	GSH（mmol/g）	MDA（μmol/g）
对照组	35.16±3.56	239.68±23.96	4.68±0.46
H₂O₂组	16.25±1.62^a	126.26±12.52^a	14.52±1.45^a
RES-L组	22.36±2.24^b	153.65±15.36^b	8.34±0.83^b
RES-M组	28.68±2.87^bc	182.35±18.24^bc	6.65±0.62^bc
RES-H组	35.18±3.51^bcd	233.64±23.36^bcd	4.72±0.47^bcd
ML385组	10.59±1.05^b	93.36±8.53^b	21.21±2.12^b
RES-H+ML385组	16.35±1.63^ef	127.18±12.72^ef	14.48±1.45^ef
F	89.533^＊＊	63.216^＊＊	160.027^＊＊

组别	Nrf2	HO-1	GPX4
对照组	0.76±0.07	0.89±0.09	0.94±0.09
H₂O₂组	0.28±0.03^a	0.31±0.03^a	0.35±0.03^a
RES-L组	0.42±0.04^b	0.56±0.05^b	0.54±0.05^b
RES-M组	0.59±0.06^bc	0.72±0.07^bc	0.73±0.07^bc
RES-H组	0.73±0.07^bcd	0.87±0.08^bcd	0.92±0.09^bcd
ML385组	0.11±0.01^b	0.18±0.02^b	0.16±0.02^b
RES-H+ML385组	0.26±0.02^ef	0.28±0.03^ef	0.37±0.03^ef
F	160.317^＊＊	149.344^＊＊	147.147^＊＊

Influence of resveratrol on H₂O₂-induced ferroptosis in alveolar epithelial cells by regulating the Nrf2-GPX4 pathway

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