Study On The Anti-influenza Virus Effect And TLR/IFN Signal Pathway Mechanism Of Volatile Oil Of Ramulus Cinnamomi And Cinnamaldehyde

Objective:To study the anti-influenza A virus (H1N1) activity of the volatile oil of Ramulus Cinnamomi (VORC) and cinnamaldehyde both in vitro and in vivo, and reveal its mechanism of TLR/IFN signaling pathway. Expecting to illustrate the scientific meaning of "relieving the exterior syndrome".Methods:①The median toxic concentration (TC50) and maximum non-toxic concentration (TCo) of VORC and cinnamaldehyde on MDCK cells were determined by MTT assay. The susceptibility of mouse-adapted influenza virus A/PR/8/34strain on MDCK cells were determined by hemagglutination test The IC50and TI of VORC and cinnamaldehyde in vitro were determined by MTT assay and Cytopathic effect assay.②Anti-influenza virus activity in chick embryos:the embryos were treated with different dosage of drugs, which were injected into the allantoic cavity and vitelline sac respectively in preventive and therapeutic administration way. Allantoic fluid was collected after incubating for72h, and the allantoic fluid HA on the chick embryo level was used as a measurement indicator.③Anti-influenza virus activity in mice:the mice which be infected with influenza virus were treated respectively with drugs in preventive and therapeutic administration way, and the death protection rate, lung index, lung tissue HA and pathomorphological change were adapted to evaluate the antiviral activity in mice.④Real time RT-PCR and ELIS A test were conducted to determine the serum level of IFN-β in virus infected cells.⑤The infected cells were collected after8h post-infection, and the real-time RT-PCR was used to determine the expresstion levels of TLR3, TLR7, TRAF-3, IRAK-4and IFN-β mRNA.Results:①It was found that the TC50of VORC and cinnamaldehyde on MDCK cells both were0.5×10-5ml/ml. VORC and cinnamaldehyde showed inhibition effect on virus proliferation after adsorption. Their IC50were5.894×10-8ml/ml and5.317×10"8ml/ml. The therapeutic index (TI) were84.83and90,04respectively. Both VORC and cinnamaldehyde had an direct virucidal activity; Their IC50were1.849×10-7ml/ml and5.770×10-7ml/ml. The therapeutic index (TI) were27.04and9.51respectively.②Allantoic fluid HA was significantly decreased after the chick embryos were treated with the VORC (0.1×2-14ml/ egg) and cinnamaldehyde (0.1×2-14ml/egg,0.5×2-15ml/egg,0.25×2-15ml/egg) by the allantoic cavity injection in the therapeutic administration way. In addition, allantoic fluid HA was significantly decreased after the chick embryos were treated with the cinnamaldehyde (0.1×2-14ml/egg,0.5×2-15ml/egg,0.25×2-15ml/egg) by the vitelline sac injection both in the preventive and therapeutic administration way,③The VORC (0.348ml/kg) has a significant effect on increasing death protection rate in preventive administration way, while the VORC (0.348ml/kg) and cinnamaldehyde dosage of0.264ml/kg and0.132ml/kg has a significant effect on increasing death protection rate in therapeutic administration way; In addition, VORC (0.174ml/kg) and cinnamaldehyde (0.132ml/kg) has a significant effect on decreasing lung index in therapeutic administration way.and VORC (0.348ml/kg), cinnamaldehyde (0.264ml/kg) decreasing lung tissueHA in therapeutic administration way.④ELISA tests showed VORC and cinnamaldehyde (0.25×10-5ml/ml) had a significant effect on decreasing serum level of TNF-β in virus infected cells.⑤the expression levels of TLR7, IRAK-4and IFN-B mRNA in infected MDCK cells were significantly increased after treated with VORC and cinnamaldehyde (0.25×10-5ml/ml)(p<0.01)Conclusion:This study showed that VORC and cinnamaldehyde had an good anti-influenza virus activity in cellular level, chick embryo level and in mice, and its mechanism was related to the activiation of TLR7signaling pathway and IRAK-4leading to the large amounts of IFN-B synthesis and releasefinally activiated the virus pattern recognition system.