治疗痛风性关节炎的四妙散活性成分靶基因筛选及生物学功能分析

Screening of target genes of Simiao powder in treatment of gouty arthritis and analysis of their biological functions

目的筛选治疗痛风性关节炎(GA)的四妙散活性成分靶基因,并分析其生物学功能。方法采用中药系统药理学数据库(TCMSP)收集四妙散的成分,然后根据ADME参数(OB≥30%、DL≥0.18)筛选其可能的活性成分及其靶基因;利用Gene Cards、OMIM和DrugBank数据库获取GA发病相关的靶基因;将四妙散活性成分调控的靶基因与GA发病相关的靶基因进行映射取交集,得到四妙散治疗GA的潜在作用靶基因。利用Cytoscape3.7. 2软件分析获得关键活性成分;根据蛋白互作(PPI)网络筛选出关键靶基因;使用DAVID数据库对作用靶基因进行基因本体(GO)功能和京都基因与基因组百科全书(KEGG)通路富集分析;采用Autodock1.5. 6软件对四妙散治疗GA的关键活性成分与关键靶基因进行分子对接以评价网络分析预测的可靠性。结果共筛选四妙散活性成分65个,活性成分调控靶基因197个;GA发病相关靶基因211个;四妙散治疗GA的作用靶基因23个;"单味药—活性成分—作用靶基因"网络共包括47个节点(25个活性成分和22个作用靶基因)和93条边,该网络中度值排名前5位的活性成分分别是槲皮黄素、吴茱萸次碱、汉黄芩素、山柰酚、黄芩素;PPI网络关键靶基因5个,包括泛素连接酶CUL7蛋白(CUL7)、β-微管蛋白(TUBB)、生长因子受体结合蛋白2(GRB2)、核因子κB1(NF-κB1)、重组人泛素结合酶E2I(UBE2I);GO富集分析得到P<0.05条件下条目共936条,其中生物过程共有888条,细胞组成共有15条,分子功能共有33条,涉及脂多糖的细胞反应、对细菌来源分子的反应、细胞对生物刺激的反应等生物学过程;KEGG信号通路分析共获得信号通路82条,包括IL-17、TNF、AGE-RAGE、NOD样受体、C型凝集素受体、NF-κB、Toll样受体等信号通路;关键活性成分与关键靶基因间的结合能远小于-5.0 kJ/mol。结论四妙散治疗GA的关键活性成分为槲皮黄素、吴茱萸次碱、汉黄芩素、山柰酚、黄芩素,其可有效通过CUL7、TUBB、GRB2等关键靶基因作用于脂多糖的细胞反应,对细菌来源分子的反应等生物学过程,及IL-17、TNF等信号通路参与调控免疫—炎症反应、软骨细胞增殖分化与凋亡、机体抗氧化应激反应等分子机制,协同发挥治疗作用。

Objective To screen out the effective genes of Simiao powder in the treatment of gouty arthritis(GA),and to analyze the biological functions of the acting genes.Methods We used TCM System Pharmacology Database(TCMSP) to collect the ingredients of Simiao powder,and then screened its possible active ingredients and target genes according to ADME parameters(OB≥30%,DL≥0.18);Gene Cards,OMIM,and DrugBank databases were used to obtain target genes related to the pathogenesis of GA;the target genes regulated by the active ingredients of Simiao powder and the target genes related to the pathogenesis of GA were mapped and intersected to obtain potential target genes in the treatment of GA.We used Cytoscape 3.7.2 software to analyze and obtain the key active ingredients,screened out key target genes according to protein interaction(PPI) network,and used DAVID database to perform Gene Ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways on target genes for enrichment analysis.Autodock 1.5.6 software was used to molecularly dock the key active components of Simiao powder with key target genes to evaluate the reliability of network analysis prediction.Results A total of 65 active ingredients of Simiao powder were screened out,and197 target genes were regulated by active ingredients;211 target genes related to the pathogenesis of GA and 23 target genes of Simiao powder in treatment of GA were screened out;the network "Single medicine-active ingredients-target genes" consisted of 47 nodes(25 active ingredients and 22 target genes) and 93 edges,and the top 5 active ingredients in the network with a median value were quercetin,evodia,wogonin,kaempferol,and baicalein;there were 5 key target genes of PPI network,including ubiquitin ligase CUL7 protein(CUL7),β-tubulin(TUBB),growth factor receptor binding protein 2(GRB2),nuclear factor κB1(NF-κB1),and recombinant human ubiquitin-conjugating enzyme E2 I(UBE2 I);GO enrichment analysis revealed a total of 936 entries under the condition of P<0.05,including 888 biological processes,15 cellular components,and 33 molecular functions,and the biological processes involved lipopolysaccharide cell response,bacterial-derived molecules,cell response to biological stimuli,etc.;KEGG signaling pathway analysis obtained a total of 82 signaling pathways,including IL-17,TNF,AGE-RAGE,and NOD-like receptors,C-type lectin receptor,NF-κB,Toll-like receptor and other signaling pathways;the binding energy between key active ingredients and key target genes was much less than-5.0 kJ/mol.Conclusions The key active ingredients of Simiao powder in treatment of GA are quercetin,evodia,wogonin,kaempferol,and baicalein,which can effectively play a synergistic therapeutic effect by acting on the cell response of lipopolysaccharide through key target genes such as CUL7,TUBB,and GRB2,biological processes such as the response to bacteria-derived molecules,and signaling pathways such as IL-17 and TNF which are involved in the regulation of immune-inflammatory response,chondrocyte proliferation and differentiation and apoptosis,and the body’s anti-oxidative stress response and other molecular mechanisms.