The Initial Study On Mechanism Of Baicalin Against H9N2 Avian Influenza Virus In Vitro

Objective: By building the pulmonary microvascular endothelial cells model and coculture cell model of type II alveolar epithelial cells and pulmonary microvascular endothelial cells infected by H9N2 subtype avian influenza virus,this paper researches the influence of baicalin on the expression of type I interferon and anti-viral protein,to explore immune mechanism of baicalin in vivo.Methods: The virus was propagated by the inoculation method in chicken embryo allantoic cavity and the virus titer was estimated by agglutination test and plaque test.MTT assay was used to select concentration of baicalin on cell.The pulmonary microvascular endothelial cells model and coculture cell model of type II alveolar epithelial cells and pulmonary microvascular endothelial cells infected by H9N2 subtype avian influenza virus was eatablished in vitro.After treated with baicalin at different concentrations and at different time periods,the expression of type I interferon and anti-viral protein was detected with Real-time fluorescent quantitative PCR,Western Blotting and enzyme-linked immunoabsorbent assay methods.Results:(1)After pulmonary microvascular endothelial cell was infected by H9N2 subtype avian influenza virus,the expression of IFN-? and IFN-? was increased.The expression of IFN-? increased significantly(P<0.01)at 24 hours.the expression of IFN-? was increased significantly(P<0.05)at 48 hours.In low-dose baicalin group,the expression of IFN-? was significantly reduced(P<0.05)at 12?24 and 36 hours,but the expression of IFN-? changed not significantly.In middle-dose baicalin group,the expression of IFN-? and IFN-? was reduced significantly at 24 hours and 6 hours respectively(P<0.05).In high-dose baicalin group,the expression of IFN-? was significantly increased at 6 hours and 36 hours(P<0.05),the expression of IFN-? was significantly increased at 12 hours and 24 hours(P<0.05).(2)After pulmonary microvascular endothelial cells was infected with virus,the relative quantitative expression of PKR and Mx1 m RNA was increased significantly(P<0.01).In low-dose baicalin group,the expression of PKR m RNA was significantly reduced(P<0.05)at 12 hours,but the expression of Mx1 m RNA was increased significantly at 12 hours and24 hours(P<0.05).In middle-dose baicalin group,the expression of PKR and Mx1 m RNA was increased significantly(P<0.05)at 12 hours,but substantially reduced(P<0.05)at 36 hours and 48 hours.In high-dose baicalin group,the expression of PKR m RNA and Mx1 m RNA was declined markedly(P<0.05)at all time points.After virus infection,the expression of PKR was increased significantly(P<0.05)in every period of time,but the expression of Mx1 was significantly reduced at24?36 and 48 hours(P<0.05).In low-dose baicalin group,the expression of PKR was significantly reduced(P<0.01)at 12 hours and he expression of Mx1 was increased significantly(P<0.05)at 24 hours.In middle-dose baicalin group,the expression of PKR and Mx1 was significantly increased(P<0.05)at 12 hours,but the expression of PKR was significantly reduced(P<0.05)at 36 hours and 48 hours.In high-dose baicalin group,the expression of PKR was significantly reduced(P<0.05)in every period of time,the expression of Mx1 was significantly increased(P<0.01)at 12 hours and reduced(P<0.05)at 36 hours.(3)After the coculture cell model of PMVECs/ATs II was infected with virus,the relative quantitative expression of PKR and Mx1 m RNA was increased significantly(P<0.01).In low-dose baicalin group,the expression of PKR m RNA was significantly reduced(P<0.01)at 12 hours,but the expression of Mx1 m RNA was increased significantly(P<0.01)at 12 hours.In middle-dose baicalin group,the expression of PKR m RNA was significantly reduced(P<0.01)at 12?36 and 48 hours,but the expression of Mx1 m RNA was increased significantly(P<0.05)at 12 and 24 hours.In high-dose baicalin group,the expression of PKR m RNA was increased significantly(P<0.01)in every period of time,the expression of Mx1 m RNA was increased significantly(P<0.05)at 12 hours and decreased significantly(P<0.05)at 36 hours.After virus infection,the expression of PKR was increased to some extent at various points in time,the expression of Mx1 was significantly increased(P<0.05)at 36 hours.In low-dose baicalin group,the expression of Mx1 was significantly increased(P<0.05)at 12 and 36 hours.In middle-dose baicalin group,the expression of Mx1 was significantly increased(P<0.05)at 12 hours and significantly reduced(P<0.01)at 48 hours.In high-dose baicalin group,the expression of Mx1 was significantly increased(P<0.05)at 12?24 hours and significantly reduced(P<0.01)at 48 hours.In every period of time,there was no difference in the expression of PKR.(4)After the coculture cell model of ATs II/PMVECs was infected with virus,the relative quantitative expression of PKR and Mx1 m RNA was increased significantly(P<0.01)in every period of time.In low-dose baicalin group,the expression of PKR and Mx1 m RNA was significantly reduced(P<0.05)at 12 and 24 hours,and the expression of PKR m RNA was decreased(P<0.01)in very significant difference at 48 hours.In middle-dose baicalin group,the expression of PKR m RNA was remarkable decreased(P<0.01)at 12?24 and 48 hours,but the expression of Mx1 m RNA was increased significantly(P<0.05)at 36 and 48 hours.In high-dose baicalin group,the expression of PKR and Mx1 m RNA was decreased in very significant difference(P<0.01)at 12 and 24 hours,the expression of Mx1 m RNA was increased significantly(P<0.05)at 48 hours.After virus infection,the expression of PKR was significantly increased(P<0.05)at 12?24 and 48 hours,the expression of Mx1 was significantly increased(P<0.05)at 12 hours but significantly reduced(P<0.05)at 24 and 48 hours.In low-dose baicalin group,the expression of Mx1 was significantly increased(P<0.05)at 12 ? 36 and 48 hours.In middle-dose baicalin group,the expression of Mx1 was significantly increased(P<0.05)at every time point.In high-dose baicalin group,the expression of PKR was significantly reduced(P<0.05)at 12 hours,but the expression of Mx1 was significantly increased(P<0.05)at 12 and 36 hours.Conclusion:(1)After pulmonary microvascular endothelial cell was infected by H9N2 subtype avian influenza virus,high concentration Baicalin could improve expression level of IFN-? and IFN-?.(2)After pulmonary microvascular endothelial cell was infected by H9N2 subtype avian influenza virus,baicalin could improve expression level of PKR and Mx1,improve the anti-virus activity of cell.(3)Type II alveolar epithelial cells and pulmonary microvascular endothelial cells can interact with each other.After infected with virus,baicalin could improve expression level of Mx1,but there are no influence on the expression of PKR.