柯麗青 成文翔 林結(jié)桃 陳建海 鞏勛 姜泉 張鵬
摘要?目的:清熱活血在治療類風(fēng)濕性關(guān)節(jié)炎(RA)中效果顯著,但其成分多樣,作用機(jī)制不明,通過網(wǎng)絡(luò)藥理學(xué)方法探討清熱活血方抗RA的作用機(jī)制。方法:利用中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫與分析平臺(TCMSP)篩選藥物成分并利用SwissTargetPrediction數(shù)據(jù)庫預(yù)測藥物靶點,同時通過藥物數(shù)據(jù)庫(Drugbank)和在線人類孟德爾遺傳數(shù)據(jù)庫(OMIM)收集疾病靶點。將藥物靶點與疾病靶點取交集,拓展并拓?fù)浍@取關(guān)鍵靶點,利用DAVID數(shù)據(jù)庫對關(guān)鍵靶點進(jìn)行基因本體論(GO)分類富集分析和京都基因和基因組百科全書(KEGG)通路富集分析,通過Centiscape計算獲取hub節(jié)點。結(jié)果:從清熱活血方中篩選出活性成分31個,預(yù)測靶點419個,數(shù)據(jù)庫挖掘疾病靶點共430個。將藥物靶點與疾病靶點取交集、拓?fù)浞治霁@得清熱活血方治療類風(fēng)濕性關(guān)節(jié)炎的關(guān)鍵靶點113個,GO分析共獲得生物過程、分子功能、細(xì)胞組分共479個富集結(jié)果。KEGG富集表明清熱活血方可能通過MAPK、Estrogen、PI3K-AKT、Thyroid hormone、Neurotrophin、HIF-1、ErbB、FoxO、B cell receptor等通路干預(yù)RA。拓?fù)涞玫紺ULI、EP300、HSP90AA1、TP53、HSPA8、CDC5L、UBC、RPS27A共8個干預(yù)RA的Hub節(jié)點。結(jié)論:清熱活血方可能通過多靶點、多通路進(jìn)行調(diào)控,達(dá)到治療類風(fēng)濕性關(guān)節(jié)炎的作用。
關(guān)鍵詞?清熱活血方;類風(fēng)濕性關(guān)節(jié)炎;網(wǎng)絡(luò)藥理學(xué);信號通路;作用機(jī)制;土茯苓;金銀花;丹參
Abstract?Objective:Qingre Huoxue Formula has significant effects in the treatment of rheumatoid arthritis,but its components are diverse and the mechanism of action is unknown.Therefore,the mechanism of Qingre Huoxue Formula′s anti-RA effect was explored through network pharmacology.Methods:The Chinese medicine system pharmacology database and analysis platform (TCMSP) was used to screen drug components and used SwissTargetPrediction database to predict drug targets.Meanwhile,disease targets were collected through Drugbank and online human Mendelian genetic database (OMIM).Intersect drug targets with disease targets,expand and topologically obtain key targets,use the DAVID database to carry out gene ontology (GO) taxonomic enrichment analysis and Kyoto gene and genome encyclopedia (KEGG) pathway enrichment analysis for key targets,and finally obtain hub targets through Centiscape calculation.Results:A total of 31 active ingredients were selected from Qingre Huoxue Formula,419 targets were predicted,and 430 disease targets were mined in the database.The key targets of Qingre Huoxue Formula for the treatment of rheumatoid arthritis were obtained by intersecting drug targets with disease targets and topological analysis.GO analysis obtained a total of 479 enrichment results of biological processes,molecular functions,and cellular components.KEGG enrichment indicated that Qingre Huoxue Formula may interfere with RA through MAPK,Estrogen,PI3K-AKT,Thyroid hormone,Neurotrophin,HIF-1,ErbB,F(xiàn)oxO,B cell receptor and other pathways.A total of 8 Hub nodes interfering with RA were obtained from the topology by CULI,EP300,HSP90AA1,TP53,HSPA8,CDC5L,UBC,and RPS27A.Conclusion:Qingre Huoxue Formula may play an anti-rheumatoid arthritis role by regulating multi-target and multi-pathway.
Keywords?Qingre Huoxue Formula; Rheumatoid arthritis; Network pharmacology; Signal pathway; Mechanism of action; Smilacis Glabrae Rhixoma; Lonicerae Japonicae Flos; Radix Salviae
中圖分類號:R285;R255.6;R593.22文獻(xiàn)標(biāo)識碼:Adoi:10.3969/j.issn.1673-7202.2021.22.005
類風(fēng)濕性關(guān)節(jié)炎(Rheumatoid Arthritis,RA)是常見的風(fēng)濕免疫病,全世界發(fā)病率為0.5%~1.0%,造成嚴(yán)重的個人和社會負(fù)擔(dān)[1]。目前常用非甾體抗炎藥、糖皮質(zhì)激素、甲氨蝶呤以及各類生物制劑等臨床干預(yù)[2],然而通常一種藥物只針對單個因子或者某條信號通路,而RA這類復(fù)雜疾病需要多靶點組合治療,隨著現(xiàn)代中醫(yī)藥學(xué)研究的發(fā)展,選用療效確切的中藥已成為RA治療用藥的重要途徑,前期臨床觀察表明清熱活血方抗RA療效顯著且不良反應(yīng)較少[3-4],但其活性成分眾多,作用機(jī)制不明,本研究基于網(wǎng)絡(luò)藥理學(xué)理念,輔助生物信息學(xué)手段,通過“藥物-靶點-通路”方法預(yù)測清熱活血方抗RA的潛在活性成分及作用機(jī)制,為深入機(jī)制研究及臨床應(yīng)用提供依據(jù)。
1?資料與方法
1.1?篩選清熱活血方活性成分?通過中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫與分析平臺(TCMSP)[5]對清熱活血方中土茯苓、金銀花、丹參等中藥進(jìn)行活性成分的檢索,根據(jù)成分藥動學(xué)吸收、分布、代謝、排泄(ADME)參數(shù)[6]進(jìn)行篩選,其中口服生物利用度(OB)≥30%、類藥性(DL)≥0.18,篩選出化合物并利用Pubchem獲取其結(jié)構(gòu)[7]。
1.2?預(yù)測清熱活血方活性成分作用靶點并進(jìn)行通路富集分析?以上結(jié)構(gòu)導(dǎo)入SwissTargetPrediction數(shù)據(jù)庫[8],根據(jù)其結(jié)構(gòu)預(yù)測藥物可能存在的作用靶點,整合去重后進(jìn)行京都基因和基因組百科全書(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析并利用Cytoscape對有效成分及其對應(yīng)預(yù)測靶點進(jìn)行可視化[9]。
1.3?檢索RA發(fā)病靶點并挖掘清熱活血方治療RA的關(guān)鍵靶點?以“Rheumatoid arthritis”為關(guān)鍵詞,檢索藥物數(shù)據(jù)庫(Drugbank)[10]和在線人類孟德爾遺傳數(shù)據(jù)庫(OMIM)[11],獲取已發(fā)現(xiàn)的RA發(fā)病機(jī)制相關(guān)靶點。將其與預(yù)測的藥物作用靶點依次進(jìn)行映射、擴(kuò)展、拓?fù)鋄12],得到清熱活血方作用RA的關(guān)鍵靶點。
1.4?構(gòu)建藥物-疾病靶點蛋白質(zhì)-蛋白質(zhì)相互作用PPI網(wǎng)絡(luò)?為驗證清熱活血方作用RA的關(guān)鍵靶點的挖掘準(zhǔn)確性,將上述獲取的關(guān)鍵靶點導(dǎo)入蛋白質(zhì)-蛋白質(zhì)相互作用網(wǎng)絡(luò)(PPI)數(shù)據(jù)庫(String)[13],選擇最高置信度的數(shù)據(jù),挖掘關(guān)鍵靶點的直接、間接調(diào)控關(guān)系,并據(jù)此構(gòu)建靶點PPI網(wǎng)絡(luò)。
1.5?關(guān)鍵靶點富集分析?將獲取的關(guān)鍵靶點蛋白導(dǎo)入DAVID數(shù)據(jù)庫,標(biāo)識符選擇“official_gene_symbol”,靶點背景和物種背景都選擇“Homo Sapiens”,進(jìn)行關(guān)鍵靶點的基因本體(Gene Ontology,GO)分析和KEGG分析,以此獲取清熱活血方治療RA的關(guān)鍵靶點蛋白在基因功能和信號通路中的作用。
1.6?篩選Hub節(jié)點?為提高靶點的挖掘精確性,將已獲取的關(guān)鍵靶點蛋白導(dǎo)入Centiscape,根據(jù)拓?fù)浜蜕飳W(xué)屬性,應(yīng)用“plot by centrality”找出高分靶點,即發(fā)生了強(qiáng)烈的相互作用的靶點蛋白。
2?結(jié)果
2.1?清熱活血方活性成分?清熱活血方根據(jù)OB和DL等ADME參數(shù)共篩選出49個化合物,再根據(jù)活性結(jié)構(gòu)和預(yù)測靶點,共篩出31個有潛在靶點的活性成分(去重后的)。見表1。
2.2?預(yù)測清熱活血方活性成分作用靶點并進(jìn)行通路富集分析?對31個活性化合物進(jìn)行靶點預(yù)測,剔重整合后共得到419個潛在靶點。將其預(yù)測關(guān)系導(dǎo)入Cytoscape軟件構(gòu)建化合物-靶點網(wǎng)絡(luò)圖(C-T圖)。見圖1。紅色代表活性化合物,淺藍(lán)色代表預(yù)測靶點。將活性成分作用靶點導(dǎo)入DAVID數(shù)據(jù)庫進(jìn)行KEGG分析,共富集出80條信號通路,按P值從小到大排序,P值越小越顯著,把廣泛通路剔除后篩選P值前10個,得到以下通路:HIF-1 signaling pathway、Nitrogen metabolism、Prolactin signaling pathway、Calcium signaling pathway、TNF signaling pathway、Type Ⅱ diabetes mellitus、Cholinergic synapse、Sphingolipid signaling pathway、Inflammatory mediator regulation of TRP channels、Acute myeloid leukemia、ErbB signaling pathway、PI3K-AKT signaling pathway、Rap1 signaling pathway、Arachidonic acid metabolism。
2.3?檢索RA發(fā)病靶點并挖掘清熱活血方治療RA的關(guān)鍵靶點?通過OMIM、Drugbank數(shù)據(jù)庫檢索、去重后共得到430個RA發(fā)病相關(guān)靶點。將藥物預(yù)測靶點與疾病靶點進(jìn)行映射、擴(kuò)展、拓?fù)浜蟮玫?13個關(guān)鍵靶點。
2.4?構(gòu)建藥物-疾病靶點PPI圖?構(gòu)建清熱活血方與RA靶點蛋白的PPI網(wǎng)絡(luò)圖,共得到705條相互作用關(guān)系。見圖2。節(jié)點代表RA靶點,邊代表靶點間關(guān)系,不同顏色對應(yīng)不同的相互作用類型。將上述111個存在相互作用的靶點與清熱活血方預(yù)測靶點和RA靶點構(gòu)建韋恩圖分析。其中,14個靶點屬于清熱活血方預(yù)測靶點,5個靶點屬于RA靶點。見圖3。
2.5?GO富集與KEGG通路富集分析?將獲取的關(guān)鍵靶點蛋白導(dǎo)入DAVID數(shù)據(jù)庫進(jìn)行GO分析和
KEGG分析,KEGG共富集出62條信號通路,按P值從小到大排序,P值越小越顯著,把廣泛通路剔除后篩選P值前15個,即得關(guān)鍵靶點基因的注釋分類信息以及其參與的信號通路,其中大多數(shù)為RA發(fā)病密切相關(guān)通路。見圖4。GO分析共獲得479個富集結(jié)果,其中生物過程(Biological Process,BP)319項,分子功能(Molecular Function,MF)93項,細(xì)胞組分(Cellular Component,CC)67項,排名前15的GO富集結(jié)果見圖5。
2.6?拓?fù)渫诰騂ub節(jié)點?將已獲取的關(guān)鍵靶點蛋白導(dǎo)入Cytoscape中的Centiscape,利用參數(shù)Betweenness中介性、Degree自由度等進(jìn)行計算并作圖,由此篩選出CULI,EP300,HSP90AA1,TP53,HSPA8,CDC5L,UBC,RPS27A共8個具顯著作用的Hub節(jié)點,越靠近右上角的靶點越重要,提示這些Hub節(jié)點可能是清熱活血方作用RA的重要靶點。見圖6。
3?討論
RA屬于侵蝕性、自身免疫性疾病,有“不死的癌癥”之稱,嚴(yán)重影響患者的生命質(zhì)量,其發(fā)病機(jī)制不明,涉及免疫反應(yīng)、下游炎癥細(xì)胞浸潤、滑膜增生、血管翳形成和骨破壞等病理進(jìn)程,現(xiàn)有治療藥物如DMARDs、NSAIDs等治療靶點較為單一,多是延緩病理進(jìn)程且不良反應(yīng)較大[14],通過多靶點調(diào)節(jié)免疫、抗炎、抑制滑膜血管翳增生、抗骨質(zhì)疏松、保護(hù)軟骨等聯(lián)合治療更為有效,逐漸引起了研究者們的注意[15]。
清熱活血方具有清熱利濕活血功效,前期臨床觀察表明此方劑對于RA具有明顯的療效[16]。而清熱活血方內(nèi)含多種有效成分,已被證實可通過阻斷多種病理過程延緩RA進(jìn)展:體內(nèi)外研究表明山柰酚(kaempferol)能夠通過多種途徑抑制RA的發(fā)展[17-18]。槲皮素(quercetin)可明顯降低炎癥發(fā)病的嚴(yán)重性,并且干預(yù)后發(fā)病程度與TNF-α、IL-1β、IL-17和MCP-1水平的降低相關(guān)[19]。落新婦苷(Astilbin)與低劑量的甲氨蝶呤(MTX)聯(lián)用能有效改善膠原誘導(dǎo)的DBA小鼠關(guān)節(jié)炎等[20]。清熱活血方的C-T網(wǎng)絡(luò)圖表明,一種化合物具有多個靶點,符合RA多靶點的治療需求,同時多種化合物作用于相同靶點,說明不同化合物間可發(fā)揮協(xié)同作用,增強(qiáng)干預(yù)療效;而這些重合靶點可能是清熱活血方干預(yù)RA的關(guān)鍵靶點。將419個化合物預(yù)測靶點進(jìn)行KEGG分析,發(fā)現(xiàn)多數(shù)排名靠前的通路均與RA有關(guān),如HIF-1 signaling pathway、PI3K-AKT signaling pathway、VEGF signaling pathway、T cell receptor signaling pathway、ErbB signaling pathway、Inflammatory mediator regulation of TRP channels、TNF signaling pathway[21-22],說明清熱活血方可通過多通路達(dá)到治療RA的目的。PPI網(wǎng)絡(luò)靶點的韋恩圖表明清熱活血方可能通過直接或間接靶點共同作用于RA,其中屬于清熱活血方的有YWHAG、EGF、ABL1、HSPA8、SRC、CDK2、AKT1、ESR1、GAPDH、AR、VCP、APP PARP1、HSPA5,屬于RA靶點的有SMURF1、FN1、TP53、EEF1A1,而同時屬于清熱活血方和RA的是HSPA5,表明清熱活血方可能直接或者間接通過以上靶點干預(yù)RA。
傳統(tǒng)中藥復(fù)方具有多成分、多靶點、多通路、多功能的作用特點,本研究通過數(shù)據(jù)挖掘,分別獲得RA發(fā)生相關(guān)基因430個及清熱活血方有效成分預(yù)測靶點419個,進(jìn)而整合出小復(fù)方抗RA藥效相關(guān)靶點,建立小復(fù)方抗RA效應(yīng)基因相互作用網(wǎng)絡(luò)圖,通過網(wǎng)絡(luò)拓?fù)溆嬎愫凸δ芡诰?,進(jìn)一步印證小復(fù)方抗RA的關(guān)鍵候補(bǔ)靶點參與RA進(jìn)展相關(guān)的“炎癥-免疫”通路:如MAPK信號通路參與RA炎癥細(xì)胞浸潤和血管翳形成,阻斷MAPK通路可有效降低RA動物模型炎癥浸潤和血管形成[23]。PI3K-AKT信號通路參與RA中成纖維滑膜細(xì)胞異常激活,并導(dǎo)致其過度增殖、遷移和侵襲[24]。在RA病理過程中,滑膜和血管內(nèi)皮細(xì)胞周期失衡并過度增殖既是病理發(fā)展結(jié)果也加速了病理惡化進(jìn)程[1],通過調(diào)控細(xì)胞周期可能具有延緩RA進(jìn)展的作用。B細(xì)胞通路在RA發(fā)病的起始階段,促進(jìn)炎癥介質(zhì)聚集以及破骨細(xì)胞活化過程中發(fā)揮了重要作用[25-26]。Hippo信號通路參與成纖維滑膜的侵襲表型[27],在RA缺氧關(guān)節(jié)環(huán)境中,HIF-1信號通路促進(jìn)滑膜血管翳增生、炎癥反應(yīng)及骨侵蝕等病理過程[28-29],RA患者滑膜細(xì)胞中EGFR和HER2高表達(dá),而通過干預(yù)HIF-1通路可能有助于RA的治療[30],此外,小復(fù)方抗RA的關(guān)鍵候選靶點還參與了細(xì)胞凋亡通路、黏著斑激酶途徑、FoxO信號通路,這些通路都直接或者間接影響RA的進(jìn)程。GO富集分析表明清熱活血方可能通過調(diào)節(jié)轉(zhuǎn)錄過程、蛋白質(zhì)異構(gòu)化、細(xì)胞凋亡等BP過程干預(yù)RA,MF層面顯示清熱活血方抗RA可能與調(diào)節(jié)各種蛋白質(zhì)結(jié)合、酶結(jié)合、核酸結(jié)合等途徑相關(guān);CC層面表明細(xì)胞核、核糖核蛋白復(fù)合物、細(xì)胞質(zhì)、細(xì)胞核、核小體等細(xì)胞組分可能與清熱活血方抗RA相關(guān)。
對關(guān)鍵靶點進(jìn)行中心值的計算,篩選出8個Hub節(jié)點,有研究表明這些Hub節(jié)點與RA發(fā)病密切相關(guān):其中HSP90AA1可阻斷核因子κB通路,降低炎癥介質(zhì)水平,通過降低HSP90AA1表達(dá)水平可減輕滑膜炎癥和關(guān)節(jié)軟骨破壞[31]。TP53基因異常表達(dá)與RA炎癥細(xì)胞浸潤和滑膜組織異常增生以及骨與軟骨的進(jìn)行性破壞密切相關(guān)[32]。EP300抑制劑可降低RA并發(fā)癥模型的指標(biāo)[33-34]。進(jìn)一步驗證了本研究預(yù)測的準(zhǔn)確性,這些靶點可能是清熱活血方發(fā)揮抗RA作用的關(guān)鍵靶點。
RA是多因素影響、多基因相關(guān)、多條通路參與的復(fù)雜病理過程的,本研究表明,清熱活血方中31個活性化合物通過113個有效靶點及其作用網(wǎng)絡(luò)調(diào)控多條信號通路和生物學(xué)過程發(fā)揮抗RA作用,拓?fù)浞治龀鲇行О悬c中的8個Hub節(jié)點,其中3個已被證明與RA相關(guān),表明清熱活血方其有效成分通過多靶點、多通路來發(fā)揮治療RA的藥效。從系統(tǒng)的角度初步揭示了清熱活血方抗RA的作用機(jī)制,體現(xiàn)了中藥多成分-多靶點-多途徑的作用特點,一定程度反映了中醫(yī)藥治療的整體觀念,為后續(xù)闡述清熱活血方功效的生物學(xué)內(nèi)涵打下基礎(chǔ),同時也為針對發(fā)病機(jī)制復(fù)雜的RA治療思路及開發(fā)新藥物提供了方向。
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(2020-03-31收稿?責(zé)任編輯:王明)