桂枝汤含药血清对小鼠巨噬细胞Toll样受体3、4型及其下游信号转导通路元件的影响

Effect of Guizhi Tang drug serum on the expression of Toll-like receptors and downstream signaling components in RAW 264.7 cells

目的:研究桂枝汤含药血清对LPS和Poly(I:C)刺激小鼠巨噬细胞RAW264.7的Toll样受体(TLR)及其下游主要信号转导通路元件的影响,从mRNA和蛋白水平探讨桂枝汤含药血清防治感染性疾病的免疫学基础。方法:用LPS或Poly(I:C)分别刺激RAW264.7细胞,加桂枝汤含药血清干预24h后,取细胞上清液测定炎症因子TNF-α和IFN-β的含量;荧光定量PCR方法测定TLR3、TLR4和下游信号转导通路元件MyD88、TRAF-6、TRAM和TRIF mRNA的表达;Western blotting法分析TLR3、TLR4蛋白的表达;细胞免疫荧光法分析MyD88、TRAF-6蛋白表达的强弱。结果:桂枝汤含药血清可显著降低LPS刺激诱导的MyD88、TRAM和TRIF的高表达;Poly(I:C)刺激后,桂枝汤含药血清显著降低TLR3、MyD88和TRAM的高表达。结论:桂枝汤能直接作用于TLR3,抑制其高表达,阻断TLR3胞内信号转导的MyD88依赖和非依赖两条途径,抑制相关基因表达产物TNF-α、IFN-β过度分泌,具有TLR3拮抗剂样作用。桂枝汤也可直接作用于接头蛋白MyD88、TRAM和TRIF,影响TLR4信号转导的MyD88依赖和非依赖性途径,抑制TNF-α、IFN-β的过度分泌。

Object： To investigate the influence of Guizhi Tang drug serum on expression of TLRs and downstream signaling components in RAW264.7cell. Methods： RAW264.7 cell line was stimulated with LPS and Poly（ I： C） respectively, then treated with the drug serum of Guizhi Tang, 24 hours later, collected the supernatant and measured the inflammatory factors such as TNF -α and IFN - β, measured the expression of TLR3, TLR4, MyD88, TRAF - 6, TRAM and TRIF mRNA with real - time PCR, using western blotting method to analyze the expression of TLR3, TLR4 protein, using cell immumofluorescence method to analyze the expression of MyD88 and TRAF -6 protein. Resuits： Guizhi Tang drug serum decreased the high expression of MyD88, TRAM and TRIF（ vs LPS stimulated group, P 〈 0.05 or P 〈 0.01 ） when the cells were stimulated by LPS, simultaneously, it weakened the high expression of TLR3, MyD88 and TRAM when the cells were stimulated by Poly（ I： C） （ vs Poly（ I： C） - stimulated group, P 〈 0.01 ）. Conclusion： Guizhi Tang blocked the high expression of TLR3, blocked both MyD88 - dependent and MyD88 - independent signaling pathways, inhibited the excess secretion of related gene expression products such as TNF - α, IFN - β, showed TLR3 - antagon - like actions. Guizhi Tang also influenced the junction protein such as MyD88, TRAM and TRIF directly, influenced the MyD88 - dependent and independent signaling pathways of TLR4, as a result, it inhibited the excess secretion of TNF - α and IFN - β.