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目的:观察并比较银翘散在不同煎煮时间下体外抑制甲型流感病毒FM1株的作用,及其含药血清对FM1刺激小鼠巨噬细胞Ana-1株Toll样受体及其下游信号转导通路的影响,探讨银翘散的抗病毒作用及其最佳煎煮时间。方法:采用血凝试验观察银翘散在不同煎煮时间、不同浓度下体外抑制流感病毒及对流感病毒感染鸡胚的预防和治疗作用;用FM1刺激巨噬细胞Ana-1,同时给予不同煎煮时间的银翘散含药血清进行干预,培养24 h后提取细胞RNA,应用实时定量PCR方法测定TLR3、TLR4及其下游信号转导通路My D88、TRAF-6、TRAM和TRIF m RNA的表达。结果:与病毒对照组比较,银翘散在不同煎煮时间、不同浓度下均可直接抑制FM1株的活性,其抑制作用时间从1 h持续到24 h,其中以3 min高、中浓度组抑制作用最强;银翘散各组对流感病毒感染鸡胚均有一定的预防和保护作用,其中以3 min高、中浓度组和6 min高浓度组的作用最强(P<0.05)。银翘散在不同煎煮时间下的含药血清均能显著降低FM1诱导的巨噬细胞TLR3、TLR4、My D88、TRAF-6、TRAM和TRIF的高表达(P<0.05),尤以3 min高、中、低浓度组和6 min高浓度组最为显著(P<0.05)。结论:银翘散在不同煎煮时间、不同浓度下均可有效抑制甲型流感病毒,尤以3~6 min高浓度组的作用最强;不同煎煮时间下的银翘散均能阻断TLR3和TLR4的高表达,并能阻断TLR4信号转导的My D88依赖和非依赖途径,尤以3 min和6 min组最显著,由此推断银翘散的最佳煎煮时间可能为煮沸后3~6 min。
OBJECTIVE: To observe and compare the effect of Yinqiao powder in inhibiting influenza A virus FM1 strain in vitro under different decocting time, and its drug-containing serum on Toll-like receptor and downstream signal transduction of FM1-stimulated mouse macrophage Ana-1 strain The effect of conduction path, to explore the anti-virus effect of Yinqiaosan and its best boiling time. Methods: The hemagglutination test was used to observe the preventive and therapeutic effects of Yinqiaosan on preventing and treating influenza virus infection in vitro at different boiling time, different concentrations and in different concentrations. FMD-1 stimulated the expression of Ana-1 in macrophages, Time of Yinqiaosan medicated serum were taken and cultured for 24 hours. Cell RNA was extracted and the expression of TLR3, TLR4 and its downstream signal transduction pathway My D88, TRAF-6, TRAM and TRIF m RNA were detected by real-time PCR. Results: Compared with the virus control group, Yinqiao powder could directly inhibit the activity of FM1 strain at different decocting time and different concentrations, and the inhibitory effect time was prolonged from 1 h to 24 h, of which, 3 min high and medium concentration groups inhibited The group of Yinqiaosan had the best preventive and protective effects on the chicken embryos infected by influenza virus, and the effect was the strongest (P <0.05) at 3 min, medium and 6 min. Yinqiaosi serum at different decoction time could significantly reduce the high expression of TLR3, TLR4, My D88, TRAF-6, TRAM and TRIF in FM1-induced macrophages (P <0.05) , Middle and low concentration group and 6 min high concentration group were the most significant (P <0.05). Conclusion: Yinqiao powder can effectively inhibit influenza A virus at different decocting time and concentration, especially in 3 ~ 6 min high concentration group; Yinqiao powder can block TLR3 under different decocting time And TLR4 overexpression, and can block the My D88-dependent and independent pathways of TLR4 signaling, especially in the 3 min and 6 min groups, suggesting that the optimal decoction time of Yinqiao Powder may be after boiling 3 ~ 6 min.