Effect Of H1N1 Influenza Virus Hemagglutinin On The Expression Of ICAM-1,VCAM-1 And E-selectin In Endothelial Cells

Obejective:During the past century,influenza A virus has been caused emergence of four influenza pandemics,which include 1918 Spanish influenza(H1N1),1957 Asian influenza(H2N2),1968 Hong Kong influenza(H3N2),and 2009 Mexico influenza(H1N1);The emergence of influenza A pandemic has caused serious damage to human health and the social economy.At present,Influenza A viruses cause seasonal epidemics and sporadic pandemics,And continue to pose a significant threat to public health and the economy worldwide.The influenza A virus belongs to the orthomyxoviridae family and Its genome is comprised of eight negative sense,single-stranded RNA segments.Due to the instability of its genome,antigen drift and antigen transformation occur,finally leading to the emergence of seasonal influenza and pandemic.In the future,the next emergence of influenza pandemics is unpredictable and therefore we must make adequate preparations for its arrival.Currently the main drugs for prevention and treatment of influenza virus infections are Influenza virus vaccine and the M2 ion channel blockers(amantadine)and the neuraminidase inhibitors(oseltamivir)etc.M2 ion channel blocker,as the earliest anti-influenza drug,plays an important role in the treatment of influenza virus infection.As influenza viruses Constantly mutate,the majority of strains have resistance to the drug.At present,neuraminidase inhibitors are used as the first choice of anti-influenza drugs.but drug resistance has also been reported in some areas of China.neuraminidase inhibitors are effective in the treatment of influenza virus infection within 48 hours,and have little effect on the treatment of various irreversible pathological injuries in severe influenza patients.Therefore,it is of great importance to explore the pathogenesis of influenza and develop effective drugsThe H1N1 influenza virus enter the body through the respiratory tract,and causing a series of pathological changes.Light cases might only develop symptoms of cough,expectoration,fever,and other similar symptoms of the upper respiratory tract cold;but severe cases might have pneumonia,respiratory failure,acute respiratory distress syndrome(ARDS),and Multiple organ dysfunction syndrome(MODS).The severe acute lung injury induced by influenza A was associated with apoptosis and necrosis of lung epithelial cells,activation of immune system function,abnormal coagulation,and micro thrombosis.However,the activation of immune system function plays a critical role in acute lung injury by inducing excessive inflammatory cell infiltration and hypercytokinemia formation.The study confirmed H1N1 influenza virus can promote inflammatory infiltrates including mononuclear,lymphocytes and macrophages of lung tissue;in addition,neutrophils and mononuclear macrophage infiltration is closely related to the severity of lung injury.A clinical study found that elevated levels of various cytokines and chemokines can be detected in the serum of influenza virus infected patients compared to healthy people.Moreover,elevated levels of cytokines(IL-6,CCL-2,CCL-4,CXCL-8,CXCL-9 and CXCL-10)are related to the pathogenicity of the influenza virus;levels of Chemokines(CCL-2,CXCL-8,CXCL-9,and CXCL-10)were also associated with mortality of infected people.Cytokines and chemokines are important mediators of the immune response.They can kill pathogens by activating various enzyme activities in cells directly or inducing infiltration of inflammatory cells.However,influenza virus causes a vicious cycle of the inflammatory response and the aggravation of tissue damage by Promoting Synthesis and release of cytokines and chemokinesInfluenza virus hemagglutinin(HA)is a spike protein on the surface of the influenza virus,which is an important component to ensure the survival of the influenza virus.Moreover,HA promotes adhesion between targeted cells and viruses and virus-to-cell membrane fusion.In addition,HA is also an antigen that could be recognized by the immune system,and which plays an important role in influenza virus-mediated pathological injury.A study has shown that influenza virus(H5N1)recombinant hemagglutinn can abnormally activate the innate immune system in mice,leading to the excessive release of various cytokines and chemokines,and then inducing severe pathological changes in the lungs.Although the study demonstrated the pathogenicity of HA,the complicated pathogenic mechanism is still not clear.In this study,the effects of HA on the expression of intercellular adhesion molecule-1(ICAM-1),Vascular adhesion molecule-1(VCAM-1),and E-selectin in vascular endothelial cells were investigated,and the relationship between viral hemagglutinin and inflammatory response to investigate possible mechanisms of HA pathogenesisResearch contents and methods:Part ?: The effect of H1N1 influenza virus hemagglutinin on the expression of vascular endothelial cell adhesion molecules(ICAM-1,VCAM-1,and E-selectin)1.1 Human umbilical vein endothelial cells(HUEVC)were cultured and randomly divided into three groups of group 1(0ug/ml HA),group2(1.25ug/ml HA),group 3(2.5ug/ml HA).After being treated with HA at different concentrations for 3 h,the total protein of cells in each group was extracted,and the protein levels of ICAM-1,VCAM-1,and E-selectin were detected by Western blotting.1.2 HUEVC were cultured and randomly divided into three groups of group 1(0ug/ml HA),group 2(1.25ug/ml HA),group 3(2.5ug/ml HA).After being treated with HA at different concentrations for 3 h,the total RNA of cells in each group was extracted,and the expressions of ICAM-1,VCAM-1,and E-selectin at the gene level were detected by real-time PCR.Part ?: Adhesion between monocytes and HA-treated vascular endothelial cells1.3 HUEVC were cultured and randomly divided into three groups of group 1(0ug/ml HA),group 2(1.25ug/ml HA),group 3(2.5ug/ml HA).After being treated with HA at different concentrations for 3 h and then cultured with human myeloid leukemia mononuclear cells(THP1)stained with calcium anthocyanin for 1h.The adhesion of mononuclear cells to endothelial cells in each group was observed under the fluorescence microscope.Results:Part ?: The expression of ICAM-1,VCAM-1,and E-selectin in vascular endothelial cells induced by H1N1 virus hemagglutinin.Results of Real-time PCR and Western blotting showed that the mRNA and protein expressions of ICAM-1 and VCAM-1 increased along with increased of the HA concentration.Treatment of HUEVC with HA(2.5 ug/ml)significantly increased the mRNA and protein expressions of ICAM-1 and VCAM-1 compared with the blank control group(P<0.05).Treatment of HUEVC with HA(1.25 ug/ml)significantly increased the mRNA and protein expressions of E-selectin were significantly increased compared with the blank control group(P<0.05).Part ?: H1N1 virus hemagglutinin promotes the adhesion of vascular endothelial cells to monocytes.Results of Monocyte adhesion assay: Treatment of HUEVC with HA(1.25 ug/ml,2.5ug/ml)significantly increased the adhesion number of monocytes compared with the blank control group,(P<0.05),and treatment of HUEVC with HA(2.5ug/ml)significantly increased the adhesion number of cells compared with the HA(1.25ug/ml)(P<0.05).Conclusion1.Influenza virus hemagglutinin can promote the expression of ICAM-1,VCAM-1,and E selectin in endothelial cells2.Influenza virus hemagglutinin can promote the adhesion reaction between monocytes and endothelial cells.