大黄抗肾脏纤维化的网络药理学及其活性成分的作用机制

Active Components and Mechanism of Rhei Radix et Rhizoma on Renal Fibrosis Based on Network Pharmacology

目的:通过网络药理学方法,预测中药大黄抗肾脏纤维化的活性成分、潜在靶点及通路,选取活性成分,对筛选出的靶基因进行体外实验验证。方法:检索中药系统药理学数据库与分析平台(TCMSP)与中药分子机制分析综合数据库(TCMID)获取并筛选大黄主要活性成分,利用相似度系综算法数据库(SEA),瑞士生物信息学研究所(SIB),Gene Cards数据库预测和筛选大黄抗肾脏纤维化的潜在作用靶点。采用String Version 10.5数据库构建靶点蛋白相互作用(PPI)网络;运用David 6.8软件对关键靶点进行基因本体论(GO)富集分析、京都基因与基因组百科全书(KEGG)通路分析。采用Cytoscape Version 3.6.0软件对关键蛋白相互作用网络、活性成分-关键靶点网络、活性成分-靶点-信号通路网络进行可视化分析。结合Malarcards数据库筛选出与肾脏纤维化高关联性信号通路。进一步采用细胞实验验证:选取HK-2细胞,利用转化生长因子-β1(TGF-β1)诱导细胞纤维化模型,予大黄酸干预细胞48 h,蛋白免疫印迹法(Western blot)检测缺氧诱导因子1α(HIF-1α),血管内皮生长因子(VEGF),血小板衍生生长因子受体-α(PDGFR-α),免疫荧光检测E-钙黏蛋白(E-cadherin),α-平滑肌肌动蛋白(α-SMA)蛋白表达,流式细胞术检测细胞凋亡。结果:筛选得到大黄活性成分17个,大黄抗肾脏纤维化潜在靶点424个,关键靶点5个,依次为蛋白激酶B(Akt)1,丝裂原激活的蛋白激酶(MAPK)3,EGFR,白细胞介素-6(IL-6),VEGFA;GO富集生物学过程主要涉及信号转导、细胞增殖、凋亡等生物过程;KEGG通路富集结果发现磷脂酰肌醇3-激酶(PI3K)/Akt,HIF-1α,VEGF,叉头转录因子(FoxO)等通路与大黄抗肾脏纤维化作用机制相关。体外实验验证表明,大黄酸抑制E-cadherin,α-SMA,HIF-1α,VEGF,PDGFR-α的表达水平,同时抑制肾小管上皮细胞凋亡,验证了网络药理学部分预测结果。结论:本研究体现了大黄多成分、多靶点、多途径的作用特点,其抗肾脏纤维化的作用机制可能与其抑制HIF-1α/VEGF/PDGFR-α信号转导途径,抑制细胞凋亡及肾小管上皮细胞上皮间质转化(EMT)有关。

Objective:To explore the active components,potential targets and signaling pathways of Rhei Radix et Rhizoma in the treatment of renal fibrosis based on the network pharmacology method,and then to verify the target genes in vitro.Method:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and Traditional Chinese Medicine Integrated Database(TCMID)were retrieved to obtain the main active ingredients of Rhei Radix et Rhizoma.The potential anti-renal fibrosis targets of Rhei Radix et Rhizoma were predicted by similarity ensemble approach(SEA),Swiss Institute of Bioinformatics(SIB)and GeneCards Database.Target protein-protein interaction(PPI)network was constructed by using String Version 10.5 database.David 6.8 software was used for gene ontology(GO)enrichment analysis and the Kyoto Encyclopedia of genes and genomes(KEGG)pathway enrichment analysis of the key targets.Cytoscape Version 3.6.0 software was used for visualized analysis of PPI network,active ingredient-key target network and the ingredient-target-signal pathway network.In combination with Malachards database,the signal pathways with high correlation with renal fibrosis were screened.Then,cell experiments were used for verification:HK-2 cells were selected to establish fibrosis model by transforming growth factor-β1(TGF-β1)stimulation.The cells were treated with rhein for 48 hours.Western blot assay was used to detect the protein expression level of hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),and platelet-derived growth factor receptor-α(PDGFR-α).Protein expression levels of E-cadherin andαsmooth muscle actin(α-SMA)were detected by immunofluorescence.Apoptosis was detected with flow cytometry.Result:Totally 17 active ingredients of Rhei Radix et Rhizoma and424 targets of anti-renal fibrosis effect were screened out,including five key targets:protein kinase B(Akt)1,mitogen activated protein kinases 3(MAPK3),epidermal growth factor receptor(EGFR),interleukin(IL)-6 and VEGFA in turn.The biological process of GO enrichment mainly involved signal transduction,cell proliferation and apoptotic process.The results of KEGG pathway enrichment showed that phosphatidylinositol 3-kinase(PI3 K)/Akt,HIF-1,VEGF,and forkhead transcription factor(FoxO)pathways were related to the anti-renal fibrosis mechanism of Rhei Radix et Rhizoma.Results of the in vitro experiment proved that rhein could inhibit the expression of E-cadherin,α-SMA,HIF-1α,VEGF and PDGFR-α.In addition,rhein inhibited apoptosis induced by TGF-β1 in HK-2 cells.Part of the prediction results of network pharmacology were verified.Conclusion:This study reflects the multi-component,multi-target and multi-pathway mechanism characteristics of Rhei Radix et Rhizoma.The mechanisms of its anti-renal fibrosis effects may be related to inhibiting HIF-1α/VEGF/PDGFR-αsignaling pathway,apoptosis and epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells.