天然产物库中体外颉颃A型塞内卡病毒化合物的筛选及作用途径研究

Screening and Action Pathway of Compounds Antagonistic Senacavirus A in vitro from Natural Product Library

【目的】寻找对A型塞内卡病毒(SVA)具有抑制活性的天然产物,并深入研究其颉颃途径。【方法】将天然产物浓度统一稀释至10μmol/L,与荧光素酶重组塞内卡病毒(rSVA-NLuc)相结合,在天然产物小分子库中筛选出具有荧光素酶抑制活性的化合物,采用实时荧光定量RT-PCR方法验证其活性,通过乳酸脱氢酶(LDH)法进行细胞毒性试验确定最大无毒浓度,结合病毒滴度测定、实时荧光定量RT-PCR、Western blotting、间接免疫荧光试验(IFA)等技术,在病毒感染周期的不同阶段,对其颉颃SVA的效果及途径进行研究。【结果】通过初筛,从136种天然产物中筛选出莫能霉素钠盐、孕酮、叶下珠次素、4-羟基德里辛和2-甲基雌酮5种能体外颉颃SVA的化合物。进一步通过实时荧光定量RT-PCR复核验证和细胞毒性检测,筛选出一种能体外颉颃SVA的天然产物——孕酮。与空白对照组相比,其在体外可导致SVA mRNA表达水平极显著下降(P<0.01),最大无毒浓度达50μmol/L。进一步研究表明,孕酮能在感染后8、12、24、36 h持续抑制SVA增殖,与对照组相比,在感染后24和36 h病毒滴度显著下降(P<0.05)。与对照组相比,不同浓度(10、20和50μmol/L)孕酮在吸附阶段导致SVA吸附水平极显著下降(P<0.01);在复制阶段使SVA VP3水平显著下降且使病毒滴度极显著下降(P<0.01);在释放阶段使释放胞外的病毒比例极显著下降(P<0.01);但对其他途径无显著影响。说明孕酮能在体外抑制SVA吸附、复制和释放,但对其入胞、组装途径无明显作用。【结论】本研究从天然产物小分子库中筛选出一种具有低细胞毒性且对SVA具有较强颉颃作用的天然产物——孕酮。该产物对SVA吸附、复制和释放阶段具有抑制作用,从而颉颃SVA的感染。本研究为促进抗SVA药物的开发和研究SVA的感染偏好性提供了科学参考。

【Objective】The experiment was aimed to search for natural products with inhibitory activity against Senecavirus A(SVA)and further investigate its antagonistic pathway.【Method】The concentration of the natural products were uniformly diluted to 10μmol/L.Combined with the recombinant Senecavirus A with luciferase(rSVA-NLuc),compounds with luciferase inhibitory activity were screened from the natural products library,and their activity was further verified using Real-time quantitative RT-PCR.The maximum non-toxic concentration was determined by cytotoxicity testing using lactate dehydrogenase(LDH)method.Virus titer determination,Real-time quantitative RT-PCR,Western blotting,IFA and other techniques were used to study the effect and pathway of antagonism against SVA.【Result】Five compounds,monensin sodium salt,progesterone,hypophyllanthin,4-hydroxyderricin and 2-methoxyestrone,which could antagonize SVA in vitro,were screened from 136 natural products.Progesterone,a natural product that could antagonize SVA in vitro,was screened by Real-time quantitative RT-PCR and cytotoxicity detection.Compared with blank control group,the mRNA expression level of SVA was extremely significantly decreased in vitro(P<0.01),and the maximum non-toxic concentration reached 50μmol/L.Further study showed that progesterone could continuously inhibit the proliferation of SVA at 8,12,24 and 36 h after infection,and the virus titer was significantly decreased at 24 and 36 h after infection compared with control group(P<0.05).Compared with control group,different concentrations of progesterone(10,20 and 50μmol/L)extremely significantly decreased the adsorption level of SVA at the adsorption stage(P<0.01).In the replication stage,the level of SVA VP3 and virus titer were extremely significantly decreased(P<0.01).The proportion of released extracellular virus extremely significantly decreased(P<0.01).But there was no significant effect on other approaches.These results indicated that progesterone could inhibit the adsorption,replication and release of SVA in vitro,but it had no obvious effect on its induction and assembly pathway.【Conclusion】In this study,progesterone,a natural product with low cytotoxicity and excellent antagonistic effect against SVA,was screened from the small molecule library of natural products.The product could inhibit the adsorption,replication and release of SVA,thus preventing the infection of SVA.This study provided a scientific reference for promoting the development of anti-SVA drugs and studying the infection bias of SVA.