Andrographolide Anti-H1N1 Infection In Vitro And Effect To RLH Signal Pathway

OBJECTIVE:To observe the anti-virus effect of andrographolide(AD) and changes of cytokines in RLH signal pathway when dendritic cells, macrophages and monocytes were infected by H1N1 in order to explicate the mechanism of AD for anti-virus effect.METHODS:H1N1 was amplified through the chicken embryo. Leukomonocyte were obtained from umbilical cord blood by Ficoll density gradient centrifugation and immunological cells were havested through different cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with 16HBE. The optimal concentration of AD was defined by MTT assay. After the immunological cells and virus infected cells co-cultured, AD was also added into the medium as a treatment intervention. After 2 h co-culture, immunological cells was collected for RT-PCR experiments to determine the expression levels of cytokines which related to RLH pathway.RESULTS:Amplification of influenza virus was successful by chicken embryo and the RBC agglutinatingtiter was 1:640 and TCID50 is 10-4.56. Mature immunological cells could be obtained by different stimulating cytokines factor.100TCID50 H1N1 infected with 16HBE for 2h could establish an infected cell model successfully. The optimal concentration of AD for anti-virus effect was 250μg/ml by MTT assay. After infection for 24 h, RT-PCR analysis showed that cytokines in RLH pathway increased when cells was infected by virus, RIG-1, IPS-1, IRF-7, IRF-3 and NF-κB increased significantly(P<0.05). When AD was added into co-culture medium, their expression levels decreased, and RIG-1, IPS-1, IRF-7, IRF-3 and NF-κB decresed significantly in each group with significant statistic difference(P<0.05).CONCLUSION:1.The cytokines of RLH pathway increased after virus infection, suggesting that virus could activate the immune system through this pathway and take effect by increasing their expression levels, such as RIG-1, IPS-1, IRF-7, IRF-3, NF-κB.2.AD, used as an anti-viral medicine, could enhance the activity of immnologcal cells, and downregulate the expression level of RLH pathway cytokines, such as RIG-1, IPS-1, IRF-7, IRF-3 and NF-κB. And this anti-viral effect maybe related to RLH pathway.3. Data analysis showed that the expression level of RIG-1, IPS-1, IRF-7, IRF-3 and NF-κB in each group increased significantly when virus attacked, with statistical significance (P<0.05). And this suggested that these factors played an important role in identifying the virus signal recognition mechanism in the RLH and induced the production of interferon of the RLH signaling pathway.4. Data analysis showed that the cytokines of RLH pathway in DC and monocytes group were significantly higher than macrophages group, suggesting that DC and monocytes had a stronger immune recognition. This phenomenon also showed that different immunological cell had different effects to virus attack, and maybe related to their functional characteristics.