目的 建立甲型H1N1流感病毒（H1N1）继发金黄色葡萄球菌Staphylococcus aureus感染模型，探讨金银花水提物（LJAE）对该模型药效及机制。方法 小鼠按体质量随机分为对照组、模型组、磷酸奥司他韦（OT，抗病毒阳性药，11 mg·kg^-1，临床等效剂量）组、头孢克洛缓释片（CF，抗菌阳性药，106.38 mg·kg^-1，临床等效剂量）组和LJAE低、中、高剂量（6.5、13.0、26.0 mL·kg^-1）组，除对照组外，建立致死型H1N1继发S.aureus模型，连续给药5 d，观察死亡率；除对照组外，建立亚致死性H1N1继发S.aureus模型，连续给药5 d，测定肺指数，通过实时荧光定量PCR（qRT-PCR）法检测肺病毒量，平板法检测肺菌量，苏木精-伊红（HE）染色观察肺损伤；瑞氏-吉姆萨染色法测定肺泡灌洗液中巨噬细胞和中性粒细胞数目；通过qRT-PCR法检测肺单核细胞趋化蛋白-1（MCP-1）、肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）、白介素-4（IL-4）、趋化因子（C-X-C基序）配体1（CXCL-1） mRNA表达量； ELISA法检测肺组织MCP-1、TNF-α、IFN-γ水平；流式细胞术测定外周血CD4⁺IFN-γ⁺（Th1）和CD4⁺IL-4⁺（Th2）细胞比例。结果 与模型组比较，LJAE显著降低致死型H1N1继发S.aureus模型小鼠的死亡率（P＜0.05、0.01、0.001），显著降低亚致死型H1N1继发S.aureus模型小鼠的肺指数（P＜0.001）、肺病毒量（P＜0.001）和肺菌量（P＜0.001），减轻肺病理损伤； LJAE显著减少肺部中性粒细胞和巨噬细胞的募集（ P＜0.001），显著降低趋化因子MCP-1和促炎细胞因子TNF-α和IFN-γ mRNA和蛋白的表达（P＜0.001），降低外周血单核细胞CD4⁺IFN-γ⁺/CD4⁺IL-4⁺（P＜0.001）。结论 LJAE具有显著的抗H1N1继发S.aureus感染的活性，能够通过调节过激的Th1型免疫反应，抑制Th1型细胞因子MCP-1、TNF-α和IFN-γ的释放，减轻肺部炎症细胞的过度浸润，修复H1N1继发S.aureus引发的肺损伤。

Objective To establish a secondary Staphylococcus aureus (S. aureus) infection model of influenza A H1N1 virus (H1N1), and explore the efficacy evaluation and research of Lonicera japonica aqueous extract (LJAE) on this model. Methods Mice were randomly divided into the control group, model group, oseltamivir phosphate (OT, antiviral positive drug, 11 mg·kg^-1, clinical equivalent dose) group, Cefaclor Sustained-Release Tablets (CF, antibacterial positive drug, 106.38 mg·kg^-1, clinical equivalent dose) group and low, medium and high dose LJAE (6.5, 13.0, 26.0 mL·kg^-1) groups according to body weight. Except for the control group, lethal H1N1 secondary S. aureus models were established, and continuous administration was given for five days to observe the mortality rate. Except for the control group, sublethal H1N1 secondary S. aureus models were established, and continuous administration was given for five days. The lung index was measured, the lung viral load was detected by real-time fluorescence quantitative PCR (qRT-PCR), the lung bacterial load was detected by plate method, and lung injury was observed by hematoxylineosin (HE) staining. The number of macrophages and neutrophils in the alveolar lavage fluid was determined by Wright-Giemsa staining. The mRNA expression levels of lung monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-4 (IL-4), and chemokine (C-X-C motif) ligand 1 (CXCL-1) were detected by qRT-PCR. The levels of MCP-1, TNF-α, and IFN-γ in lung tissue were detected by ELISA. The proportions of CD4⁺IFN-γ⁺ (Th1) and CD4⁺IL-4⁺ (Th2) cells in peripheral blood were determined by flow cytometry. Results Compared with the model group, LJAE significantly reduced the mortality rate of mice in the lethal H1N1 secondary S. aureus model (P < 0.05, 0.01, 0.001), significantly reduced the lung index (P < 0.001), lung viral load (P < 0.001), and lung bacterial load (P < 0.001) of mice in the sublethal H1N1 secondary S. aureus model, and alleviated lung injury. LJAE significantly reduced the recruitment of neutrophils and macrophages in the lungs (P < 0.001), significantly decreased the mRNA and protein expression of chemokine MCP-1 and pro-inflammatory cytokines TNF-α and IFN-γ (P < 0.001), and decreased the ratio of CD4⁺IFN-γ⁺/CD4⁺IL-4⁺ in peripheral blood mononuclear cells. Conclusion LJAE has significant anti-H1N1 secondary S. aureus activity, by inhibiting the excessive Th1 immune response, it inhibits the release of Th1 cytokines MCP-1, TNF- α, and IFN-γ, reduces excessive infiltration of lung inflammatory cells, and alleviates lung injury caused by H1N1 secondary S. aureus.

R285.5

山东省重点研发计划项目（2021CXGC010511）；校企合作项目-金银花口服液技术开发项目（KYC2022035）；2023年全国中药特色技术传承人才培训项目（T20234832005）