虎杖苷与白藜芦醇体外抗肠道病毒71型的初步研究

Preliminary study on the antiviral activities of polydatin and resveratrol against enterovirus 71 in vitro

目的检测虎杖苷与白藜芦醇体外抗肠道病毒71型(EV71)活性,分析其与NF-κB信号通路的关系。方法采用噻唑蓝(MTT)比色法检测虎杖苷与白藜芦醇对人横纹肌肉瘤(RD)细胞的毒性以及抗EV71作用;western blot分析EV71感染RD细胞后EV71/衣壳蛋白VP1、NF-κBp50、NF-κBp65蛋白表达及磷酸化水平,Luminex流式液相芯片技术检测RD细胞分泌IL-6、肿瘤坏死因子-α(TNF-α)、干扰素-α(IFN-α)和IFN-β的水平。结果虎杖苷与白藜芦醇的50%病毒抑制细胞毒性浓度(CC50)分别为542.76μg/mL和85.73μg/mL,50%病毒抑制浓度(IC50)分别为979.66μg/mL和21.36μg/mL,治疗指数(TI)为0.55和4.01。虎杖苷对EV71的抑制作用较弱,250μg/mL时的病毒抑制率为(23.57±3.64)%。白藜芦醇可抑制RD细胞EV71/VP1合成,作用8、12、24 h的VP1表达量差异具有统计学意义(t值分别为5.968、5.364和4.922,P均<0.05)。与EV71感染组比较,EV71+白藜芦醇组NF-κBp50磷酸化水平降低,差异有统计学意义(8、12、24 h的t值分别为11.642、12.284和28.844,P均<0.05);NF-κBp65磷酸化水平降低,也具有统计学意义(4、8、12、24 h的t值分别为9.017、16.883、10.287和6.922,P均<0.05)。白藜芦醇可显著降低EV71感染的RD细胞分泌IL-6、TNF-α和IFN-β(t值分别为23.589、34.564和13.296,P均<0.05)。结论白藜芦醇可通过抑制RD细胞的NF-κB信号通路干扰EV71复制。

Objective To investigate the antiviral activities of polydatin and resveratrol against enterovirus 71 （EVT1） in vitro and its correlation with nuclear factor kappa B （NF-KB） signaling pathway. Methods The cytotoxicity and antiviral effect of polydatin and resveratrol on EV71 infected-human embryonic rhabdomyosarcoma （RD） cells were detected by MTT（3- [ 4,5-dimethylthiazol-2-yl ] -2, 5-diphenyl tetrazolium bromide） assay. The EV71/viral protein 1 （VP1） expression and the total and phosphorylation levels of NF- κBp50 and NF-κBp65 in RD cells were detected by western blot. In addition, the concentrations of interleukin 6 （IL-6）, tumor necro- sis factor α（ TNF-α）, interferon α （IFN-α） and INF-β in RD cells were detected by Luminex Liquid chip. Results The cytotoxic concentrations of 50% （ IC50 ） of polydatin and resveratrol were 542.76 μg/mL and 85.73 μg/mL, respectively. Meanwhile, the inhibition concentrations of 50% （ IC50 ） of polydatin and resveratrol were 979.66μg/mL and 21.36 μg/mL, respectively, and the therapeutic index（TI） were 0.55 and 4.01, respectively. There was a weaker inhibitory rate of polydatin against EV71 replication, with （23.57±3.64） % of inhibition for 250 μg/mL of polydatin. However, resveratrol was able to inhibit the synthesis of EV71/VP1 in RD cells, and there was significant difference in the VP1 expression in RD cells with EV71 infection and resveratrol pretreatment for 8, 12 and 24 h（ t = 5. 968, 5. 364 and 4. 922, respectively, P 〈 0.05 ）. Compared with EV71-infected RD ceils, the phosphorylation levels of NF-KBp50 （t = 11. 642, 12. 284 and 28. 844 for 8, 12 and 24 h, respectively, P 〈 0.05 ） and NF-κBp65 （t = 9. 017, 16. 883, 10. 287 and 6. 922 for 4, 8, 12 and 24 h, respectively, P 〈0.05） in RD ceils with EV71 infection and resveratrol pretreatment decreased significantly. In addition, resveratrol could significantly inhibit EV71-infected RD cells to secret IL-6, TNF-α and IFNβ（t = 23. 589, 34. 564 and 13. 296, respectively, P 〈 0.05 ）. Conclusion Resveratrol may interfere EV71 replication via inhibiting the NF-κB signaling pathway of RD ceils.