Study On The Antiviral Action And TLR/IFN Signal Pathway Mechanism Of Serum Containing Volatile Oil Of Schizonepeta And Ramulus Cinnamomi In Vitro

Objective:To study the influence of volatile oil of Schizonepetae and Ramulus Cinnamomi (VOSR) medicated serum on proliferation of mouce adapted strain of influenza A virus (IAV) A/PR/8/34(H1N1) via serum pharmacology in vitro, and to discuss the mechanism of antiviral activity through TLR/IFN singal pathway with the purpose of providing scientific experimental basis for elucidating and applying the "dispersing and relieving the exterior syndrome" efficacy of diaphoretics.Methods:(1) Anti-IAV effect of medicated serum in vitro:①The TC0of VOSR medicated serum on MDCK cells was detected by CPE assay.②The infectivity of IAV on MDCK cells was determinated by hemagglutination test.③VOSR medicated serum at different time points was prepared, meanwhile, its efficacy was compared with each other by CPE and hemagglutination test to screen the best time for serum preparation.④The inhibitory and exterminate action of VOSR medicated serum on IAV proliferation in cells was determinated through hemagglutination test and MTT assay, meanwhile, the best time of administration for medicated serum affect virus proliferation was discussed.(2)Anti-IAV mechanism of medicated serum:⑤The IFN-β level of infected cells was detected by ELISA test.②The influence of medicated serum on TLR7, IFN-β, TRAF6and MyD88mRNA expression levels in infected cells was determinated by RT-PCR.③The influence of medicated serum on TRAF6protein expression level of infected cells was determinated by Wesrtern Blot.Results:①The TCo of VOSR medicated serum was5%(with5%serum in culture).②The TCID50of IAV A/PR/8/34(H1N1) was10-3.5/0.1ml on MDCK cells.③The time point of1h after the last administration was confirmed as the best time for VOSR medicated serum preparation.④The VOSR medicated serum (concentration of5%,3.5%,2.5%) could inhibit IAV proliferation(10TCID50) significantly, furthermore, concentration of5%,3.5%has an exterminated action on IAV.⑤The VOSR medicated serum(concentration of5%) could significantly up-regulate the IFN-β level in supernate of infected MDCK cells and the TLR7, IFN-β mRNA expression level of MDCK cells (P<0.01or P<0.05), meanwhile, could down-regulate the expression level of TRAF6, MyD88mRNA and TRAF6protein (P<0.01).Conclusion:VOSR medicated serum has a significant inhibitory action on IAV proliferation in MDCK cells, and has some extermination action at the same time. The results were consistent with the study of two kinds of volatile oil both in vivo and in vitro, in addition, the anti-IAV effect was further confirmed. The anti-IAV mechanism might be related to activation of TLR7pathway, improvement of the IFN-β excretion, up-regulation of IFN-β, TLR7mRNA and TRAF6protein expression, and down-regulation of MyD88mRNA expression of infected cells.