San Wu Huangqin Decoction Inhibits Influenza A/PR/8/34(H1N1) Virus Infection And Its Mechanism In Vitro And In Vivo

BackgroundInfluenza is a kind of zoonotic and acute respiratory infectious diseases caused by influenza virus,which usually occurs in the winter and spring and serious harm to human health.And it belongs to "epidemic disease" in the traditional Chinese diseases.In particular,it's very difficult to prepare an effective vaccine for the influenza A virus because of its mutable feature.What's more,the anti-influenza western medicines have strong side effects and easy induction of drug resistance.Traditional Chinese medicine has the advantages of eliminating pathogenic righting,simultaneous treatment,and so on.It plays an increasingly important role in the prevention and control of influenza viruses.The San Wu Huangqin Decoction(SWHD),a classic formula for the treatment of cold caused by exposure to wind-pathogen with hyperpyrexia and deficiency of Yin-blood,was first prescribed by Sun Simiao in "Beiji Qianjin Yao Fang".SWHD is a herbal remedy for replenishing Yin and removing heat and toxic materials.It is composed of Sophora flavescens,Scutellaria baicalensis and Rehmannia glutinosa.It showed that SWHD and its constituent herbs had the functions of anti-tumor,anti-virus and enhancing the body immune.Actually,SWHD was initially used for colds and fever in hemopenia puerpera,and its contemporary clinical application has only been observated in clinical effect,however,the effect of SWHD on the influenza virus(IFV)and its mechanism remain unknown.The aim of this study was to evaluate,for the first time,whether SWHD could be used to treat influenza in vivo and in vitro and its mechanism,which could provid the experimental basis for the clinical application and it is important for the prevention and treatment of influenza virus..Methods and results1.Study on quality control of Sanwu Huangqi DecoctionTo determinate the eight active constituents of baicalin,wogonoside,baicalein,matrine,sophocarpine,oxymatrine,and oxysophocarpine on San Wu Huangqi Decoction by HPLC.The content of each component was 82.83 mg/g,21.38 mg/g,2.11 mg/g,8.77 mg/g,2.33 mg/g,0.63 mg/g and 1.91 mg/g,respectively.2.In vitro inhibition studyMTT assays were used to detect the inhibitory effect of Sanwu Huangqi Decoction on different infections of influenza virus,and western blot,qPCR and immunofluorescence were used to detect the inhibition of viral protein expression.The results showed that Sanwu Huangqi Decoction could effectively inhibit the proliferation and adsorption of virus in cells,and could effectively prevent invasion into cells by viruses.What's more,it could significantly inhibit the expression of NP,HA,NA,and M2 viral proteins in a dose-and time-dependent manner.3.In vivo inhibition study3.1 To observe the therapeutic effect and mechanism of the model after administration.The results showed that Sanwu Huangqi Decoction could effectively inhibit the mortality of infected mice,prolong the survival time of mice,reduce the degree of pneumonia injury in mice,and reduce the lung index and virus titer in the lung tissue.3.2 The MTT assays,Western Blot,qPCR,and ELISA assays were used to detect the regulation of the drugs of the immune mechanisms.The results showed that Sanwu Huangqi Decoction could significantly promote the proliferation activity of T,B lymphocytes and promote the the killing function of the NK cells and phagocytosis of macrophages.What's more,it could reduce the expression of IL-1?,TNF-?,IL-6 and IFN-y in the lung tissue,serum and bronchoalveolar lavage fluid of mice,and promote the expression of IL-4,respectively.3.3 The Effect of Sanwu Huangqi Decoction on IKK/I?B/NF-?B Signaling PathwayWestern Blot,qPCR and immunohistochemical were used to detect the regulation of IKK/I?B/NF-?B signaling pathways.The results showed that the IKK/I?B/NF-?B signaling pathway was activated after viral infection,and the expression of the four proteins IKK?/?,NF-?B p65,p-NF-?B p65 and p-I?B? were significantly increased,while the protein level of IkappaBalpha was significantly inhibited.After the intervention of Sanwu Huangqin decoction,it could significantly inhibit the expression of mRNA and proteins of NF-?B p65,IKK?/? and I?B?,and promote the expression of the p-I?B? protein,which achieved the effect of anti-influenza virus.ConclusionThe results suggested that San Wu Huangqin Decoction had a significant inhibitory effect on influenza virus infection in vitro and in vivo.It not only inbibited the antiviral effect directly,but also could regulate viral infection by regulating the immune function,the secretion of cytokines and the IKK/I?B/NF-?B signal pathway disorders,which could inhibit influenza virus indirectly.