Preliminary Study On The Protective Mechanism Of Recombinant Human Interferon α1b On Lung Injury Induced By Influenza A Virus H1N1 In Mice

IntroductionInfluenza is a highly contagious acute respiratory disease caused by influenza virus,with the epidemiological characteristics of wide spread,acute onset,and great harm.Influenza viruses are transmitted through aerosols and cause acute febrile respiratory diseases in humans,especially in children,elderly and people who with low immune function.It is estimateed by the World Health Organization（WHO）that influenza viruses lead to one billion influenza cases,35 million severe illnesses,and 300,000 to500,000 deaths worldwide each year.Numerous studies have demonstrated that influenza virus induced inflammatory lung injury is considered as the most related factor for causing people death.Pathological studies and animal experiments in severe cases have found that the influenza A H1N1 virus isolated in 2009 possesses the ability to directly cause severe pneumonia.Previous studies have found that the severity of lung inflammation caused by influenza virus is closely related to host complement activation and"cytokine storm"induced by virus.It has been suggested that recombinant human interferon-α1b（rhIFN-α1b）may play an important role in the treatment of pneumonia caused by influenza A,but the molecular mechanism of pneumonia caused by influenza virus is not very clear at present,and its regulatory role needs further explored.ObjectiveIn order to investigate the therapeutic effect and the protective mechanism of rhIFN-α1b in lung inflammatory injury induced by influenza virus,Influenza A virus H1N1 was used to infect mice animal models and cell models,which may give insight into the novel treatment of influenza A virus in clinic.Methods1.Establishment of mice lung adaptive strain of influenza A virus H1N1 and construction of mice infection model.In order to establish a mouse lung adaptive strain of influenza A virus H1N1nymcx-179a,nymcx-179a strain was first inoculated into BALB/c mice by dropping nose.Mice with obvious flu like clinical symptoms were selected to obtain lung tissue homogenate,and the supernatant was taken for RT-PCR detection.It was confirmed as influenza A virus H1N1.After the supernatant was proliferated in chicken embryo,ha titer was determined by hemagglutination test,and mice were inoculated again.After 5generations of culture in the animal body repeatedly,the lung adaptive strain which can cause all the mice to die was prepared.The lung tissue of the final infected mice was taken,and HE staining,immunohistochemistry staining and electron microscopy were used to observe the pathological changes and virus distribution in the lungs of the mice.2.Effect of rhIFN-α1b on mice infected with influenza A virus H1N1Mice infected with influenza A virus H1N1 were divided into three groups and treated with rhIFN-α1b nebulization,rhIFN-α1b intramuscular injection,and oral oseltamivir,respectively.The death protection of rhIFN-α1b against influenza A infected mice was studied through statistical analysis of mice deaths.ELISA,HE staining,immunohistochemical staining,and q RT-PCR were used to determine whether rhIFN-α1b has anti-inflammatory and anti-viral effects,such as alleviate tissue damage,reduce virus content in tissues,and inhibit viral expansion with different route.3.Effect of rhIFN-α1b on MDCK cells infected by influenza A virus H1N1In order to determine the protective effect of rhIFN-α1B on MDCK cell infection induced by influenza A virus H1N1,TCID50 of influenza A virus H1N1 on MDCK cell was determined by CPE method.The IC50 of rh INF-α1b in MDCK cells was determined by MTT method,and three concentrations of rh INF-α1b,1μM,2μM and4μM,were selected according to the results to further test whether rh INF-α1b can protect MDCK from influenza A virus infection.ELISA,q RT-PCR,Western blot and flow cytometry were used to study the inhibitory effect of rh INF-α1b on MDCK cell infection caused by influenza A virus H1N1.4.Molecular mechanism of rhIFN-α1b against influenza A virusOn the basis of confirming that rh INF-α1b can protect animals after influenza A virus H1N1 infection and inhibit the infection of MDCK cells caused by influenza A virus H1N1,Western blot and immunohistochemical staining were used to detect the activation protein of JAK/STAT signal pathway,meanwhile,the JAK/STAT signal pathway inhibitor AG490 was used to block and detect the signal pathway,to clarify whether rhIFN-α1b can protect cells by activating JAK/STAT signal pathway.Results1.A mice lung-adapted strain of influenza A virus H1N1 NYMCX-179A was successfully constructed and clearly identified,with titer of 2⁷,LD₅₀=10^-1.5/50μL.2.A mice model of influenza A virus H1N1 pneumonia was established:Mice infected with influenza A virus H1N1 showed obvious inflammation of lung tissues,and virus particles was observed under the electron microscope.3.When rh INF-α1b atomization,intramuscular injection and clinical drug oseltamivir were used to treat influenza A infected mice,the mortality of mice decreased,the inflammatory factors in serum decreased,the IFN-γcontent increased,and the inflammatory injury of lung tissue in each group improved to some extent.Among them,rh INF-α1b aerosol inhalation can reduce the degree of lung inflammatory injury most obviously,the distribution of influenza A virus H1N1 in lung tissue is the least,the amplification of influenza A virus is the most obvious,the virus content is the lowest,and the antiviral ability of rh INF-α1b aerosol inhalation is the strongest.There was significant difference between groups（P<0.05）4.The MDCK cell model of mice lung adaptive strain of influenza A H1N1 virus was successfully established,with a TCID₅₀ of 10^-4.25/0.1ml.The IC₅₀ of rh INF-α1b in MDCK cells was 7.587μM.5.rh INF-α1b has significant antiviral effect in MDCK cell influenza A virus infection model.rh INF-α1b can inhibit the expansion of influenza A virus H1N1 in cells and reduce the apoptosis caused by influenza A virus.The higher the concentration of rh INF-α1b,the stronger the effect.The differences between the groups were statistically significant（P<0.05）.6.Western blot showed that after rh INF-α1b interfered with MDCK cells infected with influenza A virus,the expression of JAK/STAT signal pathway related protein activation forms p-jak2 and p-STAT3 was up-regulated,With the increase of rhinf-α1b concentration,the up regulation effect is more obvious,immunohistochemical staining showed that rh INF-α1b intervention treatment significantly increased the distribution of p-JAK2 and p-STAT3 expressions in lung tissues in animal model.The differences between the groups were statistically significant（P<0.05）.7.rh INF-α1b can promote the activation of key pathway related proteins in JAK/stat,reduce the content of inflammatory factors and the rate of apoptosis.AG490intervention could attenuate the inhibitory effect of rh INF-α1b on virus expansion in MDCK cells,suppress the activation of JAK/STAT signaling pathway via inhibiting the expressions of p-JAK2,p-STAT3,ISGF3γand2’,5’-OAS,recovery of inflammatory factor expressions,enhance cell apoptosis.The differences between the groups were statistically significant（P<0.05）.Conclusions1.rhIFN-α1b has the effects of reducing mortality,lung injury,anti-inflammatory and antiviral in mice infected with influenza A virus.2.rhIFN-α1b treatment shows anti-inflammatory and antiviral effects and reduces apoptosis during influenza A infection in MDCK cells in a dose-dependent mammer.In a certain concentration range,with the increase of dose,the effect is enhanced.3.The protective effect of rhIFN-α1b against influenza A infection might be partly through activating JAK/STAT signaling pathway.