鹿衔草活性成分对裂谷热病毒的抑制作用及其机制研究

Study on the inhibitory effect and mechanism of the active ingredients from Pyrola decorate Andres against Rift Valley fever virus

关键词:  鹿衔草  裂谷热病毒  槲皮素  增强型绿色荧光蛋白  分子对接

Key words:  Pyrola decorate Andres  RVFV  Quercetin  EGFP  Molecular docking

摘要:

目的：探究鹿衔草中活性化合物对裂谷热病毒（RVFV，Rift Valley fever virus）的抑制活性及作用机制。 方法：通过表达增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein，EGFP）的裂谷热病毒模型（RVFV-EGFP）对鹿衔草成分进行抗病毒活性筛选，并通过CCK-8法评价活性化合物的安全性。利用高内涵分析检测在不同时间点或不同阶段给药对宿主细胞EGFP阳性率的影响；通过RVFV-Gc蛋白介导的细胞-细胞融合评价化合物对RVFV膜融合的抑制作用。利用计算机分子对接技术预测活性化合物与RVFV糖蛋白Gn和Gc之间的相互作用。 结果：从鹿衔草中筛选出积雪草酸、铁冬青酸和槲皮素对RVFV有抑制效果，SI分别为1.52、0.78和4.68。槲皮素在预处理病毒、吸附期给药和感染后给药均能降低细胞EGFP阳性率（P＜0.01），其中感染后0 h给药抑制率达83.6%（P＜0.0001），而预处理细胞组无显著变化，进一步实验表明槲皮素能够抑制RVFV-Gc介导的合胞体的形成，提示槲皮素主要作用于病毒感染细胞的早期，即吸附和膜融合阶段。分子对接显示槲皮素与Gn和Gc的对接能量分别为-8.5和-5.2 kcal/mol，与Gn蛋白的GLY 10、THR 30、ASP 217、CYS 221、PHE 244，和Gc蛋白的CYS 691、LYS 748、ASN 1077氨基酸残基存在氢键作用。 结论：本研究从鹿衔草中筛选出抗RVFV活性化合物，对抑制RVFV潜在药物槲皮素进行了评价和机制研究，为天然产物抗RVFV药物研发提供了新思路。

Abstract:

Objective: To investigate the inhibitory activity and mechanism of active compounds from Pyrola decorate Andres against RVFV. Methods: The antiviral activity of compounds from Pyrola decorate Andres was screened by expressing enhanced green fluorescent protein RVFV model (RVFV-EGFP). Meanwhile the safety of active compounds was evaluated by CCK-8 assay. And then, high-content analysis was used to detect the effect of drug administration at different time points or stages on the EGFP positive rate of host cells. Furthermore, the inhibitory effect of compounds on RVFV membrane fusion was evaluated by interfering with RVFV-GC protein-mediated cell-cell fusion. Additionally, computer molecular docking was used to predict the interaction between the active compounds and RVFV-Gc and Gn protein. Results: Asiatic acid, rutundic acid and quercetin showed inhibitory effects on RVFV with SI of 1.52, 0.78 and 4.68 respectively. And pretreated virus, adsorption period administration and post-infection administration with quercetin could significantly reduce the EGFP-positive rate of the cells (P < 0.01), while there was no significant change observed in the pretreated cells. Moreover, the inhibition rate was 83.6% at 0 h after infection (P < 0.0001). Further experiments showed that quercetin was able to inhibit RVFV-Gc protein-mediated cell-cell membrane fusion, suggesting that quercetin mainly acts in the early stages of virus infection, namely adsorption and membrane fusion stages. Molecular docking showed that the docking energy of quercetin with Gn and Gc was -8.5 kcal/mol and -5.2 kcal/mol, respectively. Besides, quercetin had hydrogen bond with GLY 10, THR 30, ASP 217, CYS 221, PHE 244 of Gn protein and CYS 691, LYS 748, ASN 1077 of Gc protein. Conclusions: In this study, the inhibitory effect and mechanism of the active ingredients from Pyrola decorate Andres against Rift Valley fever virus were screened, among which quercetin was chosen as a potential drug to inhibit RVFV. It provides a new idea for the research and development of natural products anti-RVFV drugs.