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.

Reduning Injection  /  secondary bacterial infection following influenza  /  alveolar macrophages  /  Th1/Th2 homeostasis  /  inflammation