Liu Shen Wan inhibits influenza virus-induced secondary Staphylococcus aureus infection in vivo and in vitro

Liu Shen Wan (LSW) is a traditional Chinese medicine (TCM) with detoxification and antiphlogistic activity; it is composed of bezoar, toad venom, musk, pearl powder, borneol and realgar. In recent years, LSW has been widely used in traditional medicine for the treatment of influenza, tonsillitis, pharyngitis, mumps, cancer and leukaemia. The anti-influenza virus properties of LSW and its inhibition of the inflammatory response was demonstrated in our previous research; however, the effect and potential mechanism of LSW against influenza induced secondary bacteria have remained obscure. Therefore, in the present study, a model of influenza virus PR8 with secondary infection by Staphylococcus aureus (S. aureus) in vitro and in mice was established to examine the effect and potential mechanism by which LSW inhibits bacterial adhesion and subsequent severe pneumonia after viral infection. We investigated the effect of LSW on the PR8-induced adhesion of live S. aureus in A549 cells. RT-qPCR was used to detect the expression of adhesion molecules. Western blotting was used to determine the expression of CEACAM1, RIG-1, MDA5, p–NF–κB, and NF-κB in A549 cells. Inflammatory cytokines were detected using a Bio-Plex Pro Human Cytokine Screening Panel (R&D) in A549 cells and Mouse Magnetic Luminex Assays (R&D) in mice infected with PR8 virus and secondarily with S. aureus, respectively. Moreover, the survival rate, lung index, viral titre, bacterial loads and pathological changes in the lung tissue of mice infected with PR8 and S. aureus were investigated to estimate the effect of LSW in inhibiting severe pneumonia. LSW significantly decreased S. aureus adhesion following influenza virus infection in A549 cells, which may have occurred by suppressing expression of the adhesion molecule CEACAM1. In addition, treatment with LSW dramatically suppressed the induction of proinflammatory cytokines (CCL2/MCP-1 and CXCL-9/MIG) and chemokines (IL-6 and TNF-α) by PR8 infection following secondary LPS stimulation in A549 cells. Upregulation of related signalling proteins (RIG-I, MDA5 and NF-κB) induced by viruses and bacteria was suppressed by LSW in A549 cells. LSW significantly decreased the viral titres and bacterial load, prolonged survival time, and ameliorated lung inflammation and injury in mice with S. aureus infection secondary to PR8 infection. We demonstrated that LSW prevents S. aureus adherence to influenza virus-infected A549 cells, perhaps by inhibiting the expression of the adhesion molecule CEACAM1. The upregulation of proinflammatory cytokines and related signalling proteins induced by viruses and bacteria was suppressed by LSW in A549 cells. LSW significantly ameliorated lung injury caused by viral and secondary bacterial infection. These findings provide a further evaluation of LSW and suggest a beneficial effect of LSW for the prevention of secondary bacterial infection and related complications.