基于网络药理学和体外实验研究橙皮苷抗轮状病毒的作用机制

Anti-rotavirus mechanism of hesperidin based on network pharmacology and in vitro experiments

目的基于网络药理学和体外实验揭示橙皮苷抗轮状病毒(RV)的作用及机制。方法采用RV感染Caco-2、MA104两种不同细胞株评价橙皮苷的抗RV吸附、直接抑制RV以及抗RV生物合成作用;利用TCMSP、GeneCards、SwissTarget Prediction、OMIM以及DisGeNET获取橙皮苷和RV的潜在靶点,得到橙皮苷-RV交集靶点后进行STRING分析,导入Cytoscape后得到抗RV的关键靶点,然后进行KEGG富集分析并构建靶点-通路图;利用AutoDock Vina进行分子对接,预测橙皮苷与关键作用靶点的结合度;RT-qPCR法检测PIK3CA、Akt1 mRNA的表达;Western blot检测细胞中PI3K、Akt蛋白表达水平。结果细胞实验结果显示,橙皮苷具有抗RV生物合成作用,无抗RV吸附和直接抑制RV作用。通过网络药理学分析,最终获得橙皮苷的潜在靶点361个,RV相关靶点445个,共同靶点38个。从蛋白质-蛋白质相互作用(PPI)网络中鉴定橙皮苷抗RV的核心靶点为CASP3、EGFR、FN1、PIK3CA等。分子对接结果显示橙皮苷和关键靶标有较好的结合能力。RT-qPCR表明橙皮苷各药物组浓度均可上调PIK3CA、Akt1 mRNA的表达。Western blot结果表明橙皮苷显著上调细胞p-PI3K、p-Akt蛋白的表达,而PI3K和Akt蛋白表达量无明显变化。结论通过网络药理学分析、分子对接验证和实验研究,表明橙皮苷不仅具有抗RV生物合成作用,而且可能通过调控PI3K/Akt信号通路发挥抗RV的作用。

Objective To determine the action and mechanism of hesperidin against rotavirus(RV)based on network pharmacology and in vitro experiments.Methods Two cell lines,RV-infected Caco-2 and MA104,were used to evaluate the inhibition of RV attachment,direct inhibition of RV and inhibition of RV replication effects of hesperidin.TCMSP,GeneCards,SwissTarget Prediction,OMIM and DisGeNET were used to obtain the potential targets of hesperidin and RV,and hesperidin-RV crossover targets were analyzed by STRING and imported into Cytoscape to obtain the key targets against RV.KEGG enrichment analysis was performed and the target-pathway map was established.Molecular docking was performed with AutoDock Vina to predict the binding of hesperidin to the key targets of action.The expression of PIK3CA and Akt1 mRNA was detected by RT-qPCR.The expression levels of PI3K and Akt proteins in the cells were detected by Western blot.Results The cellular assay showed that hesperidin had anti-RV replication effect without anti-RV adsorption and direct inhibition of RV.The network pharmacological analysis revealed 361 potential targets of hesperidin,445 RV-related targets and 38 common targets.The core targets of hesperidin against RV were identified from the protein-protein interaction(PPI)network(CASP3,EGFR,FN1,and PIK3CA).The molecular docking showed that hesperidin and key targets had good binding ability.RT-qPCR showed that hesperidin upregulated the expression of PIK3CA and Akt1 mRNA at all concentrations.Western blot showed that hesperidin significantly upregulated the expression of p-PI3K and p-Akt protein,while the expression of PI3K and Akt protein did not change obviously.Conclusion Based on network pharmacology,molecular docking validation and experimental studies,hesperidin not only has anti-RV replication effect,but also exerts anti-RV effect through regulating PI3K/Akt signaling pathway.