目的 从抑制肺泡巨噬细胞（alveolar macrophages，AMs）耗竭角度探究热毒宁注射液抗甲型H1N1流感病毒继发金黄色葡萄球菌Staphylococcus aureus感染的药效机制。方法 建立H1N1继发S. aureus致死性和亚致死性感染模型，给予药物干预后，检测小鼠死亡率、体质量变化率、肺指数；qRT-PCR法检测肺病毒量，平板菌落计数法检测肺菌量；qRT-PCR和ELISA法检测肺组织γ干扰素（interferon-γ，IFN-γ）、CXC趋化因子配体-1（C-X-C motif chemokine ligand-1，CXCL-1）、单核细胞趋化蛋白-1（monocyte chemoattractant protein-1，MCP-1）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）表达；ELISA法检测支气管肺泡灌洗液（bronchoalveolar lavage fluid，BALF）中髓过氧化物酶-DNA复合物（myeloperoxidase-DNA，MPO-DNA）和中性粒细胞弹性蛋白酶-DNA复合物（neutrophil elastase-DNA，NE-DNA）水平；流式细胞术检测肺单细胞悬液中AMs、中性粒细胞和单核细胞比例，检测外周血1型辅助性T细胞（T helper 1，Th1）/2型辅助性T细胞（T helper 2，Th2）细胞比例。结果 与继发感染组相比，高剂量的热毒宁注射液可降低小鼠死亡率、抑制体质量下降、降低肺指数（P＜0.01、0.001）；热毒宁注射液高、低剂量组均可显著降低肺病毒量和肺菌量（P＜0.05、0.001）；且高剂量的热毒宁注射液在抑制体质量下降、降低肺指数和肺病毒量方面优于头孢克洛（P＜0.05、0.01）。热毒宁注射液高、低剂量组肺组织IFN-γ、CXCL-1水平显著降低，AMs比例显著升高（P＜0.05、0.01、0.001），热毒宁注射液高剂量组MCP-1水平显著降低（P＜0.05、0.01）；且热毒宁注射液高剂量组AMs比例高于磷酸奥司他韦组和头孢克洛组（P＜0.05、0.001），CXCL-1和MCP-1水平低于头孢克洛组（P＜0.05、0.01）。热毒宁注射液高、低剂量组中性粒细胞和单核细胞浸润均显著减少（P＜0.001），且热毒宁注射液高剂量组均优于磷酸奥司他韦组和头孢克洛组（P＜0.05、0.001）。热毒宁注射液高剂量组TNF-α水平显著下降（P＜0.01），热毒宁注射液高、低剂量组MPO-DNA和NE-DNA水平显著下降（P＜0.05、0.001）；且热毒宁注射液高剂量组TNF-α和NE-DNA水平低于头孢克洛组（P＜0.01）。热毒宁注射液高、低剂量组Th1/Th2比值显著下降（P＜0.01、0.001），且热毒宁注射液高剂量组低于头孢克洛组（P＜0.05）。结论 热毒宁注射液具有显著的抗H1N1继发S. aureus感染活性，通过修正过激的Th1型免疫极化，减少IFN-γ表达，恢复AMs比例，减少效应细胞浸润，减轻了肺部炎症损伤。

Objective To investigate the mechanism by which Reduning Injection (热毒宁注射液, RDN) against secondary Staphylococcus aureus infection following H1N1 influenza A virus infection by inhibiting alveolar macrophages (AMs) depletion. Methods Lethal and sub lethal infection model of H1N1 secondary S. aureus were established and drugs were given for intervention, mortality, body weight change rate and lung index of mice were determined. Lung viral load was detected by qRT-PCR and lung bacterial load was detected using plate colony counting. The expressions of interferon-γ (IFN-γ), C-X-C motif chemokine ligand-1 (CXCL-1), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in lung tissue were analyzed by qRT-PCR and ELISA, levels of myeloperoxidase-DNA (MPO-DNA) and neutrophil elastase-DNA (NE-DNA) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. Flow cytometry was used to assess the proportions of AMs, neutrophils and monocytes in lung single-cell suspensions and the proportion of T helper 1(Th1)/T helper 2 (Th2) cells in peripheral blood. Results Compared with the secondary infection group, high-dose of RDN reduced mortality and inhibited body weight loss and lung index (P < 0.01, 0.001). Both high-and low-dose of RDN significantly reduced lung virus and bacteria levels (P < 0.05, 0.001). High-dose of RDN were superior to cefuroxime in inhibiting weight loss, reducing the lung index and decreasing lung virus level (P < 0.05, 0.01). RDN high-and low-dose groups significantly reduced the levels of IFN-γ and CXCL-1 in lung tissue and significantly increased the proportion of AMs (P < 0.05, 0.01, 0.001). RDN high-dose group significantly reduced MCP-1 level (P < 0.05, 0.01); The proportion of AMs in RDN high-dose group was higher than that of oseltamivir phosphate group and cefaclor group (P < 0.05, 0.001), while the levels of CXCL-1 and MCP-1 were lower than those of cefaclor group (P < 0.05, 0.01). The infiltration of neutrophils and monocytes in RDN high-and low-dose groups was significantly reduced (P < 0.001), and RDN high-dose group was superior to oseltamivir phosphate group and cefaclor group (P < 0.05, 0.001). The level of TNF-α was significantly decreased in RDN high-dose group (P < 0.01), while the levels of MPO-DNA and NE-DNA were significantly decreased in RDN high-and low-dose groups (P < 0.05, 0.001); The levels of TNF-α and NE-DNA in RDN high-dose group were lower than those in cefaclor group (P < 0.01). The Th1/Th2 ratio was significantly decreased in RDN high-and low-dose groups (P < 0.01, 0.001), and RDN high-dose group was lower than cefaclor group (P < 0.05). Conclusion RDN shows significant activity against secondary S. aureus infection following H1N1 infection. RDN alleviates lung inflammatory damage by correcting excessive Th1-type immune polarization, reducing IFN-γ expression, restoring the AMs ratio and reducing effector cell infiltration.

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国家自然科学基金青年基金资助项目（81904103）；山东省自然基金青年基金资助项目（ZR2020QH328）；国家级大学生创新创业训练计划（202310440211）；国家级大学生创新创业训练计划（202510440001）