Liu Shen Wan inhibits influenza a virus and excessive virus-induced inflammatory response via suppression of TLR4/NF-κB signaling pathway in vitro and in vivo

Liu Shen Wan (LSW), first prescribed in "Lei Yunshang Song Fen Tang Fang", traditional Chinese medicine (TCM), is used to cure influenza, tonsillitis, pharyngitis and mumps for more than one hundred years.LSW was proved extensive pharmacological properties, for instance, anti-inflammatory, anticancer, antiviral, analgesic, antibacterial and immunomodulatory activities. Nevertheless, the mechanism of this process and the evaluation of this product is still ambiguous. Hence, the study was designed to investigate the antiviral and anti-inflammatory activities of LSW against the influenza virus in vitro and vivo.The antiviral activities of LSW were assayed in virus-infected cells and mice. To study the antiviral effects of LSW against influenza A/PR/8/34 virus (PR8), we employed CPE inhibition assay with different concentrations of LSW at different times of infection in vitro. The mice were intranasally infected with virus to induce viral pneumonia, then treated with different doses of LSW. The death protection of the mice, the lung index, virus titer and pathological changes in the lung tissue of mice were investigated to estimate the anti-virus effect of LSW. Moreover, RT-qPCR was used to determine the mRNA expression of TNF-α, IL-1β, IL-6, and IFN-γ in the A549 cells and the supernatant of lung tissues, and the concentrations of these four cytokines in serum of mice were determined with ELISA. Western blot was used to determine the expression of TLR4, p-NF-κB p65, NF-κB p65, p-IκBα and IκBα in the A549 cells and lung tissues, which are the key targets of TLR4/NF-κB pathway. Moreover, the immunohistochemical assay was used to determine the expression of the NF-κB p65 in the mice lungs.LSW could significantly inhibit influenza virus at different stages of viral replication (at the process of the pre-, post-, and co-virus infection) in vitro. And LSW (100 mg/kg and 50 mg/kg) could effectively increase the survival time of mice. The virus titres, lung index, pathological changes in the mice lungs also decreased. Moreover, LSW could significantly reduce the contents of IL-1β, TNF-α, IFN-γ and IL-6 in the infected cells and the infected-mice. In addition, LSW could significantly reduce the expression of TLR4, p-NF-κB p65, NF-κB p65 and p-IκBα, while increase the IκBα in the infected cells and in the lung of mice.LSW could significantly not only inhibit virus replication and proliferation in vitro, but also ameliorate pneumonia damage in vivo. The antiviral effect was attributed to down-regulating the expression of inflammatory cytokines induced by influenza virus via regulating the activity of TLR4/NF-кB signaling pathway.