| H5N1 and H9N2 AIVs greatly affect the farming economy around the world and cause great threat to human health.Inflammation is one of the most important modes of the body against pathogen infection and is also the core indicator of pathogenicity evaluation.Chemokines and cytokines play important roles in the inflammatory response,because they can activate inflammatory cells to fight and eliminate pathogens,and recruit inflammatory cells to the infected parts.Why are the number and types of inflammatory cells recruited at the infected parts significantly different after different types of AIVs infect the host? Whether it is possible to find some candidate drugs to intervene this process,the further research of these issues will provide direct scientific data for influenza inflammation mechanisms and comprehensive prevention and control.In this study,the chemokines and cytokines were screened dynamically after DF-1 cells were infected respectively with H5N1 and H9N2 AIVs,and the dynamic expression data of IL8L1?IL8L2?CX3CL1?CCL5?CCL20?K203?SCYA4?CCLi10?CCL19?XCL-1?IFN-??IFN-??IL-1??IL-6 and TNF-? were obtained.Based on the above test data,we explored the effect of Forsythia suspensa in interfering with the chemokine and cytokines of AIVs and found that it had good anti-inflammatory and anti-viral effects,which provides basic data for the search for unique anti-inflammatory and antiviral double-resistance highly effective anti-influenza virus drugs.The content of this study are showed as following.1.The influence to the expression of chemokines and cytokines in DF-1 cells infected by H5N1 and H9N2 AIVs.Supernatant and cells were collected at different time points after DF-1 cells were infected by H5N1 and H9N2 AIVs.The virus growth curve was plotted by the viral titer of the supernatant at different time points,while technique of real-time PCR were used to detect the changes of chemokines and cytokines gene expression in cells at each time point.The results showed that the expression of CCLi10,CCL19 and XCL-1 wasn't detected at each time point after infection of DF-1 cells with H5N1 and H9N2 AIVs,but other chemokine genes showed varying degrees of distinct increase trend.The expression level of chemokines in DF-1 cells infected by H9N2 AIV was significantly higher than that of H5N1 AIV infected,and K203,CX3CL1,SCYA4 and CCL20 are the specific chemokines response to H5N1 AIV infection,while K203,CCL20 for H9N2 AIV.The results also showed that all the cytokines tended a significant increase.The expression level of cytokines in DF-1 cells infected by H5N1 AIV was significantly higher than that of H9N2 infected,and IFN-?,IL-1? and TNF-? are the specific cytokines response to H5N1 AIV infection,while IFN-?,IL-1? and IL-6 for H9N2 AIV.2.The influence of Forsythia suspensa on anti-inflammatory activity and anti-viral activity in DF-1 cells infected by H5N1 and H9N2 AIVs.To explore the safe concentration range of water extract solution of Forsythia suspensa to DF-1 cells,CCK-8 was used to examine the toxic effects of different concentrations of the extract in DF-1 cells.Accroding to the possible mechanism of Forsythia suspensa,three treatment methods were established: virus were pretreated by the extract and the infected;the extract was add into cell maintenance fluid after cells were infected by virus;cells were pretreated by the extract and then infected.MTT method was used to detect the virus inhibition rate to ensure the best administration method and the best concentration of the extract.Technique of real-time PCR were used to detect the changes of chemokines and cytokines gene expression in cells at each time point under the optimal conditions of the extract.The results showed that the safe concentration range is 0~4 mg/mL and the best concentration is 4 mg/mL.DF-1 cells were infected by H5N1 and H9N2 AIVs and then added the extract,which is the best treatment method.The anti-AVIs activity of the extract by showing inhibited infections in DF-1 cells of H5N1 and H9N2 AIVs in a concentration-dependent manner.Treatment with the extract results in the decline of proliferation rate and reduction of progeny virus titers.The expression of CX3CL1,IL8L1,CCL5,SCYA4,IL-1?,IL-6 and TNF-? showed significantly decreased compared with control group in H5N1 AIV group,while CX3CL1,IL8L1,CCL5,IFN-?,IL-6 and TNF-? for H9N2 AIV group.The above results showed that water extract solution of Forsythia suspensa has good anti-inflammatory and anti-viral activity. |
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