目的:运用肝肾同源理论及网络药理学研究肉苁蓉–巴戟天干预迟发性性腺功能减退症可能存在的作用机制。方法:通过中药系统药理学数据库与分析平台(TCMSP)对肉苁蓉–巴戟天药对中活性成分靶点进行筛选,通过GeneCards、OMIM数据库检索迟发性性腺功能减退症靶点,取得交集基因后使用Cytoscape3.9.0建立化合物–疾病–靶点调控网络,基于生物学信息注释数据库(DAVID)对核心靶点数据信息进行下载,并用R软件绘制气泡图取得(GO)功能富集分析与基因组百科全书(KEGG)通路富集分析。结果:得到肉苁蓉–巴戟天药对的22个活性成分和271个潜在靶点,相关的疾病靶点2462个。GO富集分析主要包括正向调节凋亡过程、细胞因子介导的信号通路、药物反应、线粒体、蛋白质结合、酶结合等功能途径。KEGG通路分析主要包括丝裂原活化蛋白激酶(MAPK)信号通路、肿瘤坏死因子(TNF)信号通路、缺氧诱导因子1 (HIF-1)信号通路、糖尿病并发症中的RAGE信号通路等。结论:基于网络药理学及肝肾同源理论初步揭示出肉苁蓉–巴戟天药对干预LOH具有活性成分及作用靶点多样、作用途径广泛的特点,其作用机制可能与有效成分槲皮素、β谷甾醇,核心靶点及相关通路对睾丸间质细胞及支持细胞的调节,影响睾酮分泌有关。 Objective: Study the possible mechanism of Cistanches Herba and Morindae Officinalis Radix inter-vening late-onset hypogonadism (LOH) by using the theory of “homogeny of liver and kidney” and network pharmacology. Methods: The active component targets of Cistanches Herba and Morindae Officinalis Radix were screened through Traditional Chinese Medicine Systems Pharmacology Da-tabase and Analysis Platform (TCMSP). The targets of late-onset hypogonadism were searched through Gene Cards and OMIM databases. After obtaining the intersection genes, the com-pound-disease-target regulation network was established by using Cytoscape 3.9.0, and the core target data information was downloaded based on the database for annotation, visualization and integrated discovery (DAVID). The bubble diagram was drawn with R software to obtain (GO) func-tional enrichment analysis and genomic encyclopedia (KEGG) pathway enrichment analysis. Results: 22 active components and 271 potential targets of Cistanches Herba-Morindae Officinalis Radixwere were obtained, and 2462 related disease targets were obtained. GO enrichment analysis mainly in-cludes functional pathways such as positive regulation of protein phosphorylation, cyto-kine-mediated signaling pathway, angiogenesis, mitochondrion, zincion binding, transcription fac-tor binding and so on. KEGG pathway analysis mainly includes MAPK signaling pathway, TNF sig-naling pathway, HIF-1 signaling pathway, and RAGE signaling pathway in diabetic complications. Conclusion: Based on the network pharmacology and the theory of “homogeny of liver and kidney”, it is preliminarily revealed that Cistanches Herba and Morindae Officinalis Radix have the charac-teristics of diverse active components, action targets and wide action pathways in the intervention of LOH, and its action mechanism may be related to the effective components quercetin, β Sitosterol, core targets and related pathways regulating Leydig cells and Sertoli cells, affecting testosterone secretion.
目的:运用肝肾同源理论及网络药理学研究肉苁蓉–巴戟天干预迟发性性腺功能减退症可能存在的作用机制。方法:通过中药系统药理学数据库与分析平台(TCMSP)对肉苁蓉–巴戟天药对中活性成分靶点进行筛选,通过GeneCards、OMIM数据库检索迟发性性腺功能减退症靶点,取得交集基因后使用Cytoscape3.9.0建立化合物–疾病–靶点调控网络,基于生物学信息注释数据库(DAVID)对核心靶点数据信息进行下载,并用R软件绘制气泡图取得(GO)功能富集分析与基因组百科全书(KEGG)通路富集分析。结果:得到肉苁蓉–巴戟天药对的22个活性成分和271个潜在靶点,相关的疾病靶点2462个。GO富集分析主要包括正向调节凋亡过程、细胞因子介导的信号通路、药物反应、线粒体、蛋白质结合、酶结合等功能途径。KEGG通路分析主要包括丝裂原活化蛋白激酶(MAPK)信号通路、肿瘤坏死因子(TNF)信号通路、缺氧诱导因子1 (HIF-1)信号通路、糖尿病并发症中的RAGE信号通路等。结论:基于网络药理学及肝肾同源理论初步揭示出肉苁蓉–巴戟天药对干预LOH具有活性成分及作用靶点多样、作用途径广泛的特点,其作用机制可能与有效成分槲皮素、β谷甾醇,核心靶点及相关通路对睾丸间质细胞及支持细胞的调节,影响睾酮分泌有关。
网络药理学,肝肾同源,迟发性性腺功能减退症,肉苁蓉,巴戟天
Huijie Ren1, Liwen An1,2, Yuyan Guo1, Hongtao Li2, Xinxin Chen2, Shan Gao1,2*
1Heilongjiang University of Traditional Chinese Medicine, Harbin Heilongjiang
2The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin Heilongjiang
Received: Jul. 11th, 2022; accepted: Aug. 8th, 2022; published: Aug. 15th, 2022
Objective: Study the possible mechanism of Cistanches Herba and Morindae Officinalis Radix intervening late-onset hypogonadism (LOH) by using the theory of “homogeny of liver and kidney” and network pharmacology. Methods: The active component targets of Cistanches Herba and Morindae Officinalis Radix were screened through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The targets of late-onset hypogonadism were searched through Gene Cards and OMIM databases. After obtaining the intersection genes, the compound-disease-target regulation network was established by using Cytoscape 3.9.0, and the core target data information was downloaded based on the database for annotation, visualization and integrated discovery (DAVID). The bubble diagram was drawn with R software to obtain (GO) functional enrichment analysis and genomic encyclopedia (KEGG) pathway enrichment analysis. Results: 22 active components and 271 potential targets of Cistanches Herba-Morindae Officinalis Radixwere were obtained, and 2462 related disease targets were obtained. GO enrichment analysis mainly includes functional pathways such as positive regulation of protein phosphorylation, cytokine-mediated signaling pathway, angiogenesis, mitochondrion, zincion binding, transcription factor binding and so on. KEGG pathway analysis mainly includes MAPK signaling pathway, TNF signaling pathway, HIF-1 signaling pathway, and RAGE signaling pathway in diabetic complications. Conclusion: Based on the network pharmacology and the theory of “homogeny of liver and kidney”, it is preliminarily revealed that Cistanches Herba and Morindae Officinalis Radix have the characteristics of diverse active components, action targets and wide action pathways in the intervention of LOH, and its action mechanism may be related to the effective components quercetin, β Sitosterol, core targets and related pathways regulating Leydig cells and Sertoli cells, affecting testosterone secretion.
Keywords:Network Pharmacology, Homogeny of Liver and Kidney, Late-Onset Hypogonadism, Cistanches Herba, Morindae Officinalis Radix
Copyright © 2022 by author(s) and Hans Publishers Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).
http://creativecommons.org/licenses/by/4.0/
迟发性性腺功能减退症(late-onset hypogonadism, LOH)是雄性激素缺乏导致的一系列临床综合征 [
肝藏血,肾藏精,精聚为髓,髓化生为血,精血同源与肝肾,故肝肾同源;中医学肝肾同源和现代医学研究肝肾疾病与“神经–内分泌–免疫系统”及生殖轴基本相似 [
据报道中药补阳药有类雄性激素样作用,“肉苁蓉–巴戟天”相须为用能提高补阳药疗效 [
通过TCMSP (http://tcmspw.com/tcmsp.php.2.3)分别以“肉苁蓉巴戟天”为关键词检索其化学成分,类药性(DL)是测定药物研发过程中,化合物是否符合成药标准的依据,口服药物主要通过小肠上皮细胞吸收,而小肠上皮细胞的通透性在口服生物利用度(OB)中至关重要,从文献 [
以“Late-onse thypogonadism”为关键词,分别在GeneCards (https://www.genecards.org/, 4.12)、OMIM (https://omim.org/)数据库中检索迟发性性腺功能减退症潜在靶点并下载相关数据信息,两组基因取交集并去除重复值。
将“肉苁蓉–巴戟天”药对与迟发性性腺功能减退症的交集靶点,导入STRING (https://string-db.org/cgi/input.pl, 11.2)选择“multiple proteins”,种属设定为“homosapiens”,进行蛋白质-蛋白质相互作用(protein-protein interaction, PPI)分析,结合文献,试验将置信度评分设置为 > 0.9“high confidence”(0.900)“hided is connected nodes in the network”并剔除蛋白相互作网络图(PPI网络)中无关联节点其余参数默认,构建交集靶点相互作用网络拓扑。建立调控网络以便于“肉苁蓉–巴戟天”对迟发性性腺功能减退症的作用机制可视化呈现。
将肉苁蓉–巴戟天干预迟发性性腺功能减退症交集靶点导入DAVID (https://david.ncifcrf.gov/home.jsp, 6.8),选择智人“Homosapiens”和函数注释图表“Functional Annotation Chart”数据库下载相关信息,通过UltraEdit导入题录更改储存位置,导入R (R version 4.1.0)语言运行即可得到气泡图。
获取各活性成分的对应靶点并在中药相关靶点不足时,利用BATMAN数据库、TCMIP数据库对药物成分及靶点进行补充。对所获得的化学成分、靶点、和对应的中药名进行统计,结果见表1。在TCMSP检索得出肉苁蓉-巴戟天活性成分24个,排除未获得对应靶点的2个有效成分,最终获得活性成分22个;有效成分所对应的靶点271个,其中肉苁蓉175个,巴戟天96个。
MOLID | 化合物 | OB (%) | Caco-2 | DL | 药物 |
---|---|---|---|---|---|
MOL000358 | Beta-sitosterol | 36.91 | 1.32 | 0.75 | 肉苁蓉 |
MOL005320 | Arachidonate | 45.57 | 1.27 | 0.2 | 肉苁蓉 |
MOL005384 | suchilactone | 57.52 | 0.82 | 0.56 | 肉苁蓉 |
MOL007563 | Yangambin | 57.53 | 0.67 | 0.81 | 肉苁蓉 |
MOL000098 | Quercetin | 46.43 | 0.05 | 0.28 | 肉苁蓉 |
MOL008871 | Marckine | 37.05 | 0.86 | 0.69 | 肉苁蓉 |
MOL000358 | beta-sitosterol | 36.91 | 1.32 | 0.75 | 巴戟天 |
MOL000359 | Sitosterol | 36.91 | 1.32 | 0.75 | 巴戟天 |
MOL001506 | Supraene | 33.55 | 2.08 | 0.42 | 巴戟天 |
MOL002879 | Diop | 43.59 | 0.79 | 0.39 | 巴戟天 |
MOL002883 | Ethyloleate(NF) | 32.4 | 1.4 | 0.19 | 巴戟天 |
MOL006147 | Alizarin-2-methylether | 32.81 | 0.62 | 0.21 | 巴戟天 |
MOL009495 | 2-hydroxy-1,5-dimethoxy-6-(methoxymethyl)-9,10-anthraquinone | 95.85 | 0.54 | 0.37 | 巴戟天 |
MOL009496 | 1,5,7-trihydroxy-6-methoxy-2-methoxymethylanthracenequinone | 80.42 | 0.27 | 0.38 | 巴戟天 |
MOL009500 | 1,6-dihydroxy-5-methoxy-2-(methoxymethyl)-9,10-anthraquinone | 104.54 | 0.37 | 0.34 | 巴戟天 |
MOL009503 | 1-hydroxy-3-methoxy-9,10-anthraquinone | 104.33 | 0.59 | 0.21 | 巴戟天 |
MOL009504 | 1-hydroxy-6-hydroxymethylanthracenequinone | 81.77 | −0.04 | 0.21 | 巴戟天 |
MOL009513 | 2-hydroxy-1,8-dimethoxy-7-methoxymethylanthracenequinone | 112.3 | 0.46 | 0.37 | 巴戟天 |
MOL009519 | (2R,3S)-(+)-3′,5-Dihydroxy-4,7-dimethoxydihydroflavonol | 77.24 | 0.13 | 0.33 | 巴戟天 |
MOL009524 | 3beta,20(R),5-alkenyl-stigmastol | 36.91 | 1.36 | 0.75 | 巴戟天 |
MOL009525 | 3beta-24S(R)-butyl-5-alkenyl-cholestol | 35.35 | 1.36 | 0.82 | 巴戟天 |
MOL009537 | AmericaninA | 46.71 | −0.08 | 0.35 | 巴戟天 |
MOL009551 | Isoprincepin | 49.12 | −0.08 | 0.77 | 巴戟天 |
MOL009562 | OhioensinA | 38.13 | 0.6 | 0.76 | 巴戟天 |
表1. 肉苁蓉–巴戟天活性成分基本信息
在OMIM、GeneCards数据库中获得相关疾病靶点分别为647、1817个;剔除重复基因后获得与迟发性性腺功能减退症相关的2462个靶点。利用Venny2.1.0在线平台将肉苁蓉、巴戟天有效靶点与迟发性性腺功能减退症相关的疾病靶点进行映射建立Venn图,筛选交集靶点“肉苁蓉–巴戟天”迟发性性腺功能减退症的潜在靶点。获取交集靶点将肉苁蓉-巴戟天的271个作用靶点与LOH的2462个作用靶点进行匹配取交集,经Uniprot校对后得到74个共有基因,其中肉苁蓉和迟发性性腺功能减退的潜在基因有71个,巴戟天和迟发性性腺功能减退的潜在基因有14个,“肉苁蓉–巴戟天”迟发性性腺功能减退症的潜在作用基因有74个,见图1。
图1. 肉苁蓉–巴戟天与LOH的交集基因
在Cytoscape3.9.0软件安装的CytoHubba插件,计算各网络节点的拓扑性质参数(介数、自由度、接近中心性、平均最短路径长度),以所有节点这四个参数的中值作为筛选条件,取大于其二倍中位数度值12的靶点作为中心靶点。结果显示,该蛋白互作网络中74个靶点通过224条边相连接,关键靶点13个,包括JUN、MAPK1、TP53、AKT1、FOS、TNF、ESR1、IL6、EGFR、MYC、STAT1、HIF1A、RB1,以此为基础建立PPI网络图,以节点颜色深浅表现连接度大小,可视化结果见图2,关键靶点信息具体情况见表2。
图2. 肉苁蓉–巴戟天药对干预LOH核心靶点PPI网络图
靶点 | Degree | Betweenness | Closeness | MOLID | 活性成分 | 来源药物 |
---|---|---|---|---|---|---|
JUN | 24 | 487.97134 | 0.5673077 | MOL000358 | beta-sitosterol | 肉苁蓉 |
MOL000098M | quercetin | 肉苁蓉 | ||||
OL000358 | beta-sitosterol | 巴戟天 | ||||
MAPK1 | 20 | 323.49426 | 0.5315315 | MOL000098 | quercetin | 肉苁蓉 |
TP53 | 18 | 337.0527 | 0.50427353 | MOL000098 | quercetin | 肉苁蓉 |
AKT1 | 18 | 415.23752 | 0.5315315 | MOL000098 | quercetin | 肉苁蓉 |
FOS | 17 | 182.46278 | 0.5 | MOL000098 | quercetin | 肉苁蓉 |
TNF | 16 | 423.4698 | 0.5086207 | MOL000098 | quercetin | 肉苁蓉 |
ESR1 | 16 | 316.04947 | 0.50427353 | MOL006147 | Alizarin-2-methylether Estrogen | 巴戟天 |
MOL009537 | americaninA | 巴戟天 | ||||
IL6 | 14 | 178.12787 | 0.48360655 | MOL000098 | quercetin | 肉苁蓉 |
EGFR | 13 | 122.105415 | 0.47580644 | MOL000098 | quercetin | 肉苁蓉 |
MYC | 13 | 119.147064 | 0.51304346 | MOL000098 | quercetin | 肉苁蓉 |
STAT1 | 13 | 169.52452 | 0.5086207 | MOL000098 | quercetin | 肉苁蓉 |
HIF1A | 12 | 37.311474 | 0.4876033 | MOL000098 | quercetin | 肉苁蓉 |
RB1 | 12 | 49.88481 | 0.472 | MOL000098 | quercetin | 肉苁蓉 |
表2. “肉苁蓉–巴戟天”干预迟发性性腺功能减退中心靶点的网络拓扑学参数
在Cytoscape3.9.0软件绘制中药–成分–潜在作用靶点肉苁蓉–巴戟天网络图,该网络有90个节点,包括槲皮素(quercetin)、β谷甾醇(beta-sitosterol)、内酯(suchilactone)等16个有效成分;钠离子通道α (SCN5A)、类视黄醇X受体(RXRA)、特异性钾离子通道蛋白抗体(KCNH2)、等16个药物靶点,并通过106条边相连接,充分说明肉苁蓉巴戟天具有多成分、多靶点共同作用,以对迟发性性腺功能减退症达到干预的目的,见图3。
图3. “肉苁蓉巴戟天”药对–化学成分–潜在靶点网络图
将肉苁蓉-巴戟天干预迟发性性腺功能减退潜在作用靶点导入DAVID6.8数据库,对74个交集靶点进行GO富集分析以P < 0.05为筛选条件得到493条相关通路。其中生物学过程(BP)通路375条;细胞组成(CC)通路37条;分子功能(MF)通路81条。以P值排序,各取其前20条通路,用R软件绘制气泡图,见图4~6。
图4. GO-BP富集分析
图5. GO-CC富集分析
图6. GO-MF富集分析
生物学过程,细胞组分,分子功能结果前10个条目主要包括:蛋白质磷酸化的正向调控(positive regulation of protein phosphorylation);缺氧反应(response to hypoxia);染色质(chromatin);高分子复合物(macromolecular complex);线粒体(mitochondrion);锌离子结合(zinc ion binding);转录因子结合(transcription factor binding)等。
利用DAVID6.8数据库对74个“肉苁蓉–巴戟天”的潜在作用靶点进行KEGG富集分析,共得到159条相关通路,以P < 0.05为筛选条件,得到151条相关通路。取其前20条,用R软件绘制气泡图,见图7,各数值表现方法同GO通路表现。
KEGG部分前20位信号通路包括:癌症的途径(Pathways in cancer);化学致癌–受体激活(Chemical carcinogenesis-receptor activation);脂质与动脉粥样硬化(Lipid and atherosclerosis);丝裂原活化蛋白激酶信号通路(MAPK signaling pathway);糖尿病并发症中的RAGE信号通路(RAGE signaling pathway in diabetic complications);缺氧诱导因子1信号通路(HIF-1 signaling pathway);肿瘤坏死因子信号通路(TNF signaling pathway);非洲锥虫病(African trypanosomiasis)等。
临床上部分LOH患者血清睾酮水平降低程度与症状体征严重程度并不完全吻合,目前对睾酮补充治疗(testosterone supplementation therapy, TST)的标准值尚未达到共识,这些都为中医药干预LOH提供了可能 [
图7. GO-KEGG富集分析
本研究初步筛选肉苁蓉巴戟天有效成分22个,干预LOH的核心靶点有13个,化合物–疾病–靶点调控网络表明肉苁蓉–巴戟天药对干预LOH作用机制复杂多样且靶点众多。药理研究表明肉苁蓉具有缓解衰老、抗疲劳、平衡免疫、保护肝功能、促进生殖等方面的作用 [
GO分析表明“肉苁蓉–巴戟天”干预LOH涉及多个生物过程,如药物反应、线粒体、蛋白质结合、酶结合等。KEGG通路富集分析显示丝裂原活化蛋白激酶信号通路;糖尿病并发症中的RAGE信号通路,丝裂原活化蛋白激酶(MAPK)信号通路、肿瘤坏死因子(TNF)信号通路、缺氧诱导因子1 (HIF-1)信号通路、糖尿病并发症中的RAGE信号通路等,这些通路都可能是“肉苁蓉–巴戟天”干预LOH的主要通路。糖尿病睾丸间质细胞中miR-504、miR-935过度表达,通过抑制MAPK通路调节间质细胞增殖、凋亡,从而影响雄性激素分泌、精子形成 [
LOH为本虚标实之症,肾精不足为本,肝郁为标。肾藏精,主脏腑气化,肾精化肾气,肾气分阴阳,肾精肾气充盛则筋骨强,身体壮实;精生髓,肾精充足则髓海得养,思捷敏锐,精力充沛。肝藏血,肝主疏泄,条畅情志,《灵枢平人绝谷》曰:“血脉和利,精神乃居。”肝气疏泄,气血调和则情志舒畅 [
综上所述,基于网络药理学及肝肾同源中医学理论发现肉苁蓉–巴戟天药对干预LOH具有活性成分及作用靶点多样、作用途径广泛的特点,主要靶点包括:JUN、MAPK1、TP53、AKT1、FOS、TNF、ESR1、IL6等,涉及生物学通路包括丝裂原活化蛋白激酶(MAPK)信号通路、肿瘤坏死因子(TNF)信号通路、缺氧诱导因子1 (HIF-1)信号通路、糖尿病并发症中的RAGE信号通路等可通过对睾丸间质细胞及支持细胞的作用而影响睾酮分泌,本研究为后续针对肝肾同源及“肉苁蓉–巴戟天”干预LOH研究提供了有力的依据及参考方向。
值得注意的是,此研究不足之处在于:① 条件受限,无法测出药物在体内的代谢过程以及发生的一系列化学反应,② 数据库可能不够完备,有些成分可能尚未发现,③ 药物的含量及浓度和挥发度欠缺,因此仅作为机制预测方向,具体药物机制与中医理论相结合还需要进一步实验研究。
黑龙江省中医药学会青年中医药科技创新项目(ZHY19-014)。
任慧杰,安立文,郭玉岩,李洪涛,陈昕昕,高 山. 基于网络药理学及肝肾同源理论探讨肉苁蓉-巴戟天干预迟发性性腺功能减退症作用机制Study on the Mechanism of Cistanches Herba and Morindae Officinalis Radix Intervening Late-Onset Hypogonadism Based on Network Pharmacology and the Theory of “Homogeny of Liver and Kidney”[J]. 临床医学进展, 2022, 12(08): 7449-7461. https://doi.org/10.12677/ACM.2022.1281076