| Influenza A virus is the causative agent of the most common and highly infectious human disease. This cosmopolitan pathogen is responsible for a significant morbidity and mortality in humans as well as animals. Despite extensive clinical and pathological studies on influenza,our understanding of the mechanisms of disease development is still incomplete.The mammalian innate immune system recognizes the presence of invading pathogens by a family of receptors belonging to the Toll-like receptors (TLRs). Viral infection triggers an array of immune responses that effectively limit the replication and spread of viral infection. Recent studies have demonstrated that influenza viral infection up-regulates the expression of TLR7. In human immune cells,the expression of TLR7 in a given cell type is mostly to plasmacytoid dendritic cells (pDC) and monocytes. The plasmacytoid dendritic cells (pDC) and monocytes recognize ssRNA of virus mediated by MyD88 dependence pathway. The host cleared the virus by the innate immune response,and trigger the subsequent specific immune response. The recognition of these viruses by plasmacytoid dendritic cells and monocytes through TLR7 results in their activation of costimulatory molecules and production of cytokines. If the pathway lost their normal control, the inflammatory hurt will be happened. The pathogenesis of influenza belongs to the theory"toxin hurts collaterals"of our Chinese medicine,and the toxin often result in the infectious lung hurt. The development of the influenza is process of the war between the inflammatory and anti-inflammatory. In this work,we investigated the antiviral ways and effect of the drug and the possible recognization mechenism that TLR7 recognize ssRNA; We try to discover the antiviral molecular target of the Dureping injection combine with the Chinese medical theory.Objective:To observe the antiviral efficiency of different drug-added ways of Dureping Injection against influenza virus FM1 strain in vitro and its antiviral effective rate at 24h,36h,48h and 72h; We try to find the optimal antiviral time and the possible mechanism of the drug; To observe the antiviral mechanism of the drug in the immunological network by measure the kinetic change of nitrous oxide (NO) and the cytokines such as regulated upon activation normal T cell expressed and secreted (RANTES),macrophage inflammatory protein (MIP-1α),interleukin-6 (IL-6),interferon-β(IFN-β) and induced protein (IP-10) in murinal celiac macrophage(Ana-1) infected by the influenza virus FM1 strain; At the same time investigating the molecular mechanism that the drug inhibits the influenza virus via the MyD88-dependent pathway mediated the Toll-like receptor (TLR) 7 and finding the antiviral molecular targets of the Chinese medicine.Methods:1. To observe the inhibitive effect of different drug-added ways of Dureping Injection against influenza virus FM1 strain in MDCK and its antiviral effective rate at 24h,36h,48h and 72h,which were assayed by crystal violet staining method. To investigate the antiviral efficiency and the characteristics of the drug in vitro.2. To survey the effect of the Dureping Injection on the kinetic change of nitrous oxide (NO) in macrophage infected by the influenza virus FM1 strain. The murinal celiac macrophage (Ana-1) was infected by the virus,add the different concentration of the drug in the supernatant of the macrophage for 6h,12h,24h and 36h. Measure the level of the NO in the supernatant by the method of traditional Griess Reagent.3. Murinal celiac macrophage(Ana-1) cell line was infected by influenza virus FM1 strain then treated with the Dureping Injection for 6h,12h and 24h later, collected the supernatant and measured the kinetic change of RANTES,MIP-1α,IL-6,IP-10 and IFN-βrespectively by ELISA.4. Using the method Real-time-Q PCR detected the influence of the Dureping Injection on the antiviral signal transduction pathway mediated by the Toll-like receptor 7 (TLR7) in different concentration (10μg/ml group and 1μg/ml group) for 12h and 24h. To observe the level of mRNA expressed by the Toll-like receptor7(TLR7),myeloid differentiation factor 88 (MyD88),IL-1 receptor associated kinase4 (IRAK4),TNF receptor associated kinase6 (TRAF6) and nuclear factor-κB P65 (NF-κB p65) of the pathway in the murinal celiac macrophage infected by the virus; To survey the influence of the drug on the expression of NF-κB p65 of the signal transduction pathway by the method of Western-bolt.Results:1. Dureping Injection showed an obvious inhibitive effect against adsorption of the virus and inhibition on its prolongation inside MDCK. It was found that Dureping Injection had direct killing effect on the virus at high concentration (≥0.18 mg/ml). Its antiviral effect goes to the peak at 48h after infection, with the prolongation of acting time of drug the inhibitive effect of Dureping Injection kept no change at the high concentration (≥0.27 mg/ml) and weakened at low concentration.2. Six different concentration of Dureping Injection influenced the macrophage for 6h,12h,24h and 36h,the amount of NO secreted by the murinal celiac macrophage infected by the virus was down-regulated,and showed dose relationship significantly,expecially in the concentration 60μg/ml,30μg/ml and 10μg/ml.3. Secretion of cytokines such as RANTES,MIP-1α,IL-6,IP-10 steps up gradually after infected by influenza virus 6h, get the peak after 12h, then drop out after 24h,shows a kinetic change. Dureping Injection can down-regulate the amount of RANTES,MIP-1α,IL-6,IP-10 and up-regulate the amount of IFN-β, which secreted by the murinal celiac macrophage infected by the virus,and shows dose dependent relationship significantly.4. Dureping Injection can down-regulate the expression of mRNA of the signal proteins such as TLR7,MyD88,IRAK4,TRAF6 and NF-κB p65 in the signal transduction pathway mediated by the TLR7.The down-regulation is significantly and shows dose and time dependent relationship.5. The 10μg/ml group and 1μg/ml group of Dureping Injection can down-regulate the expression of the NF-κB p65 protein detected by the method of Western-blot,the regulation shows the dose and time dependent relationship significantly.Conclusion:1. Dureping Injection has an obvious effect against influenza virus FM1 strain by multiple ways. The drug can prevent the viral infection, inhibit the virus adsorb the host cells and the proliferation of the virus in the cells. The antiviral effect shows stable and significant.2. Dureping Injection can prevent the production of the inflammatory factor and the chemokines such as NO,RANTES,MIP-1α,IL-6 and IP-10 and promote the secretion of the IFN-βby inhibiting the activity of the NF-κB signal pathway. These cytokines secreted by the murinal celiac macrophage (Ana-1) was infected by the virus and the drug shows the antiviral effect by regulating the immunological network. The drug relieve the inflammatory pathological changes,control the process of the disease.3. Dureping Injection can regulate the MyD88-dependent pathway mediated by the TLR7 and inhibit the infection from the influenza virus, the inhibition shows the dose and time depedent relationship. The drug can down-regulate the expression of the mRNA of TLR7,MyD88,IRAK4,TRAF6 and NF-κB p65 and the expression of NF-κB p65 protein in the macrophage infected by influenza virus. It can provide a more effective way for study the antiviral mechanism of Chinese medicine.4. Dureping Injection inhibits the influenza virus by the direct and indirect ways. The drug regulates the host immune function such as killing or preventing the virus or inducing the production of the IFN-β. The effect of the drug shows the unity of the Chinese medicine theory"drive away evil"and"strengthening body resistance". So this effect reveals the action mechanism of relieving the toxin and dredging the collateral of Dureping Injection. The result showed the new ideas about the compatibility of the drug, according to theory of"toxin hurt lung collaterals"of our Chinese medicine. |
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