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目的:研究板蓝根含药血清对呼吸道合胞病毒(RSV)感染RAW264.7细胞中干扰素-β(IFN-β),Toll样受体3(TLR3),TBK1,干扰素调节因子3(IRF3)表达的影响,阐释其可能的抗RSV机制,为临床使用提供实验依据。方法:本实验分为6组,分别为空白组,RSV感染细胞模型组,板蓝根含药血清低、中、高体积分数组(40%,50%,60%板蓝根含药血清),利巴韦林含药血清组(70%利巴韦林含药血清)。除空白组外,其余各组接种RSV造成病毒感染模型,采用板蓝根含药血清或利巴韦林含药血清进行干预,12 h后收集细胞及其上清液,酶联免疫吸附测定(ELISA)法检测细胞上清液中IFN-β含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测RAW264.7细胞中TLR3,TBK1,IRF3和IFN-βmRNA的表达;蛋白质免疫印迹(Western blot)法检测TLR3,TBK1,IRF3和p-IRF3蛋白的表达。结果:RSV感染RAW264.7细胞12 h后,与空白组比较,模型组TLR3,TBK1和IFN-βmRNA的表达显著升高,IFN-β,TLR3,TBK1和p-IRF3蛋白的表达也显著升高(P<0.01),IRF3 mRNA和蛋白均无明显变化;与模型组比较,板蓝根含药血清中、高体积分数组能够显著下调RSV诱导的TLR3,TBK1和IFN-βmRNA的高表达(P<0.05,P<0.01),同时明显下调RSV诱导的TLR3,TBK1和p-IRF3蛋白的高表达(P<0.05,P<0.01),板蓝根含药血清各体积分数组的下调作用低于利巴韦林含药血清组,两者对IRF3 mRNA和蛋白均无明显调控作用。结论:RSV能够诱导TLR3信号转导通路的激活从而促进IFN-β的表达,而板蓝根含药血清通过下调TLR3信号通路关键信号分子TLR3,TBK1,p-IRF3使IFN-β适度表达。
OBJECTIVE: To investigate the effects of Banlangen-containing serum on interferon-β, TLR3, TBK1, IRF3 in RAW264.7 cells infected with respiratory syncytial virus (RSV) Expression of the impact of its possible anti-RSV mechanism to provide experimental evidence for clinical use. Methods: The experiment was divided into 6 groups: blank group, RSV infected cell model group, low, medium and high volume fraction of Radix Isatidis (40%, 50%, 60% Radix isatidis containing serum), Ribavir Lin containing serum group (70% Ribavirin containing serum). In addition to the blank group, the remaining groups inoculated with RSV virus infection caused by the model, using Radix containing medicated serum or ribavirin serum intervention, after 12 h cells and supernatants were collected, enzyme-linked immunosorbent assay (ELISA) Method to detect the content of IFN-β in the supernatant of the cells. The expression of TLR3, TBK1, IRF3 and IFN-βmRNA in RAW264.7 cells was detected by real-time PCR. Western blot ) Method was used to detect the expression of TLR3, TBK1, IRF3 and p-IRF3 protein. Results: Compared with the blank group, the expression of TLR3, TBK1 and IFN-βmRNA in the model group was significantly increased and the expression of IFN-β, TLR3, TBK1 and p-IRF3 protein were also significantly increased in RSV-infected RAW264.7 cells (P <0.01), IRF3 mRNA and protein had no significant change. Compared with model group, the medium and high volume fraction of Radix isatidis could significantly down-regulate RSV-induced high expression of TLR3, TBK1 and IFN-βmRNA , P <0.01). At the same time, the downregulation of TLR3, TBK1 and p-IRF3 proteins induced by RSV was significantly decreased (P <0.05, P <0.01) Drug-containing serum group, both on IRF3 mRNA and protein no significant regulatory role. Conclusion: RSV can induce the activation of TLR3 signal transduction pathway and promote the expression of IFN-β. Banlangen-containing serum can moderately express IFN-β by down-regulating TLR3, TBK1 and p-IRF3 signaling molecules.