Study On The Mucosal Immune Of Bing-xiang-san And The Adjuvant Activities Of 1,8-Cineol As Influenza Vaccine Mucosal Adjuvants For Prevention Of Influenza Virus Infection In Mice

Influenza caused by influenza virus,can resulte respiratory infectious diseases and high morbidity and mortality during the outbreaks.In recent years,the appearance of the highly pathogenic avian influenza because of viral antigen variation has led to the increase of infections and deaths in humans.Respiratory tract mucous membrane as the first barrier against influenza virus plays an important role in influenza virus infection.blocking viruses on the mucous is an effective means to inhibition virus replication.Vaccination is a major means to prevent infection and pandemic.Vaccine adjuvants can activate the innate immune response and improve the capacity of antigen-taked and stimulate the adaptive immune response.Bing-Xiang-San composited borneol,ageratum,wild chrysanthemum flower and AiYe.We preliminary studies has proved that Bing-Xiang-San can inhibit the influenza virus in vitro and can relieve viral pneumonia caused by influenza viruses in vivo,reduce lung viral load in the infected mice and promote antigen presenting function of dendritic cells.1,8-cineol is the one of the composition of Bing-Xiang-San.Our previous study proved that it can inhibits the NF-κB,reduces pro-inflammatory cytokines,and relieves the pathological changes of viral pneumonia in infected-mice with influenza virus.This study is to explore the immune mechanism of Bing-Xiang-San against influenza virus by intranasal.At the same time,the one of main component 1,8-cineol,as flu vaccine adjuvants,co-adnimistration with influenza vaccine to observe the prevention effect against different subtypes of influenza virus.ObjectiveTo study of mucous immunization of Bing-Xiang-San;And to evaluate the effective of inhibittion inflamation by 1,8-cineol in the infected-mice;To observe the fight against the different subtypes influenza virus of 1,8-cineol,as vaccine adjuvant,co-administration with influenza vaccine,by intranasal.Methods1.The evaluation of immune response of Bing-Xiang-San on the influenza virus(1)The influenza virus infected mice model in this study was used to assess the fighting against influenza virus.(2)the level of s-lgA、lgG in the nasal lavage fluids and IL-4、IFN-γ in serum were measured by ELISA;(3)The percente of CD4+ and CD8+ T cells in NALT were determined by immunocytochemistry;(4)Lymphocyte proliferation study was determined by CCK-8.2.The Study of 1,8-cineol on influenza viral pneumonia caused by influenza virus(1)The influenza virus infected mice in this study were used to asses the fighting against influenza virus;(2)The level of IL-6,TNF-α,IL-Iβ,IFN-γ,MCP-1,IL-4,IL-5,IL-8 measured by ELISA at OD490.(3)Viral load in lung were measured by the PCR.(4)Lung pathology were measured by HE.(5)The protein level of NF-KB,(VCAM)-1,(ICAM)-lwere measured by WB。3.The adjuvant activities of 1,8-cineol as influenza vaccine mucosal adjuvants for prevention of influenza virus infection in mice(1)The influenza virus infected mice in this study to asses the fighting against of influenza virus.(2)using PCR to observe the viral load in infected mice.(3)Lung pathological was measured by HE.(4)Using ELISA to measure the antibodies leve of the s-IgA、IgG、IgG1b、IgG2a、IgG2b in the mice-infected.(5)The number of Dendritic cells(CDllc)and co-stimulation molecular CD40,CD80,CD86 expression were measured by flow cytometry,(6)Using immunohistochemical to detecte the expression of respiratory tract IEL.Results1.Bing-Xiang-San essential oil,by intranasal administration,can improve leve of the s-IgA and IgG,and stimulate the CD4 and CD8 T cells differentiation and maturation and improve the spleen lymphocyte proliferation and the cytokine secretion of IFN-γ,IL-4,compared with the placebo group,p<0.05.1,8-cineol can prolong the survival time of infected mice,inhibite the inflammation cytokine and the protein of NF-κB,VC AM-1,ACIM-1,compared with the IFV-C group,p<0.05,p<0.01.2.1,8-cineol co-administrate with influenza vaccine can reduce the lung index of infected mice,alleviate the pulmonary lesions of infected mice and increase the antibodies leve of IgG,IgG1 IgG2a and IgG2b,compared with vaccine group,p<0.05,p<0.01.1,8-cineol co-administrate with vaccine,can effectively induce local mucosal immune response,improve the level of s-IgA and improve the expression of respiratory tract lymphocytes,compared with vaccine group,p<0.05,p<0.01.1,8-cineol co-administrate with vaccine can stimulate the expression of dendritic cells mature and the expression of co-stimulation molecule CD40,CD80,CD86,compared with vaccine group,p<0.05,p<0.01.ConclusionBing-Xiang-San essential oil,by intranasal,can effectively induce mucosal immune response.1,8-cineol can inhabite the inflammation resopnse and as vaccine adjuvants,co-administrate with influenza vaccine,can protect the mice from the different subtypes influenza virus.