基于网络药理学和转录组学及实验验证探究菲牛蛭冻干粉抗高尿酸血症的作用机制

Mechanism of Poecilobdella Manillensis Lyophilized Powder on Hyperuricemia Based on Network Pharmacology, RNA-seq Technology and Experimental Validation

目的 基于网络药理学和转录组学的方法,结合动物实验验证,探究菲牛蛭冻干粉(Poecilobdella manillensis lyophilizedpowder,以下简称SZ)抗高尿酸血症(hyperuricemia, HUA)的多靶点作用机制。方法利用Symmap、SwissTargetPrediction、Pharmmapper数据库,获得SZ潜在活性成分以及对应的靶点;通过Gene Cards和OMIM数据库获得HUA相关靶点;取交集映射得到SZ和HUA的共同靶点,利用Cytoscape 3.9.0软件构建药物成分-疾病靶点相互作用网络,结合STRING数据库构建蛋白相互作用网络并筛选核心靶点;使用DAVID数据库对交集靶点进行GO生物功能注释和KEGG通路富集分析。采用氧嗪酸钾联合高嘌呤饮食构建HUA小鼠模型,通过生化指标检测、ELISA法探究SZ对HUA小鼠的影响,qRT-PCR法验证RNA-Seq及网络药理学富集分析结果。结果 网络药理学分析表明SZ中有11种主要生物活性物质,72种潜在靶点参与治疗HUA,涉及多种生物过程和不同的信号通路。实验表明,SZ通过抑制黄嘌呤氧化酶活性,显著降低了HUA小鼠的血清尿酸、肌酐和尿素氮水平。SZ还降低了URAT1水平,同时提高了ABCG2的水平。RNA测序分析发现,模型组与治疗组、对照组与模型组和对照组与治疗组分别有112、536和107个差异表达基因。qRT-PCR结果表明,SZ下调了Th17细胞分化相关的基因以及IL-17和PI3K/Akt信号通路上基因mRNA的表达。结论 SZ具有降尿酸作用,其作用机制可能是抑制肝脏黄嘌呤氧化酶活性,下调URAT1水平,上调ABCG2水平,影响Th17细胞的分化,进而影响IL-17信号通路,从而减轻炎症反应。

OBJECTIVE To investigate the multi-target mechanism of action of Poecilobdella manillensis lyophilized powder(SZ)against hyperuricemia(HUA)based on network pharmacology and transcriptomics approaches,combined with animal experiments.METHODS Utilizing Symmap,SwissTargetPrediction,and Pharmmapper databases,the potential active components and corresponding targets of SZ were obtained.Through the Gene Cards and OMIM databases,HUA-related targets were obtained.By taking the intersection mapping,the common targets of SZ and HUA were identified.Cytoscape 3.9.0 software was used to construct a drug component-disease target interaction network,and in combination with the STRING database,a protein interaction network was built and core targets were screened.The DAVID database was used to perform GO biological function annotation and KEGG pathway enrichment analysis on the intersecting targets.A mouse model of HUA was constructed using potassium oxyzate combined with high purine diet,and the effects of SZ on these mice were examined using ELISA and biochemical index detection.qRT-PCR was used to validate the results of RNA-Seq and network pharmacology enrichment analysis.RESULTS Network pharmacological analysis identified 11 major bioactive substances in SZ and 72 potential targets involved in the treatment of hyperuricemia,involving multiple biological processes and different signaling pathways.It was shown that SZ significantly reduced serum uric acid,creatinine and urea nitrogen levels in hyperuricemic mice by inhibiting xanthineoxidase activity.SZ also reduced the levels of URAT1 while increasing the levels of ABCG2.RNA sequencing analysis revealed that there were 112,536 and 107 differentially expressed genes in the model group vs treated group,control group vs model group and control group vs treated group,respectively.qRT-PCR results indicated that SZ downregulated the expression of genes related to Th17 cell differentiation as well as mRNA of genes on IL-17 and PI3K/Akt signaling pathways.CONCLUSION SZ has therapeutic effects on hyperuricemia. The mechanism of action maybe related to the inhibition of hepatic xanthineoxidase activity, down-regulation of URAT1 levels, up-regulation of ABCG2 levels, affecting the differentiation of Th17 cells and thus the IL-17 signaling pathway, thereby reducing the inflammatory response.