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

doi:10.12122/j.issn.1673-4254.2024.06.07

Abstract

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

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.

Figures/Tables 4

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).

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.

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.

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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