Exploring The Anti-inflammatory Mechanism Of LSW Based On NLRP3 Inflammasome Signaling Pathway Against Influenza A

BackgroundSevere influenza virus infections cause inflammatory cytokine storm,which leads to lung injury and consequently high mortality.In contrast,NLRP3 inflammasome plays an important role in influenza infection.Influenza virus can activate NLRP3inflammasome and promote pyroptosis,which in turn amplifies the inflammatory response.Liu Shen Wan（LSW）is a classical prescription of Chinese medicine with more than 100 years of history in Warm Disease,which has the efficacy of clearing heat and detoxifying,reducing swelling and relieving pain.The preliminary studies found that LSW was able to inhibit the excessive inflammatory response induced by influenza virus and various novel coronavirus epidemic strains through the NF-κB pathway,suggesting that LSW has significant pharmacological effects in regulating inflammation.However,not much research has been done on the mechanisms of how LSW modulates the excessive inflammatory response caused by influenza viruses via NLRP3 inflammasome signaling pathway.PurposeTo study how LSW reduce the level of IL-1βand other pro-inflammatory factors and reduce cellular pyroptosis via NLRP3 inflammasome signaling pathway,then partly explain the mechanism of its regulation of immune resistance to excessive inflammation caused by influenza A virus.To provide the experimental data for the modern development of LSW and the development of modern Chinese medicine drugs against respiratory infectious diseases.Method1.The acute lung injury model induced by influenza A virus PR8 strains in WT and NLRP3^-/-mice were constructed,and mice were administered orally for 7 days with LSW,and the status of mice were observed and their body weights were recorded.The expression of NLRP3 inflammasome signal pathway-related proteins was detected by Western Blot,and the expression levels of pro-inflammatory factors IL-1βand IL-18were detected by ELISA.2.By constructing an inflammatory response model in RAW264.7 cells and THP-1cells infected with influenza A virus PR8 strains,Western Blot assays was used to detect protein expression of the intracellular proteins NLRP3,ASC,Caspase1,GSDMD,Caspase11 or Caspase4 in the canonical and non-canonical NLRP3 inflammasome signal pathways and q PCR assay was used to detect the m RNA expression levels of IL-1βand other cytokines;intracellular ROS expression was detected using chemical fluorescence staining.Results1.Compared with the model group,LSW was able to alleviate the trend of weight loss,reduce lung index and lung virus titers（P<0.05 or P<0.01 or P<0.001）,and improve pathological changes such as inflammatory infiltration and collagen deposition in the lungs after influenza A virus infection in WT and NLRP3^-/-mice;2.Compared with the model group,LSW was able to reduce the protein expression levels of NLRP3,ASC,Caspase1,GSDMD and Caspase11 in canonical and non-canonical NLRP3 inflammasome signal pathway,as well as the expression levels of pro-inflammatory factors IL-1βand IL-18 in lung tissue of WT mice（P<0.05 or P<0.01or P<0.001）;3.After knocking down the NLRP3 gene of mice,compared with the model group,LSW continued to reduce the protein expression levels of ASC,Caspase1,GSDMD and Caspase11 in the non-canonical NLRP3 inflammasome signal pathway in lung tissue,as well as the expression levels of the pro-inflammatory cytokines IL-1βand IL-18（P<0.05 or P<0.01 or P<0.001）;4.Compared with the model group,LSW was able to reduce the m RNA and protein expression levels of NLRP3,the m RNA expression levels of IL-1β,IL-6,CCL-4 and TNF-αas well as the protein expression levels of Caspase11,Caspase11 p38 and GSDMD-N after influenza A virus infection of RAW264.7 cells（P<0.05 or P<0.01 or P<0.001）.5.Compared with the model group,LSW was able to reduce the protein levels of NLRP3,ASC,Caspase1,GSDMD and Caspase4,as well as the expression of ROS after influenza A virus infection in macrophage-like THP-1 cells（P<0.05 or P<0.01）.ConclusionLSW can reduce influenza A virus PR8-induced excessive inflammation by inhibiting canonical and non-canonical NLRP3 inflammasome signaling pathway.LSW has good prospects for pharmacological applications against influenza virus infection of the respiratory tract.