白藜芦醇调控Nrf2-GPX4通路对H2O2诱导肺泡上皮细胞铁死亡的影响

doi:10.11958/20221584

天津医药 ›› 2023, Vol. 51 ›› Issue (6): 568-572.doi: 10.11958/20221584

白藜芦醇调控Nrf2-GPX4通路对H₂O₂诱导肺泡上皮细胞铁死亡的影响

李艳萍¹(), 王协涛¹, 史立彬², 刘琼³

1 石家庄市急救中心院前急救科（邮编050011）
2 石家庄平安医院急诊科
3 石家庄市急救中心医学教研室

收稿日期:2022-09-27 修回日期:2022-11-10 出版日期:2023-06-15 发布日期:2023-06-20
作者简介:李艳萍（1977），女，主治医师，主要从事急救相关方面研究。E-mail：pingping_guniang77@163.com
基金资助:
河北省2020年度医学科学研究课题(20200144)

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

LI Yanping¹(), WANG Xietao¹, SHI Libin², LIU Qiong³

1 Department of Pre Hospital First Aid, Shijiazhuang Emergency Center, Shijiazhuang 050011, China
2 Department of Emergency, Shijiazhuang Ping'an Hospital
3 Medical Teaching and Research Office of Shijiazhuang Emergency Center

Received:2022-09-27 Revised:2022-11-10 Published:2023-06-15 Online:2023-06-20

李艳萍, 王协涛, 史立彬, 刘琼. 白藜芦醇调控Nrf2-GPX4通路对H₂O₂诱导肺泡上皮细胞铁死亡的影响[J]. 天津医药, 2023, 51(6): 568-572.

LI Yanping, WANG Xietao, SHI Libin, LIU Qiong. Influence of resveratrol on H₂O₂-induced ferroptosis in alveolar epithelial cells by regulating the Nrf2-GPX4 pathway[J]. Tianjin Medical Journal, 2023, 51(6): 568-572.

摘要/Abstract

摘要：

目的探索白藜芦醇（RES）调控核因子E2相关因子2（Nrf2）-谷胱甘肽过氧化物酶4（GPX4）通路对H₂O₂诱导A549细胞铁死亡的影响。方法将人肺泡上皮A549细胞随机分为对照组、H₂O₂组、RES低浓度（RES-L，50 μmol/L）组、RES中浓度（RES-M，100 μmol/L）组、RES高浓度（RES-H，150 μmol/L）组、ML385（Nrf2抑制剂，2 μmol/L ML385）组、RES-H+ML385（150 μmol/L RES+2 μmol/L ML385）组。除对照组外，其余各组均经H₂O₂处理细胞后，再使用相应剂量的药物或抑制剂进行干预。干预结束后，MTT法测定各组细胞活力，试剂盒检测细胞内铁离子含量，酶联免疫吸附试验（ELISA）检测丙二醛（MDA）、超氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）水平；透射电子显微镜观察线粒体结构；Western blot法检测Nrf2、GPX4、血红素加氧酶1（HO-1）蛋白表达水平。结果与对照组相比，H₂O₂组线粒体形态较受损严重，细胞存活率、SOD、GSH含量以及Nrf2、GPX4、HO-1蛋白表达均降低，铁离子、MDA含量增加（P<0.05）；与H₂O₂组相比，随着RES浓度的增加，A549细胞线粒体形态损伤得到改善，细胞存活率、SOD、GSH含量以及Nrf2、GPX4、HO-1蛋白表达逐渐增加，铁离子、MDA含量逐渐下降（P<0.05），但ML385组细胞线粒体损伤加重，细胞存活率、SOD、GSH含量以及Nrf2、GPX4、HO-1蛋白表达均降低，铁离子、MDA含量增加（P<0.05），且ML385处理可逆转RES对H₂O₂诱导A549细胞的保护作用。结论 RES可能通过激活Nrf2-GPX4信号通路降低H₂O₂诱导的A549细胞内氧化应激水平，抑制铁死亡。

关键词: 白藜芦醇, A549细胞, 铁死亡, 氧化性应激, NF-E2相关因子2, 谷胱甘肽过氧化酶, 线粒体

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

中图分类号:

R349.5

图/表 5

参考文献 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^＊＊

白藜芦醇调控Nrf2-GPX4通路对H₂O₂诱导肺泡上皮细胞铁死亡的影响

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

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