白皮杉醇体外抗轮状病毒的作用研究

Anti-rotavirus effect of piceatannol in vitro

目的研究白皮杉醇(PIC)体外抗轮状病毒(RV)的作用及机制。方法采用RV-Wa株、RV-SA11株感染MA104细胞模型,MTT法评价白皮杉醇抗RV吸附、直接抑制RV以及抗RV生物合成作用;TCID50法检测白皮杉醇处理前后病毒滴度的变化;利用分子对接预测白皮杉醇与RV-Wa株VP8*蛋白的结合度;RT-qPCR法检测VP6 mRNA的表达;间接免疫荧光法检测VP6蛋白的表达;Western blot检测VP6、IκBα、NF-κBp65、p-NF-κBp65蛋白的表达;ELISA法检测细胞上清液中IL-1β、IL-6、TNF-α的含量。结果白皮杉醇具有直接抑制RV和抗RV生物合成的双重作用,无抗RV吸附作用。对白皮杉醇直接抑制RV作用进行研究,结果显示,白皮杉醇处理后病毒滴度显著下降;分子对接结果显示,白皮杉醇与RV-Wa株VP8*蛋白有较好的结合能力。对白皮杉醇抗RV生物合成作用进行研究,RT-qPCR结果表明白皮杉醇可以下调VP6 mRNA的表达;Western blot结果表明,RV感染后经白皮杉醇处理可以下调VP6、p-NF-κBp65蛋白的表达,上调IκBα蛋白的表达;ELISA结果表明白皮杉醇可以降低细胞上清液中IL-1β、IL-6、TNF-α的含量。结论白皮杉醇具有直接抑制RV和抗RV生物合成的双重作用,可能通过与RV-Wa株VP8*蛋白相互作用阻止RV入侵细胞以及抑制NF-κBp65磷酸化减轻RV感染引起的炎症反应发挥抗RV作用。

Objective To determine the anti-rotavirus(RV)effect of piceatannol(PIC)in vitro and related mechanism.Methods The RV-Wa and RV-SA11 strains were used to infect the MA104 cell model,and MTT was used to assess the anti-RV adsorption,direct RV inhibition,and anti-RV biosynthesis effects of piceatannol.TCID50 was used to detect the change of virus titer before and after piceatannol treatment.Molecular docking was used to predict the degree of binding of piceatannol to the VP8*protein of the RV-Wa strain.The expression of VP6 mRNA was detected by RT-qPCR.Indirect immunoassay was used to detect the expression of VP6 protein.The expression of VP6,IκBα,NF-κBp65 and p-NF-κBp65 proteins was detected by Western blot.The levels of IL-1β,IL-6,and TNF-αin the cell supernatants were evaluated by ELISA.Results Piceatannol had a dual effect of direct RV inhibition and anti-RV biosynthesis,without anti-RV adsorption.The direct inhibition of RV by piceatannol was studied,which showed that the virus titer decreased greatly after piceatannol treatment.The molecular docking revealed that piceatannol and RV-Wa VP8*protein had a better binding capacity.The anti-RV biosynthesis effect of piceatannol was studied.RT-qPCR showed that piceatannol down-regulated the expression of VP6 mRNA.Western blot showed that RV infection treated with piceatannol down-regulated the expression of VP6 and p-NF-κBp65 proteins,and up-regulate the expression of IκBαprotein.ELISA showed that piceatannol reduced the levels of IL-1β,IL-6,and TNF-αin the cell supernatants.Conclusion Piceatannol has a dual effect of direct RV inhibition and anti-RV biosynthesis.The anti-RV effect may be achieved by limiting RV invasion into cells by association with the RV-Wa VP8*protein and lowering the phosphorylation of NF-κBp65,which attenuates the inflammatory responses generated by RV infection.