联合IFN-α与U0126的抗EV-A71作用及其机制研究

Combination therapy of IFN-α with U0126 against EV-A71 infection and its mechanism

目的在细胞学水平明确IFN-α和胞外信号调节激酶(extracellular signal-regulated kinase, ERK)通路抑制剂U0126联合用药对肠道病毒A71(EV-A71)感染的作用及其可能机制。方法利用病毒致细胞病变效应、病毒终点滴定实验以及Western blot,确定IFN-α和U0126联合用药对EV-A71抗病毒效果,对细胞干扰素(interferon, IFN)受体及其下游信号通路重要蛋白水平、ERK通路活性的影响。结果 IFN-α和U0126联合用药能有效发挥抗EV-A71增殖的作用(P<0.01),同时也能有效抑制ERK通路磷酸化活性、阻断EV-A71 2Apro介导的I型干扰素受体1(interferon alpha receptor 1, IFNAR1)表达水平下调(P<0.001),并上调IFN信号通路重要分子eIF2α磷酸化(P<0.001)。此外,利用ERK抑制剂(U0126和sorafenib)或特异性siRNA分别阻断ERK磷酸化活性后,可显著阻断肠道病毒2Apro介导的eIF4GI切割和IFNAR1表达下调的作用,同时受染细胞EV-A71复制也显著下降。结论 IFN-α和U0126联合用药可通过有效地抑制ERK通路,抑制2Apro依赖的切割eIF4GI和下调IFNAR1表达的作用,使得外源IFN-α能更有效与细胞膜上IFNAR结合,有效激活IFN抗病毒信号通路,从而发挥IFN抗EV-A71蛋白翻译及增殖作用。

Objective To clarify the effect and its possible mechanism of the combination of IFN-α with ERK pathway inhibitor U0126 on enterovirus A71 infection in RD cell model. Methods The cytopathic effect induced by the EV-A71, the end-point titration experiment for the viral titers, and the Western blot for viral and host cell protein expression, were applied in this study. Results The results showed that the combination therapy of IFN-α with U0126 played an obvious effect on anti-EV-A71 proliferation(P<0.01), coincided with the significant inhibition of the phosphorylation activity of ERK pathway, blocking the down-regulation of IFNAR1 expression by EV-A71 2 Apro(P<0.001), and up-regulated the phosphorylation of eIF2α(P<0.001), an essential molecule of the interferon signaling pathway. In addition, under the condition of blocking the ERK pathway by ERK inhibitor(U0126 and sorafenib) or specific siRNAs, the cleavage of eIF4 GI and down-regulation of interferon receptor IFNAR1 mediated by enterovirus 2 Apro were inhibited, accompanied by enterovirus replication in host cells. Conclusion Our results indicated that the combination therapy could effectively inhibit the ERK pathway and the activity of viral 2 Apro, accordingly downregulate the effect of 2 Apro regulation on eIF4 GI and IFNAR1, favoring the bind of exogenous IFN-α with IFNAR1, and then activating the interferon antiviral signal pathway, to inhibit viral protein expression and virus proliferation. Our study provides new ideas for clinical interferon treatment of severe disease caused by EV-A71 infection.