Possible mechanism of Chinese patent Kugan granules against influenza infection: inducing interferon I and suppressing inflammation

Background: Kugan granules (KGKL), a Chinese patent medicine, was used widely for the treatment of influenza in China and have demonstrated good efficacy. However, the underlying mechanism remains unclear. Objective: To investigate the anti-influenza efficacy of KGKL and to explore its mechanism of action. Materials and methods: Six-week-old C57BL/6J mice were divided into 3 groups: the blank group (control group), the infection group (model group), and the Kugan granules group (KGKL group). The latter 2 groups were administered either nonlethal or lethal doses of the influenza virus AH1N1 PR8. Tissue specimens, including those from the lungs and colon, were collected on days 1, 3, 7, and 14 postinfection (pi). The levels of IFN-β were measured on days 1 and 3 pi using ELISA, while the mRNA of interferon-related proteins was analyzed on day 1 pi using real-time polymerase chain reaction (PCR). The viral load in the lungs was quantified on days 3 and 7 pi using real-time PCR. On day 7 pi, the lung index and colon length were measured, and pathologic examinations of the lungs were conducted. In addition, multiple cytokines (IL-6, IL-1β, TNF-α, IFN-γ, CXCL-10, TGF-β, IL-4, and IL-10) in the lungs were assessed using ELISA. The expression levels of NLRP3 inflammasome-related proteins and tight junction proteins in the lungs were evaluated using Western blotting. Mouse survival was continuously monitored until day 14 pi. Results: The KGKL group exhibited significantly improved lung index ( P <0.01), colon length ( P <0.0001), and pathologic score ( P <0.05) when compared with the model group. The levels of multiple cytokines or chemokines (IL-6, IL-1β, TNF-α, IFN-γ, CXCL-10, and TGF-β) in lung of KGKL group were significantly reduced (IL-6, IL-1β, TNF-α, IFN-γ, CXCL-10: P <0.05; TGF-β: P <0.01), while the releases of IL-4 and IL-10 were significantly increased (IL-4: P <0.01; IL-10: P <0.05). The viral load in the lungs of the KGKL group was significantly reduced on day 3 ( P <0.05) and day 7 pi ( P <0.01). The levels of IFN-β in the lungs of the KGKL group were significantly elevated compared with the model group on day 1 pi ( P <0.05). The expression of interferon-related proteins was upregulated on day 1 pi. The expressions of NLRP3 inflammasome-related proteins (NLRP3, ASC, pro-caspase-1, and P20) were significantly reduced in lung ( P <0.05), while the expression levels of tight junction-related proteins (ZO-1, Occludin, and Claudin-5) were significantly increased on day 7 pi ( P <0.05). Conclusions: The primary mechanism of KGKL anti-influenza probably involved reducing viral load by directly inhibiting virus replication or inducing IFN-I in the early stage of the infection and suppressing excessive inflammatory response by down-regulating NLRP3-Caspase1-mediated pyroptosis in the late stage of the infection.