外源性白介素17A激活PLC/PRF/5细胞内PI3K/AKT信号转导通路抑制HBsAg表达的研究

Exogenous interleukin 17A activates PI3K/AKT signal transduction pathway in PLC/PRF/5 cells to inhibit HBsAg expression

目的:研究外源性白介素17A(IL-17A)抗HBV活性及其可能存在的机制。方法:以分泌乙型肝炎病毒表面抗原(HBsAg)的PLC/PRF/5细胞为模型,通过外源性IL-17A处理细胞48 h,化学发光微粒子免疫技术和细胞免疫荧光技术分析细胞上清液和细胞内HBsAg含量,并以荧光定量PCR法(FQ-PCR)和Western blot检测细胞内抗病毒基因及其蛋白含量;pISRE-TA-luc转染至细胞并以IL-17A处理细胞48 h,荧光素酶活性检测干扰素刺激反应元件(ISRE)活性;以Western blot分析IL-17A处理细胞48 h后PI3K/AKT信号转导通路分子蛋白表达水平,并以信号转导通路抑制剂LY294002进一步验证IL-17A能否通过PI3K/AKT信号转导通路发挥抗HBV活性。结果:外源性IL-17A处理PLC/PRF/5细胞后,细胞上清液和细胞内HBsAg含量显著降低,同时细胞内抗病毒蛋白MxA和OAS表达明显升高;IL-17A不能增强细胞内ISRE活性,但激活PI3K/AKT信号转导通路;通过LY294002抑制PI3K/AKT信号转导通路后,IL-17A抗HBV活性及其诱导抗病毒蛋白MxA和OAS表达效应显著被抑制。结论:IL-17A能够通过激活PLC/PRF/5细胞内PI3K/AKT信号转导通路诱导抗病毒蛋白表达,从而发挥抗HBV活性。

Objective:To investigate the anti-HBV activity of exogenous interleukin 17A(IL-17A)and its possible mecha-nism.Methods:The PLC/PRF/5 cells secreting hepatitis B virus surface antigen(HBsAg)as the model,the cells were treated with exogenous IL-17A for 48 hours.The expression level of HBsAg in cell supernatant and intracellular space was analyzed by chemilumi-nescent microparticle immunoassay and immunocytochemistry.Moreover,the expression levels antiviral genes and proteins in cells were detected by fluorescence quantitative PCR(FQ-PCR)and Western blot.pISRE-TA-luc was transfected into cells and treated with IL-17A for 48 hours,followed by luciferase activity assay to measure the activity of the interferon-stimulated response element(ISRE).The molecular protein levels of PI3K/AKT signal transduction pathway in PLC/PRF/5 cells treated by IL-17A alone or combi-nation with LY294002,a PI3K/Akt pathway inhibitor,were analyzed to further verify whether IL-17A can exert anti HBV activity through PI3K/AKT signal transduction pathway.Results:After exogenous IL-17A treated PLC/PRF/5 cells,the expression level of HBsAg in cell supernatant and intracellular space was decreased significantly,while the expression levels of intracellular antiviral pro-teins MxA and OAS increased significantly.IL-17A could not enhance ISRE activity,while activated PI3K/AKT signal transduction pathway.After blocking PI3K/AKT signal transduction pathway by LY294002,the anti HBV activity of IL-17A and its induced expres-sion of antiviral proteins MxA and OAS were significantly inhibited.Conclusion:IL-17A could induce the expression of antiviral pro-tein by activating PI3K/AKT signal transduction pathway to exert anti HBV activity in PLC/PRF/5 cells.