基于TLR7/8介导的IFN-α/β蛋白表达水平探讨麻黄先煎之麻杏石甘汤抗流感病毒的机制

Mechanism research of anti influenza virus of ephedra decocted earlier Maxing Shigan Decoction from the expression level of IFN-α/β protein mediated by TLR7/8

目的:基于TLR7/8介导的IFN-α/β蛋白表达水平探讨麻黄先煎之麻杏石甘汤抗流感病毒效应机制。方法:以TLR7/8激活剂(R848)、抑制剂(抗体TLR7和抗体TLR8)干预的小鼠巨噬细胞为对照,以酶联免疫吸附技术检测流感病毒干预小鼠巨噬细胞24、48h的IFN-α/β分泌水平。以流感病毒干预的小鼠巨噬细胞为对照,以酶联免疫吸附技术和Western Blot技术检测麻黄先煎之麻杏石甘汤含药血清干预24、48h的流感病毒感染的小鼠巨噬细胞IFN-α/β分泌水平和蛋白表达水平。结果:与正常大鼠血清组及胎牛血清组比较,TLR7/8激活剂(R848)与流感病毒干预24、48h的RAW264.7细胞IFN-α和IFN-β分泌水平和蛋白表达水平显著升高(P<0.05);与病毒干预组比较,四药同煎及麻黄先煎20、25、30min之麻杏石甘汤含药血清作用24、48h后,流感病毒感染后的巨噬细胞IFN-α/β的分泌水平和蛋白表达水平显著降低(P<0.05);与四药同煎组比较,在含药血清作用48h的时间点,麻黄先煎25min之麻杏石甘汤组IFN-α分泌水平显著降低(P<0.05)。结论:流感病毒感染对TLR7/8介导的IFN-α/β蛋白分泌水平有明显的上调作用,麻杏石甘汤能够下调流感病毒感染的巨噬细胞IFN-α/β分泌水平和蛋白表达水平,在作用48h时间点,麻黄先煎25min之麻杏石甘汤含药血清对IFN-α分泌水平的调节作用优于四药同煎。

Objective: To investigate the anti influenza virus mechanism of ephedra decocted earlier Maxing Shigan Decoction from the expression level of IFN-α/βprotein mediated by TLR7/8. Methods: Mouse macrophages treated with TLR7/8 activator(R848), inhibitor(antibody TLR7 and antibody TLR8) as control, enzyme-linked immunosorbent assay was used to detect influenza virus intervention in mouse macrophages for 24 h and 48 h IFN-α/βsecretion level. Mouse macrophages treated with influenza virus as control, ephedra decocted earlier Maxing Shigan Decoction containing drug serum intervention for 24 h and48 h, enzyme-linked immunosorbent assay and Western Blot technique were used to detect the viral-infected mouse macrophage IFN-α/βsecretion levels and protein expression levels. Results: Compared with the normal rat serum group and bovine serum group, the secretion levels and protein expression levels of IFN-α and IFN-β in RAW264.7 cells treated with TLR7/8 activator(R848) and influenza virus for 24 h and 48 h were significantly increased(P<0.05). Compared with the virus intervention group,the macrophage IFN-α/β after influenza virus infection after 24 hours and 48 hours of treatment with the serum off ephedra decocted earlier Maxing Shigan Decoction for 20 minutes, 25 minutes and 30 minutes, the secretion level and protein expression level were significantly decreased(P<0.05). Compared with the four-drug boiled simultaneously group, at the time of 48 h after drug-containing serum intervention, the secretion levels of IFN-α were significantly reduced in the ephedra decocted earlier Maxing Shigan Decoction for 25 min(P<0.05). Conclusion: Influenza virus infection has a significant up-regulation of TLR7/8-mediated IFN-α/β protein secretion, and Maxing Shigan Decoction can down-regulate IFN-α/β secretion level and protein expression level of influenza virus-infected macrophages. At the time of 48 hours of treatment, the serum containing decocted earlier Maxing Shigan Decoction for 20 min and 25 min was better than t four-drug boiled simultaneously group in the regulation of IFN-β secretion.