药学学报  2018, Vol. 53 Issue (4): 567-573   PDF    
基于网络药理学的二至丸保肝作用机制研究
翟园园, 刘其南, 徐佳, 姚卫峰, 包贝华, 曹雨诞, 张丽, 丁安伟     
南京中医药大学, 江苏省方剂高技术研究重点实验室/江苏省中药资源产业化过程协同创新中心, 江苏 南京 210023
摘要: 构建二至丸保肝的"成分-核心靶点-通路"分子调控网络,探索其"多成分-多靶点-多通路"的作用机制。采用ADME/T计算方法筛选二至丸保肝活性成分,通过网络药理学研究方法,基于反向药效团匹配的靶标识别服务平台分析预测潜在作用靶点,通过生物学信息注释数据库(DAVID)对靶点基因功能及代谢通路进行分析,采用Cytoscape软件构建二至丸保肝"成分-核心靶点-通路"网络模型。结果显示,二至丸中39个主要的保肝活性成分可能通过调控HRAS、DCK、HSD17B1和UCK2等321个靶点,干预胰岛素信号通路、FoxO信号通路、代谢通路和糖酵解过程等51条通路发挥保护肝脏作用,体现了中药多成分、多靶点和多途径的作用特点,为该复方作用机制的阐述提供了新的思路和线索。
关键词: 网络药理学     二至丸     女贞子     墨旱莲     保护肝脏     作用机制    
Network pharmacology-based study on mechanism of liver protection of Erzhi Pill
ZHAI Yuan-yuan, LIU Qi-nan, XU Jia, YAO Wei-feng, BAO Bei-hua, CAO Yu-dan, ZHANG Li, DING An-wei     
Jiangsu Key Laboratory for High Technology Research of TCM Formulae and Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing 210023, China
Abstract: This study was designed to construct a "drug-core target-pathway" network of Erzhi Pill for hepatic injury treatment in an effort to explore the "multi-components, multi-targets, multi-pathways" mechanism. ADME/T calculation method was used to screen the active components of Erzhi Pill, and then predict the potential targets according to the reverse pharmacophore matching method. Biological information annotation databases (DAVID) was used to analyze the molecular function and biological process of the action targets. The Cytoscape software was used to construct the "ingredient-core target-pathway" network of Erzhi Pill for hepatic injury treatment. It was found that 39 major active ingredients of Erzhi Pill regulated 321 targets (HRAS, DCK, HSD17B1, UCK2, et al) and affected 51 pathways, such as insulin signaling pathway, FoxO signaling pathway, metabolic pathways and glycolysis/gluconeogenesis. The method revealed the action features of traditional Chinese medicine as multi-ingredients, multi-targets, multi-pathways, providing new clues for further basic study on the hepatic injury pharmacological mechanism of Erzhi Pill.
Key words: network pharmacology     Erzhi Pill     Nvzhenzi     Mohanlian     protection of liver     mechanism    

肝脏是人体内最大的实质性腺体, 是体内新陈代谢的中心站, 被喻为“人体最大的化工厂”。肝损伤系各种肝脏疾病的病变结果, 其中物理性肝损伤、化学性肝损伤、酒精性肝损伤、环境性肝损伤及代谢性肝损伤等各种肝损伤为目前严重的多发常见病[1-3], 是脂肪肝、肝硬化、肝腹水及肝癌的直接前期病因和成因。现代医学治疗肝损伤、保护肝脏的特异性药物缺乏且易产生毒副反应, 而具有多组分、多靶点作用的中药方剂对于肝损伤有一定的促进再生和修复作用[4], 但大多作用机制尚不十分清楚。因此, 研究保肝中药治疗肝损伤的多靶点、多途径作用机制越来越受到关注和重视。

“清上补下第一方”二至丸出自明代吴旻辑的《扶寿精方》, 由女贞子(酒蒸)和墨旱莲两味中药等量(1:1)组成。两味药合用, 其性平和、偏寒, 能补阴而不滋腻, 为平补肝肾之良剂, 广泛用于临床各种肝损伤的治疗[5]。本课题组前期研究表明, 二至丸及其组成药味女贞子和墨旱莲对于多种动物肝损伤具有较好的防治作用[6, 7]。然而, 由于中药方剂化学成分多靶点的调节作用, 使得二至丸保肝作用机制仍不明确, 因此本研究运用网络药理学的思路和方法, 从系统角度和分子水平, 基于“分子(药物)-靶点-通路”多层次网络模式[8]分析二至丸保护肝脏的药效活性成分和可能的分子网络机制, 为深入开展二至丸实验研究和临床应用提供依据。

材料与方法

主要化学成分的收集 利用TCMSP[9] (http://ibts.hkbu.edu.hk/LSP/tcmsp.php), 并结合TCMIP (http://www.megabionet.org/tcmid/)及TCM-PTD (http://pharminfo.zju.edu.cn/ptd/)等数据库收集二至丸中女贞子和墨旱莲的主要化学成分, 然后分别通过二至丸、女贞子、墨旱莲、保肝关键词检索中国知网、Pubmed (http://pubmed.cn/)数据库, 筛选具有文献报道证实的主要保肝活性成分。根据TCMSP数据平台计算所得的ADME/T[10]值, 删掉不能同时满足OB≥30%和DL≥0.18且在二至丸含量不高的化学成分, 得到保肝作用成分39个, 包括10-羟基女贞苷[11]、特女贞苷[12]、女贞苷G13[11]、新女贞子苷[11]、橄榄苦苷[13]、羟基酪醇[14]、齐墩果酸[15]、熊果酸[16]、萎陵菜酸[17]、2α-羟基齐墩果酸[18]、红景天苷[19]、松果菊苷[20]、毛蕊花苷[21]、鼠李糖[22]、阿拉伯糖[22]、葡萄糖[22]D-甘露醇[23]、岩藻糖[22]、芹菜素[24]、木犀草素[25]、槲皮素[26]、花青素[27]、木犀草素-7-O-β-D-葡萄糖苷[28]β-谷甾醇[29]、亚油酸[23]、棕榈酸[23]、山奈酚[30]、紫杉叶素[31]、圣草酚[32]、蟛蜞菊内酯[33]、去甲蟛蜞菊内酯[34]、豆甾醇3-O-β-D-吡喃葡萄糖苷[35]、豆甾醇[36]、刺囊酸[37]、墨旱莲皂苷Ⅱ[37]、墨旱莲皂苷Ⅰ[38]、蒙花苷[39]、金合欢素[40]、芦丁[41]

*.mol2格式文件的建立 通过文献挖掘并利用CAS数据库、chemspider (http://www.chemspider.com)、SciFinder (https://scifinder.cas.org/scifinder/view/scifinder/scifinderExplore.jsf)以及PubChem等确定39种活性成分的化学结构。将在数据库中获得的各自成分结构图以MDL Molfile (*.mol)格式存储; 然后再导入Chem Bio 3D Ultra12.0软件, 将该成分的二维结构式转换为三维立体结构式, 通过“另存为”将MDL Molfile (*.mol)格式转换为*.mol2格式文件。

潜在作用靶点反向预测 PharmMapper服务器(http://59.78.96.61/pharmmapper)是基于配体特征的反向药效团模型, 预测活性成分潜在靶点的免费Web服务器, 具有应用范围广, 预测速度快等优点, 在中药靶点预测中得到广泛的应用。本研究将二至丸主要活性成分以*.mol2文件格式上传到系统, 其中Select Targets Set选择Human Protein Targets Only, 其余参数均为默认设置。将得到的结果筛选出分子-靶点匹配度(Fit Score)大于4.5的药物靶点(若无Fit > 4.5, 选取Fit值最高的10个靶点)。由于检索到的药物靶点存在命名不规范等问题, 本文使用NCBI (National Center for Biotechnology Information)数据库的Gene基因搜索功能(http://www.ncbi.nlm.nih.gov/gene), 并限定物种为人类, 将筛选得到的靶点校正为官方简写, 由此获取与活性成分相关的靶点信息。

靶点通路注释分析  将潜在药效靶点的官方简写上传到DAVID数据库(https://david.ncifcrf.gov/), 限定靶基因名称列表为人, 进行GO (gene ontology)生物学过程富集分析和KEGG (KEGG pathway analysis)代谢通路富集分析, 选取具有统计学意义(P < 0.05)的所有通路, 药物靶点富集的通路被认为是药物调控的主要通路, 并用GraphPad Prism 5绘图。

“成分-核心靶点-通路”网络模型构建  根据二至丸主要活性成分靶点预测结果, 采用Cytoscape软件的Merge功能构建二至丸“成分-靶点-通路”网络模型, 并利用其插件cytoNCA筛选潜在活性成分的核心靶点, 构建“成分-核心靶点-通路”网络模型。以不同颜色的节点(node)表示药效成分、潜在核心作用靶点及作用通路, 边(edge)用来连接某一药效成分和其潜在作用靶点以及作用靶点和其注释的相关通路, 由此构建网络探讨二至丸多成分、多靶点和多通路的作用机制。

结果 1 二至丸保肝作用成分的筛选

通过TCMSP、TCMID和TCM-PTD等数据库以及文献挖掘, 分别在墨旱莲和女贞子中收集到42和52个主要化学成分, 对收集到的化学成分进行ADME/T计算(OB≥30%和DL≥0.18), 并利用文献验证其是否具有保肝作用, 以进一步筛选得到活性成分。此外, 虽然女贞子中齐墩果酸、熊果酸等成分的OB和DL值较低, 但因有文献报道其具有保肝作用, 且在二至丸中的量较高, 因此将这些也纳入到活性成分中, 共得到保肝活性成分39个, 其中墨旱莲中17个, 女贞子中29个, 共有成分7个。

2 靶点的预测

经过Chem Bio 3D Ultra12.0转化操作和服务器的靶点预测结果, 共得到靶点321个。其中, 墨旱莲潜在作用靶点118个, 女贞子潜在作用靶点203个。

3 靶点生物功能分析

图 1为二至丸保肝活性成分靶点富集的GO生物学过程分析, 分析结果显示, 预测靶点在生物过程中排列靠前的有信号转导、蛋白质磷酸化、细胞增殖过程、氧化还原以及代谢过程等, 反映了肝损伤涉及体内多个生物学过程, 同时表明二至丸通过改善这些生物过程可能是其发挥保肝作用的机制之一。

Figure 1 Enriched gene ontology terms for biological processes from main active ingredients of Erzhi Pill
4 靶点-通路分析

使用DAVID进行二至丸靶点通路分析, 得到通路51条(表 1)。根据通路P值, 结合生物学信息, 选取P值相对较小且靶点富集的10条通路(图 2)。根据KEGG网站上的通路信息及相关文献, 对10条通路的生物学意义简单描述如下。

Table 1 Information of common pathways

Figure 2 Enriched KEGG pathways of potential targets from main active ingredients of Erzhi Pill

胰岛素信号通路(insulin signaling pathway):胰岛素受体与胰岛素结合后引起胰岛素受体底物(IRS)发生磷酸化, 从而激活PI3K/Akt这一主要胰岛素信号传递通路。Chen等[42]研究发现通过增强IRS-1/PI3K/Akt胰岛素信号通路和上调GLUT4的表达来改善肌肉组织中的胰岛素抵抗, 能够提高肌肉对葡萄糖的摄取, 抑制肌肉组织中胰岛素抵抗(IR), 从而降低非酒精性脂肪肝(NAFLD)的肝脏脂质水平, 改善肝功能。

甲状腺激素信号通路(thyroid hormone signaling pathway):甲状腺激素(THs)是生长、发育和代谢的重要调节因子。细胞质膜αvβ3-整合素具有T3和T4不同的结合位点。Cordeiro等[43]研究表明T3结合位点能够和信号蛋白质结合, 通过PI3K途径完成肝脂质代谢过程。

非小细胞癌途径(non-small cell lung cancer):非小细胞肺癌(NSCLC)代表一组多样的癌组织, NSCLC的改变机制包括致癌基因的活化, 如K-RAS、EGFR和EML4-ALK, 及肿瘤抑制基因的失活如p53、p16INK4a、RAR-β和RASSF1。

T细胞受体信号通路(T cell receptor signaling pathway): T细胞能够杀伤靶细胞, 辅助或抑制B细胞产生抗体, 且与特异性抗原、有丝分裂的应答反应以及产生细胞因子有关。

FoxO信号通路(FoxO signaling pathway):通过Akt/PKB、PI3K的下游磷酸化, 激活炎症因子的表达。已有研究表明, Sirt1-FoxO1信号通路的激活可以改善NAFLD[44]

代谢通路(metabolic pathways):代谢通路是细胞内发生的一系列由酶催化的化学反应, 现有文献证明[45], 通过上调肝组织中pAMP和Sirt1蛋白表达、下调UCP2蛋白表达, 调节NAFLD肝脏脂质代谢紊乱。

Fc epsilon RI信号通路(Fc epsilon RI signaling pathway):通过激发NADPH氧化酶, 蛋白激酶C和丝裂原活化蛋白激酶(MAPK)的多种细胞内信号通路, 从而增强NF-κB活性, 进而促进促炎细胞因子如IL-1、IL-6和TNF-α的表达。

GnRH信号通路(GnRH signaling pathway): GnRHR与Gq/11蛋白偶联以激活磷脂酶C, 将其信号传递到二酰基甘油(DAG)和1, 4, 5-三磷酸肌醇(IP3)。DAG激活细胞内蛋白激酶C (PKC)途径, IP3能够刺激细胞内钙的释放。PKC下游的信号传导能够激活表皮生长因子(EGF)受体和MAPKs。

VEGF信号通路(VEGF signaling pathway): VEGF最初被称为血管生长因子, 主要是通过3种特异性表面受体发挥生物学作用[46]。现有大量证据表明VEGFR-2是内皮细胞中VEGF驱动反应的主要介质, 被认为是生理和病理血管生成的关键信号转导子。

糖酵解/葡萄糖生成过程(glycolysis/gluconeogenesis):糖酵解是将葡萄糖转化为丙酮酸并产生少量ATP和NADH的过程。研究表明2-DG可减轻LPS/D-Gal诱导的致死性肝损伤、下调LPS/D-Gal激活的促凋亡信号[47]

5 二至丸“成分-核心靶点-通路”网络模型构建

采用Cytoscape软件及其插件CytoNCA构建二至丸“成分-核心靶点-通路”网络模型(图 3)。二至丸39个活性成分的核心作用靶点分布于不同的代谢通路, 相互协调, 从多种途径体现药物保肝作用机制。

Figure 3 Ingredients-core targets-pathways network of Erzhi Pill. () Active ingredient of Nvzhenzi; () Active ingredient ofMohanlian; () Common ingredients of Nvzhenzi and Mohanlian; () Targets; () Pathways
讨论

据文献报道, 二至丸中女贞子、墨旱莲主要含有甾体类、黄酮类、苯乙醇-苷类、香豆素醚类、三萜类等保肝成分, 除这些成分外, 女贞子中还含有多糖类成分[1, 48-50], 均能够对抗多种肝损伤。本研究通过文献发掘及ADME筛选得到二至丸潜在保肝活性成分, 采用Cytoscape中插件CytoNCA对核心成分及靶点进行筛选, 其中女贞子的木犀草苷(degree = 22)、10-羟基女贞苷(degree = 15)、毛蕊花苷(degree = 13)、与墨旱莲中豆甾醇(degree = 11)及其共有成分齐墩果酸(degree = 10)的degree值都均大于10, 这与大量文献报道的保肝作用成分一致[11-41]。另外, 反向预测得到HRAS (degree = 24)、UCK2 (degree = 16)、HSD17B1 (degree = 14)等核心靶点, 其中HRAS和HSD17B1是10-羟基女贞苷、特女贞苷和木犀草苷等16个潜在保肝成分的共同靶点, 这体现了二至丸多成分、多靶点的保肝作用机制。通过GO生物学过程富集分析(图 1)结果可知, 靶基因涉及信号转导、细胞增殖、代谢及蛋白质磷酸化等多个生物学过程。这些生物过程可能与肝损伤的病理和生物过程密切相关, 这和文献报道的多种原因导致的肝损伤所涉及的生物过程相吻合[2, 3, 15, 16, 19]。进一步根据DAVID数据库通路富集分析(图 2)及网络图(图 3)可知, 二至丸可能是通过调节不同的通路群起作用, 主要涉及抗炎、细胞抑制、氧化和代谢等过程, 根据靶点富集情况以及生物学信息, 整合分析相关信号通路, 包括胰岛素、甲状腺等信号通路, 糖酵解途径, FoxO、Fc epsilon RI、VEGF、ErbB、GnRH等炎症相关通路及抑制非小细胞癌、膀胱癌等间接相关通路。

通过分析二至丸组方药材中的活性成分与通路的关联性, 发现女贞子中的10-羟基女贞苷、特女贞子苷和新女贞子苷主要涉及GnRH、FoxO等相关通路, 可能通过激活PI3K/Akt和MAPK途径, 调节T细胞、TNF-α等多种细胞因子, 提高SOD抗氧化酶的活性, 清除自由基, 发挥抗炎的作用, 从而保护肝脏。Lu等[11]研究也发现女贞总苷对TNF-α、IL-6和IL-1β等炎症因子的释放导致的急性肝损伤具有缓解作用。而墨旱莲中的豆甾醇及其苷主要作用于代谢通路和胰岛素信号通路, 这可能和调节多条代谢途径、降低患者的肝脏脂质水平及改善肝功能有关。根据KEGG富集分析结果显示, 在药物代谢-细胞色素P450酶相关通路中, 涉及相关靶点6个, 该结果预示二至丸可能通过细胞色素P450酶的代谢, 达到治疗肝损伤的作用。Zhou等[51]研究表明, 豆甾醇可显著降低脂肪肝细胞中甘油三酯(TG)含量, 改善NAFLD肝细胞脂肪变性程度, 并且豆甾醇在体外能显著减小氧气的生成速率, 具有较强的抗氧化活性[52]。此外, 女贞子和墨旱莲的7个共有成分中齐墩果酸、木犀草及其苷不仅涉及胰岛素信号通路、Fc epsilon RI信号通路, 也和葡萄糖的生成过程有关。这提示齐墩果酸等成分不仅能治疗胰岛素抑制引起的肝损伤, 还和一些炎症引起的急性肝损伤密切相关。He等[15]研究表明, 齐墩果酸可以通过抑制缺血再灌注过程中IKK/I-κB/NF-κB信号传导通路的激活, 上调GLUT4的表达, 从而减轻由NF-κB介导的炎症反应。齐墩果酸还可以降低CCl4导致的MDA升高, 减轻肝脏的病理损害[53]

结论

二至丸由女贞子和墨旱莲两个配伍药材组成, 共有39个不同的保肝活性成分通过调控不同的基因靶点, 作用于不同的通路, 协同配伍从而产生保护肝脏的作用, 体现中药方剂整体性、系统性的特点。本研究发现, 二至丸可能从以下3个方面发挥保肝作用: ①参与Fc epsilon RI、FoxO等炎症信号通路, 通过调节TNF-α等多种炎症因子, 清除氧自由基, 缓解急性肝损伤; ②通过体内多种代谢途径, 降低肝脏脂质水平; ③调控HRAS、HSD17B1及非小细胞癌途径, 抑制肝星状细胞增殖和活化。预测所得二至丸成分靶点与已有文献较为接近, 表明基于网络药理学的技术手段探讨其保护肝脏的作用机制具有一定的准确性, 同时也揭示了二至丸多成分、多靶点的特点, 为后续的相关研究提供参考。

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