甘草查尔酮A通过自噬-铁死亡共调控路径减轻H3N2病毒诱导RAW264.7细胞损伤的分子机制

doi:10.5246/jcps.2026.04.024

摘要/Abstract

摘要：

本研究旨在探讨甘草查尔酮A(Licochalcone A, LA)通过调控自噬与铁死亡途径减轻H3N2流感病毒诱导巨噬细胞损伤的作用机制。建立IVA病毒感染RAW264.7巨噬细胞模型, 采用CCK-8法筛选LA最佳干预浓度(15 μM), 实验分为对照组、模型组和模型+LA干预组(IAV感染+15 μM LA)。采用透射电镜观察线粒体超微结构; Western blotting检测铁死亡相关蛋白、自噬标志蛋白及AKT/mTOR通路蛋白表达; 流式细胞术检测细胞内活性氧ROS水平; ELISA法测定炎症因子含量; 同步检测丙二醛(MDA)、游离亚铁离子(Fe²⁺)及谷胱甘肽(GSH)水平。CCK-8检测结果显示模型组细胞增殖活性显著降低, LA干预可有效恢复细胞活力(P < 0.001)。电镜结果显示模型组线粒体肿胀、嵴断裂、空泡化明显, 而LA干预组线粒体结构损伤显著改善。与对照组比较, 模型组GPX4、FTH1蛋白表达显著下调55%和58%(分别为P < 0.001和P = 0.0088), TP53表达上调26%(P = 0.011); LA干预逆转上述蛋白异常表达。模型组ROS、MDA、Fe²⁺水平显著升高, GSH降低, 经过LA干预后ROS、MDA、Fe²⁺水平分别降低29%、41%及36%, 而GSH升高42%(分别为 P < 0.001, P = 0.005, P < 0.001和P = 0.001)。模型组IL-6、IL-1β、TNF-α水平显著升高(分别为F = 116.370, P < 0.001; F = 170.659, P < 0.001; F = 37.072, P < 0.001), LA干预显著抑制炎症因子释放。模型组p-AKT1、p-mTOR及LC3-II/LC3-I表达降低(分别为P = 0.004, P < 0.001和P = 0.0097), P62表达升高(P = 0.001); LA干预显著激活AKT/mTOR通路并促进自噬流。由此可得, LA通过激活AKT/mTOR信号通路增强自噬流, 同时抑制铁死亡途径, 减轻IAV诱导的巨噬细胞线粒体损伤、氧化应激及炎症反应, 最终发挥抗流感病毒作用。

关键词: 甲型流感病毒, 自噬, 铁死亡, 甘草查尔酮A, RAW264.7巨噬细胞

Abstract:

To elucidate the mechanisms by which licochalcone A (LA) attenuates influenza A virus (IAV) H3N2–induced macrophage injury through modulation of autophagy and ferroptosis, an established IAV-infected RAW264.7 macrophage model was employed. The optimal LA concentration (15 μmol/L) was determined using a Cell Counting Kit-8 (CCK-8) assay. Cells were assigned to control, model (IAV), and model + LA (IAV + 15 μmol/L LA) groups. Mitochondrial ultrastructure was examined by transmission electron microscopy; protein expression related to ferroptosis, autophagy, and the AKT/mTOR pathway was analyzed by Western blotting analysis; intracellular ROS levels were assessed by flow cytometry; cytokine secretion was quantified by ELISA; and levels of MDA, Fe²⁺, and GSH were synchronously measured. CCK-8 assays demonstrated a marked reduction in cell proliferation in the model group, which was effectively restored following LA treatment (P < 0.001). Electron microscopy revealed pronounced mitochondrial swelling, cristae disruption, and vacuolar degeneration in IAV-infected cells, whereas LA intervention substantially alleviated these structural abnormalities. Compared with the control group, the model group exhibited significant downregulation of GPX4 and FTH1 protein expression by 55% and 58% (P < 0.001 and P = 0.0088, respectively), accompanied by a 26% increase in TP53 expression (P = 0.011); these aberrant changes were reversed by LA treatment. In addition, IAV infection resulted in significantly elevated levels of ROS, MDA, and Fe²⁺, together with a reduction in GSH content. Following LA intervention, ROS, MDA, and Fe²⁺ levels decreased by 29%, 41%, and 36%, respectively, while GSH levels increased by 42% (P < 0.001, P = 0.005, P < 0.001, and P = 0.001, respectively). The concentrations of IL-6, IL-1β, and TNF-α were markedly increased in the model group (all P < 0.001), whereas LA treatment significantly suppressed their release. Furthermore, IAV infection was associated with reduced expression of p-AKT1, p-mTOR, and the LC3-II/LC3-I ratio (P = 0.004, P < 0.001, and P = 0.0097, respectively), along with increased P62 expression (P = 0.001); these alterations were significantly reversed by LA, indicating activation of the AKT/mTOR pathway and restoration of autophagic flux. Collectively, these findings demonstrated that LA activated AKT/mTOR signaling to enhance autophagic flux while inhibiting ferroptosis, thereby alleviating IAV-induced mitochondrial damage, oxidative stress, and inflammatory responses in macrophages and ultimately exerting potent anti-influenza effects.

Key words: Influenza A virus (H3N2), Autophagy, Ferroptosis, Licochalcone A, RAW264.7 macrophages

Supporting:

黄津, 张美泉, 李声忠, 黄美珍, 林眉, 陈家卫. 甘草查尔酮A通过自噬-铁死亡共调控路径减轻H3N2病毒诱导RAW264.7细胞损伤的分子机制[J]. 中国药学(英文版), 2026, 35(4): 348-358.

Jin Huang, Meiquan Zhang, Shengzhong Li, Meizhen Huang, Mei Lin, Jiawei Chen. Licochalcone A mitigates H3N2-induced RAW264.7 cell injury via co-regulation of autophagy and ferroptosis[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(4): 348-358.

图/表 7

Figure 1. Effect of LA on the proliferative activity of RAW264.7 cells. Panels: I, Dose-response of LA (*P < 0.05 vs. 0 μmol/L group, n = 3); II, Cell viability of H3N2-infected cells. Groups: (A) Control; (B) Model; (C) Model + LA. (*P < 0.05 vs. Model group, n = 3). Data are presented as mean ± SD.

Figure 2. TEM of mitochondrial ultrastructure in RAW264.7 cells across experimental groups. Groups: (A, B, C) Control; (D, E, F) Model; (G, H, I) Model + LA. Scale bars: A/D/G = 5 μm (×3000); B/E/H = 2 μm (×8000); C/F/I = 200 nm (×20 000). Data are representative of three independent experiments (n = 3).

Figure 3. Western blotting analysis of ferroptosis-related proteins (GPX4, FTH1, and TP53) in RAW264.7 cells across experimental groups. Groups: (A) Control; (B) Model; (C) Model + LA. Data are presented as mean ± SD (n = 3). *P < 0.05 vs. Model group.

Figure 4. Flow cytometric analysis of intracellular ROS levels in RAW264.7 cells across experimental groups. Groups: (A) Control; (B) Model; (C) Model + LA. Data are presented as mean ± SD (n = 3). *P < 0.05 vs. Model group.

Table 1. Expression levels of inflammatory factors in each group (x ± s, pg/mL, n = 6).

Figure 5. Expression of autophagy-related proteins in RAW264.7 cells across experimental groups. Groups: (A) Control; (B) Model; (C) Model + LA. Data are presented as mean ± SD (n = 3). *P < 0.05 vs. Model group.

Figure 6. Comparison of MDA, Fe2+, and GSH levels among experimental groups. Groups: (A) Control; (B) Model; (C) Model + LA. Data are presented as mean ± SD (n = 6). *P < 0.05 vs. Model group.

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