5-羟基-6，7-二甲氧基黄酮抑制流感病毒诱导A549细胞炎症反应和铁死亡的作用及机制

doi:10.12122/j.issn.1673-4254.2024.06.07

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1070-1078.doi: 10.12122/j.issn.1673-4254.2024.06.07

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5-羟基-6，7-二甲氧基黄酮抑制流感病毒诱导A549细胞炎症反应和铁死亡的作用及机制

任智先¹(), 周倍贤², 王林鑫³, 李菁⁴, 张荣平¹(), 潘锡平³()

^1.云南中医药大学中药学院暨云南省南药可持续利用研究重点实验室，云南昆明 650500
^2.高州市人民医院，广东高州 525200
^3.广州实验室，广东广州 510000
^4.广州医科大学附属第一医院，广东广州 510120

收稿日期:2023-12-22 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 张荣平,潘锡平 E-mail:rzxbgyx@163.com;zrpkm@163.com;xppan116@sina.com
作者简介:任智先，在读硕士研究生，E-mail: rzxbgyx@163.com
基金资助:
国家自然科学基金(82004155);广州医科大学科研提升计划项目(2024SRP066)

Inhibitory effect of 5-hydroxy-6,7-dimethoxyflavone on H1N1 influenza virus-induced ferroptosis and inflammation in A549 cells and its possible mechanisms

Zhixian REN¹(), Beixian ZHOU², Linxin WANG³, Jing LI⁴, Rongping ZHANG¹(), Xiping PAN³()

^1.School of Chinese Materia Medica and Yunnan Key Laboratory of Southern Medicine Utilization, Yunnan University of Chinese Medicine, Kunming 650500, China
^2.Gaozhou People's Hospital, Gaozhou 525200, China
^3.Guangzhou Laboratory, Guangzhou 510000, China
^4.The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China

Received:2023-12-22 Online:2024-06-20 Published:2024-07-01
Contact: Rongping ZHANG, Xiping PAN E-mail:rzxbgyx@163.com;zrpkm@163.com;xppan116@sina.com
Supported by:
National Natural Science Foundation of China(82004155)

摘要/Abstract

摘要：

目的研究四方蒿中提取出的5-羟基-6，7-二甲氧基黄酮（5-HDF）对H1N1流感病毒引起的肺损伤的保护作用及潜在机制。方法使用H1N1流感病毒感染A549细胞制作流感病毒感染模型，检测5-HDF的细胞毒性及其对感染病毒后的炎症和铁死亡相关指标及相关信号通路蛋白的影响。采用乙醇回流提取和硅胶色谱法从四方蒿中提取分离获得5-HDF，并采用NMR和MS鉴定其结构。采用MTT法检测不同浓度的5-HDF对A549细胞的毒性；H1N1流感病毒以0.1的感染复数感染A549细胞；采用流式细胞术检测5-HDF对感染H1N1流感病毒后A549细胞中TRAIL和IL-8表达水平的影响；Western blotting检测phospho-p38 MAPK（Thr180/Tyr182）、phospho-NF-κB p65（Ser536）、cleaved caspase3、cleaved PARP、SLC7A11、GPX4等炎症、细胞凋亡和铁死亡相关蛋白表达的水平。通过鼻腔接种50 μL半数致死量（LD₅₀）的H1N1流感病毒液复制小鼠H1N1流感病毒感染模型，以体质量变化率、肺解剖结果、肺组织病理形态变化为指标，考察5-HDF（30 mg/kg、60 mg/kg）体内抗H1N1流感病毒的作用。结果 MTT结果显示5-HDF在0~200 μg/mL对A549细胞没有明显的细胞毒性（P>0.05）。流式细胞术和Western blotting结果显示，5-HDF能抑制H1N1流感病毒感染的A549细胞中phospho-NF-κB p65和phospho-p38 MAPK的活化，降低促炎因子IL-8的表达，且5-HDF能上调SLC7A11和GPX4等抗铁死亡相关蛋白表达的水平，并抑制凋亡标志物cleaved caspase3和cleaved PARP及凋亡因子TRAIL的表达（P<0.05）。5-HDF灌胃给药7 d，可提高H1N1流感病毒感染导致的小鼠体质量降低，降低因感染H1N1流感病毒而异常升高的肺指数，并减轻其肺组织病变程度（P<0.05）。结论本研究推测5-HDF具有一定的抗小鼠H1N1流感病毒感染作用，可能通过增加SLC7A11和GPX4的表达并抑制phospho-NF-κB p65和phospho-p38 MAPK的活化，降低cleaved caspase3和cleaved PARP的表达，来减弱流感病毒H1N1感染A549细胞所引起的铁死亡、炎症反应和细胞凋亡。

关键词: 四方蒿, 5-羟基-6，7-二甲氧基黄酮, 铁死亡, 抗炎, 流感病毒

Abstract:

Objective To investigate the protective effect of 5-hydroxy-6,7-dimethoxyflavone (5-HDF), a compound extracted from Elsholtzia blanda Benth., against lung injury induced by H1N1 influenza virus and explore its possible mechanism of action. Methods 5-HDF was extracted from Elsholtzia blanda Benth. using ethanol reflux extraction and silica gel chromatography and characterized using NMR and MS analyses. In an A549 cell model of H1N1 influenza virus infection (MOI=0.1), the cytotoxicity of 5-HDF was assessed using MTT assay, and its effect on TRAIL and IL-8 expressions was examined using flow cytometry; Western blotting was used to detect the expression levels of inflammatory, apoptosis, and ferroptosis-related proteins. In a mouse model of H1N1 influenza virus infection established by nasal instillation of 50 μL H1N1 virus at the median lethal dose, the effects of 30 and 60 mg/kg 5-HDF by gavage on body weight, lung index, gross lung anatomy and lung histopathology were observed. Results 5-HDF exhibited no significant cytotoxicity in A549 cells within the concentration range of 0-200 μg/mL. In H1N1-infected A549 cells, treatment with 5-HDF effectively inhibited the activation of phospho-p38 MAPK and phospho-NF‑κB p65, lowered the expressions of IL-8, enhanced the expression of anti-ferroptosis proteins (SLC7A11 and GPX4), and inhibited the expressions of apoptosis markers PARP and caspase-3 and the apoptotic factor TRAIL. In H1N1-infected mice, treatment with 5-HDF for 7 days significantly suppressed body weight loss and increment of lung index and obviously alleviated lung tissue pathologies. Conclusion 5-HDF offers protection against H1N1 influenza virus infection in mice possibly by suppressing H1N1-induced ferroptosis, inflammatory responses, and apoptosis via upregulating SLC7A11 and GPX4, inhibiting the activation of phospho-NF‑κB p65 and phospho-p38 MAPK, and decreasing the expression of cleaved caspase3 and cleaved PARP.

Key words: Elsholtzia blanda Benth., 5-hydroxy-6,7-dimethoxyflavone, ferroptosis, anti-inflammation, influenza virus

任智先, 周倍贤, 王林鑫, 李菁, 张荣平, 潘锡平. 5-羟基-6，7-二甲氧基黄酮抑制流感病毒诱导A549细胞炎症反应和铁死亡的作用及机制[J]. 南方医科大学学报, 2024, 44(6): 1070-1078.

Zhixian REN, Beixian ZHOU, Linxin WANG, Jing LI, Rongping ZHANG, Xiping PAN. Inhibitory effect of 5-hydroxy-6,7-dimethoxyflavone on H1N1 influenza virus-induced ferroptosis and inflammation in A549 cells and its possible mechanisms[J]. Journal of Southern Medical University, 2024, 44(6): 1070-1078.

图/表 4

图2 MTT法检测5-HDF对A549细胞的毒性及5-HDF的IC50值

Fig.2 MTT assay for assessing cytotoxicity of 5-HDF to A549 cells (A) and determining its IC50 (B). ***P<0.001 vs 5-HDF (0 μg/mL).

图3 5-HDF对H1N1流感病毒感染小鼠的体质量变化率、肺指数、肺解剖结构、肺组织病理形态及肺损伤评分的影响

Fig.3 Effects of 5-HDF on body weight change (A), lung index (B), gross anatomy of the lung (C), lung histopathology (D; HE staining) and lung injury score (E) in H1N1 virus-infected mice. Black arrows: Alveolitis; Red arrows: Lung vasculitis; Blue arrows: Inflammation around the bronchioles; Orange arrows: Bronchiolar epithelial sloughing. ###P<0.001 vs control; **P<0.01, ***P<0.001 vs H1N1.

图4 不同浓度的5-HDF处理24 h后A549细胞内P-p38 MAPK(Thr180/Tyr182)、P-NF-κB-p65(Ser536)蛋白水平和IL-8炎症因子水平变化

Fig.4 Effect of 5-HDF treatment for 24 h on expressions of P-p38 MAPK (Thr180/Tyr182), P-NF-κB-p65 (Ser536) and IL-8 inflammatory factors in H1N1-infected A549 cells. A-C: Expression of P-p38 MAPK and P-NF-κB-p65 detected by Western blotting. D, E: Expression of IL-8 detected by flow cytometry. ##P<0.01, ###P<0.001 vs control; *P<0.05, **P<0.01, ***P<0.001 vs H1N1.

图5 不同浓度的5-HDF处理24 h后A549细胞内cleaved caspase3、cleaved PARP、SLC7A11、GPX4蛋白水平和TRAIL凋亡因子水平变化

Fig.5 Effect of 5-HDF treatment for 24 h on expression of cleaved caspase3, cleaved PARP, SLC7A11, GPX4 and TRAIL in H1N1-infected A549 cells. A, B: Expression of cleaved caspase3 and cleaved PARP detected by Western blotting. C, D: Expression of SLC7A11 and GPX4 detected by Western blotting. E, F: Expression of TRAIL detected by flow cytometry. #P<0.05, ##P<0.01, ###P<0.001 vs control; *P<0.05, **P<0.01, ***P<0.001 vs H1N1.

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5-羟基-6，7-二甲氧基黄酮抑制流感病毒诱导A549细胞炎症反应和铁死亡的作用及机制

Inhibitory effect of 5-hydroxy-6,7-dimethoxyflavone on H1N1 influenza virus-induced ferroptosis and inflammation in A549 cells and its possible mechanisms

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