痛经（dysmenorrhea）、盆腔炎（pelvic inflammatory disease,PID）和子宫肌瘤（hysteromyoma）是妇科常见疾病，多见于月经期妇女，疼痛是其主要的临床表现，严重时会影响到患者的工作、学习以及生活质量^{[1, 2, 3, 4]}。目前用于痛经和盆腔炎治疗的药物多是一些非甾体抗炎药（NSAIDS）^[5]，用于治疗子宫肌瘤的药物主要有孕激素类、纤溶药物、NSAIDS、促性腺激素类似物/拮抗剂（GnRHanalogs/antagonists）^[6]等。

由于化学药的不良反应或疗效不理想等问题，近年来从中药中寻找治疗妇科疾病的药物日渐受到重视^{[5, 7, 8, 9]}。桂枝茯苓胶囊是由江苏康缘药业股份有限公司根据经典方桂枝茯苓丸研发的现代中药制剂，其君药桂枝、臣药桃仁、佐以牡丹皮、白芍和茯苓而成，具有活血、化瘀、消癥之功效。临床主要用于妇女瘀血阻络所致癥块、经闭、痛经、产后恶露不尽以及子宫肌瘤、慢性盆腔炎等^{[10, 11]}。虽然前期对桂枝茯苓胶囊的作用机制和化学成分有一些研究^{[12, 13]}，但由于中药成分的多样性和作用机制的多靶点、多通路，桂枝茯苓胶囊的主要活性成分和作用机制的实验研究仍面临着诸多挑战。

网络药理学是基于网络观点从系统水平分析药物作用机制^[14]、发现先导化合物或新适应症^[15]、识别新药靶^[16]等一种新的药物研发策略，其与中药整体调控机体的理念相吻合^[17]。目前网络药理学研究思路也逐渐被引入到中药中来发现药效物质基础、探索分子作用机制以及阐释其科学内涵^{[18, 19, 20, 21, 22]}，同时也能大大降低中药后续实验研究的工作量。因此，本研究将网络药理学研究思路与中药桂枝茯苓胶囊相结合来阐释其治疗痛经、盆腔炎和子宫肌瘤的药效活性成分和可能的分子机制，旨在为后续的实验研究方向选择提供一定的信息支撑。

在前期对桂枝茯苓胶囊中164个化学成分分离确认的基础上^{[23, 24]}，根据OpenBabel 2.3.2软件产生的化合物InChiKey字符串从UNPD数据库（http://pkuxxj.pku.edu.cn/UNPD/）共计筛选出172个分子（表 1），经Open Babel加氢后，选用MMFF94力场进行构象优化，优化时能量阈值设定为1×10⁻³J/mol。

表 1(Table 1)

表 1 桂枝茯苓胶囊中筛选出的化合物 Table 1 Compounds separated from GFC ID 化合物 ID 化合物 UNPD10084 3-epi-dehydrotumulosic acid UNPD187407 suffrupaeonidanin A UNPD101050 pachymic acid UNPD188804 hyperoside UNPD101054 poricoic acid AM UNPD191345 hederagenin UNPD102502 α-amyrin UNPD191562 lactoflorin UNPD102661 3-epi-dehydropachymic acid UNPD194734 α-amyrin UNPD102760 poricoic acid C UNPD195952 sodium paeoniflorin sulfonate UNPD105334 guanosine UNPD20150 hyperoside UNPD105452 betulinic acid UNPD20394 arginine UNPD106083 poricoic acid C UNPD21132 coumarin UNPD108574 catechins UNPD23348 poricoic acid DM UNPD109023 cinnamic acid UNPD25688 α-amyrin UNPD109974 trehalose UNPD28633 cinnamyl alcohol UNPD11020 β-sitosterol UNPD28717 4-hydroxybenzoic acid UNPD111541 sucrose UNPD30980 hyperoside UNPD115852 polyporenic acid C UNPD31327 α-amyrin UNPD117238 kaempferol-3-O-glucosylside UNPD31682 dehydropachymic acid UNPD118360 α-amyrin UNPD32715 sucrose UNPD120575 β-L-arabinose UNPD33147 5,7-dimethyl-3′,4′-di-O-methylene-(±)-epicatechin UNPD120855 hyperoside UNPD35192 α-D-galatose UNPD121048 caffeic acid UNPD35358 poricoic acid A UNPD12116 oleanolic acid UNPD3627 poricoic acid D UNPD121313 suffrupaeonidanin A UNPD36336 mudanpioside-F UNPD123516 citrostadienol UNPD36636 poricoic acid DM UNPD126066 mudanpioside-B UNPD37924 eburicoic acid UNPD127168 hyperoside UNPD38191 campesterol UNPD128526 mudanpioside-A UNPD38563 citrostadienol UNPD12870 trehalose UNPD41421 alanine UNPD128750 sucrose UNPD41767 benzoyloxypaeoniflorin UNPD129222 dehydroeburicoicacid UNPD45635 daucosterol UNPD129797 hyperoside UNPD46148 β-sitosterol UNPD130468 α-amyrin UNPD46267 trehalose UNPD13342 α-amyrin UNPD48168 mannitol UNPD133665 caffeic acid UNPD48910 apigenin UNPD135747 lactoflorin UNPD48939 oleic acid UNPD136822 α-amyrin UNPD49205 quercetin UNPD137580 α-amyrin UNPD49664 cinnamic aldehyde UNPD138219 hyperoside UNPD49764 4-hydroxybenzyl alcohol UNPD140050 acetoguaiacon UNPD50991 poricoic acid AM UNPD140430 ergosta-4,6,8(14),22-tetraen-3-one UNPD51284 benzoic acid UNPD140949 gallic acid UNPD52427 caffeic acid UNPD143140 dehydroeburicoicacid UNPD52570 β-sitosterol UNPD143725 paeoniflorin UNPD53479 benzoylpaeoniflorin UNPD14414 α-amyrin UNPD55450 trehalose UNPD145255 adenosine UNPD57689 epicatechin UNPD147172 mudanpioside-F UNPD57841 proline UNPD147436 mudanpioside-D UNPD58928 hyperoside UNPD147757 15a-hydroxydehydrotumulosic acid UNPD60430 protocatechuic acid UNPD14867 polyporenic acid C UNPD61258 leucine UNPD148744 2,5-dihydroxy-4-methylacetophenone UNPD6300 β-sitosterol UNPD148754 lactoflorin UNPD64073 paeonol UNPD149531 5,7,3′-Trimethyl-(-)-epicatechin UNPD64969 suffrupaeonidanin B UNPD152742 citrostadienol UNPD65144 mudanpioside-B UNPD152846 citrostadienol UNPD65702 dehydroeburicoicacid UNPD15360 α-amyrin UNPD66565 β-amyrin acetate UNPD153802 suffrupaeonidanin B UNPD66644 poricoic acid B UNPD155042 taxifolin UNPD6707 daucosterol UNPD16064 7-hydroxycoumarin UNPD68611 guanosine UNPD161634 kaempferol UNPD70084 sucrose UNPD161812 trans-O-methoxy cinnamic acid UNPD70549 oxypaeoniflora UNPD16201 albiflorin UNPD72055 dehydrotumulosic acid UNPD162992 taxifolin UNPD72395 amygdalin UNPD164160 α-amyrin UNPD72621 glucose UNPD165682 2-methoxybenzoic acid UNPD73147 α-amyrin UNPD166501 α-amyrin UNPD73469 hyperoside UNPD167245 sucrose UNPD75597 hyperoside UNPD167251 syringaresinol UNPD79460 citrostadienol UNPD167512 dehydropachymic acid UNPD80387 catechins UNPD170534 suffrupaeonidanin C UNPD80675 citrostadienol UNPD172175 poricoic acid D UNPD81316 pachymic acid UNPD172347 taxifolin UNPD82149 betulinic acid UNPD172457 5-HMF UNPD83717 glucose UNPD173098 poricoic acid DM UNPD83955 poricoic acid E UNPD173899 prunasin UNPD85202 poricoic acid CM UNPD176049 taxifolin UNPD86062 desbenzoylpaeoniflorin UNPD17684 syringic acid UNPD86478 poricoic acid A UNPD177395 3,3′-di-O-methyl ether ellagic acid UNPD89095 ergosterol UNPD180657 paeoniflorin UNPD95458 cinnamyl alcohol UNPD181583 poricoic acid D UNPD95544 methyl gallate UNPD182597 α-amyrin UNPD96215 α-amyrin UNPD183645 3,3′-di-O-methyl ether ellagic acid UNPD99873 dulcitol UNPD184720 poricoic acid C GZFL_1 3-hydroxy-tirucallon-(7)-oic acid UNPD184770 tumulosic acid GZFL_2 galloylpaeoniflorin UNPD186476 α-amyrin GZFL_3 mudanpioside-E UNPD186668 poricoic acid F GZFL_4 mudanpioside-C UNPD187258 3β-p-hydroxybenzoyl-dehydrotumulosic acid UNPD59312 5a,8a-peroxydehydrotumulosic acid UNPD77610 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose UNPD134433 β-D-glucopyranoside,phenylmethyl 6-O-β-galactopyranosyl

表 1 桂枝茯苓胶囊中筛选出的化合物 Table 1 Compounds separated from GFC

根据桂枝茯苓胶囊的临床主要用于痛经、盆腔炎、子宫肌瘤等妇科疾病的治疗情况，利用Therapeutic Target Database（TTD）、DrugBank数据库结合文献综述分析^{[6, 8, 9, 25, 26, 27, 28, 29, 30, 31]}，以dysmenorrhea、hysteromyoma、uterine fibroids（子宫平滑肌瘤）、PID为检索词共筛选与上述疾病密切相关的130个靶点，并从RCSB数据库（www.pdb.org）获取人源的且含有原配体的晶体结构（Resulation＜0.25 nm，表 2），导入Discovery Studio 2.5通过CleanProtein和Forcefiled中CHARMm力场完成靶蛋白的加氢、去水、蛋白修饰以及自动分配原子类型和确定活性位点的中心坐标。

表 2(Table 2)

表 2 与痛经、盆腔炎和子宫肌瘤相关的靶蛋白 Table 2 Target proteinsrelated with dysmenorrhea,PID,and hysteromyoma Uniprot PDB 蛋白名称 Uniprot PDB 蛋白名称 Q8IU85 2JC6 calcium/calmodulin-dependent protein kinase type 1D Q8NBQ5 1YB1 estradiol 17-beta-dehydrogenase 11 Q96NX5 2JAM calcium/calmodulin-dependent protein kinase type 1G P62508 2P7A estrogen-related receptor gamma Q16566 2W4O calcium/calmodulin-dependent protein kinase type IV P05230 3K1X fibroblast growth factor 1 P27815 3I8V cAMP-specific 3',5'-cyclic phosphodiesterase 4A P09038 1BFB fibroblast growth factor 2 Q07343 1XLX cAMP-specific 3',5'-cyclic phosphodiesterase 4B P11362 2FGI fibroblast growth factor receptor 1 Q08499 3G4K cAMP-specific 3',5'-cyclic phosphodiesterase 4D P21802 3B2T fibroblast growth factor receptor 2 P22680 3SN5 cholesterol 7-alpha-monooxygenase Q9BVM4 3JUC gamma-glutamylaminecyclotransferase P61073 3ODU C-X-C chemokine receptor type 4 P49841 3I4B glycogen synthase kinase-3 beta P00533 1XKK epidermal growth factor receptor P62993 2H5K growth factor receptor-bound protein 2 P14061 1I5R estradiol 17-beta-dehydrogenase 1 P62826 3GJ0 GTP-binding nuclear protein Ran P04629 4AOJ high affinity nerve growth factor receptor P29466 1RWX caspase-1 Q14643 1N4K inositol 1,4,5-trisphosphate receptor type 1 P00740 3LC3 coagulation factor IX P23677 1W2C inositol-trisphosphate 3-kinase A P12259 3P70 coagulation factor V Q96DU7 2A98 inositol-trisphosphate 3-kinase C P08709 2FLR coagulation factor VII P08069 3i81 insulin-like growth factor 1 receptor P00451 3HNB coagulation factor VIII Q9NWZ3 2NRU interleukin-1 receptor-associated kinase 4 P00742 3M36 coagulation factor X P29460 1F42 interleukin-12 subunit beta P03951 1ZOM coagulation factor XI P07333 3DPK macrophage colony-stimulating factor 1 receptor P45452 3ELM collagenase 3 P14174 3IJG macrophage migration inhibitory factor P00746 1DIC complement factor D P49137 3R2B MAP kinase-activated protein kinase 2 P11511 3EQM cytochrome P450 19A1 P28482 3I5Z mitogen-activated protein kinase 1 P47712 1CJY cytosolic phospholipase A2 A6NG28 3TTI mitogen-activated protein kinase 10 P14416 2HLB d(2) dopamine receptor P53779 3TTI mitogen-activated protein kinase 10 P00374 2W3A dihydrofolate reductase Q15759 3GC9 mitogen-activated protein kinase 11 P03372 2QXS estrogen receptor P53778 1CM8 mitogen-activated protein kinase 12 Q92731 2QTU estrogen receptor beta Q16539 1KV1 mitogen-activated protein kinase 14 P49888 1G3M estrogen sulfotransferase P27361 2ZOQ mitogen-activated protein kinase 3 P02751 2OCF fibronectin Q13164 4B99 mitogen-activated protein kinase 7 P04150 3H52 glucocorticoid receptor P45983 3PZE mitogen-activated protein kinase 8 Q9BY41 1T69 histone deacetylase 8 P45984 3NPC mitogen-activated protein kinase 9 P09960 3FH5 leukotriene A-4 hydrolase Q99558 4DN5 mitogen-activated protein kinase kinase kinase 14 Q16873 2UUH leukotriene C4 synthase Q99683 2CLQ mitogen-activated protein kinase kinase kinase 5 P09237 1MMP matrilysin O43318 2YIY mitogen-activated protein kinase kinase kinase 7 P51512 1RM8 matrix metalloproteinase-16 P80192 3DTC mitogen-activated protein kinase kinase kinase 9 O60882 2JSD matrix metalloproteinase-20 Q9HBL8 2WM3 nmrA-like family domain-containing protein 1 P14780 1GKC matrix metalloproteinase-9 O75469 1M13 nuclear receptor subfamily 1 group i member 2 P08235 2OAX mineralocorticoid receptor Q8NEB9 3LS8 phosphatidylinositol 3-kinase catalytic subunit type 3 P29474 1M9J nitric oxide synthase,endothelial P27986 4A55 phosphatidylinositol 3-kinase regulatory subunit alpha P35228 4NOS nitric oxide synthase,inducible P14555 1J1A phospholipase A2,membrane associated P51575 4DW1 P2X purinoceptor 1 P04626 3RCD receptor tyrosine-protein kinase erbB-2 O75051 3Q3J plexin-A2 P23921 2WGH ribonucleoside-diphosphate reductase large subunit P06401 2W8Y progesterone receptor O76054 1OLM SEC14-like protein 2 O14684 3DWW prostaglandin E synthase P42345 1FAP serine/threonine-protein kinase mTOR P23219 3N8X prostaglandin G/H synthase 1 Q9NQU5 2ODB serine/threonine-protein kinase PAK 6 P35354 3LN1 prostaglandin G/H synthase 2 P05093 3RUK steroid 17-alpha-hydroxylase/17,20 lyase Q14914 2Y05 prostaglandin reductase 1 P11474 3K6P steroid hormone receptor ERR1 Q8N8N7 2W4Q prostaglandin reductase 2 P48061 2NWG stromal cell-derived factor 1 P41222 3O19 prostaglandin-H2 D-isomerase P36897 2X7O TGF-beta receptor type-1 P17252 3IW4 protein kinase C alpha type P21731 1LBN thromboxane A2 receptor P05771 2I0E protein kinase C beta type P01375 2AZ5 tumor necrosis factor P24723 3TXO protein kinase C eta type P23458 3EYG tyrosine-protein kinase JAK1 P41743 1ZRZ protein kinase C iota type P17948 3HNG vascular endothelial growth factor receptor 1 Q04759 1XJD protein kinase C theta type P35968 2QU5 vascular endothelial growth factor receptor 2 P57735 3TSO ras-related protein Rab-25 P22303 4EY5 acetylcholinesterase P13501 1U4L C-C motif chemokine 5 P10275 3L3X androgen receptor Q00722 2ZKM 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-2 P01008 3EVJ antithrombin-III P16885 2W2W 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 P10415 2W3L apoptosis regulator Bcl-2 Q9UQM7 3SOA calcium/calmodulin-dependent protein kinase type II subunit alpha P09917 3V99 arachidonate 5-lipoxygenase Q13554 3BHH calcium/calmodulin-dependent protein kinase type II subunit beta Q96GD4 4AF3 aurora kinase B Q13557 2VN9 calcium/calmodulin-dependent protein kinase type II subunit delta P07550 3NY8 beta-2 adrenergic receptor Q13555 2V7O calcium/calmodulin-dependent protein kinase type II subunit gamma P62158 1CTR calmodulin P36507 1S9I dual specificity mitogen-activated protein kinase kinase 2 P42336 3HHM phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Q13946 P52564 1ZKL 3FME high affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A dual specificity mitogen-activated protein kinase kinase 6 P48736 3SD5 phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform P67775 2IE4 serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform Q02750 3DY7 dual specificity mitogen-activated protein kinase kinase 1 P19438 1FT4 tumor necrosis factor receptor superfamily member 1A

表 2 与痛经、盆腔炎和子宫肌瘤相关的靶蛋白 Table 2 Target proteinsrelated with dysmenorrhea,PID,and hysteromyoma

1.2 分子对接

根据上述确定的分子UNPD与靶蛋白的Uniprot，从TCMN数据库^[32]中抽提出分子与靶蛋白相互作用数据对。在TCMN数据库中，分子与靶蛋白相互作用通过DOVIS2.0平台中AutoDock4.0内核来完成分子与靶蛋白对接计算^[33]。具体对接参数如下：以靶蛋白中原配体的坐标为活性位点的中心，盒子大小为4 nm×4nm×4 nm，格点间隔为0.037 5 nm，采用拉马克遗传算法采集分子构象，初始种群为150，旋转步长为50°，平移步长为0.2 nm，交叉率为0.8，突变率为0.02，局部搜索频率为0.06，其余均为默认设置。

根据分子与蛋白的对接分值，以原配体对接得分为阈值，抽取对接得分较高（score≥5.00）的分子-靶蛋白相互作用数据对导入Cytoscape3.0^[34]构建桂枝茯苓胶囊中小分子与靶蛋白相互作用网络，并将网络中小分子进一步投射到相应的药材构建出药材-分子-靶标网络（herb-drug-target network，HDTN）。同时，针对网络中的靶蛋白，进一步投射到KEGG数据库（http://www.genome.jp/kegg/pathway. html）构建出靶蛋白-生物通路网络（target-pathwaynetwork，TPN），从而获得桂枝茯苓胶囊对生物通路的影响。上述网络的构建及网络特征的分析均采用Cytoscape 3.0及其插件NetworkAnalyzer来计算。

在构建的HDTN网络（图 1）中，包含115个分子，116个靶蛋白以及5味中药，平均每个分子靶向17.90个靶蛋白，每个蛋白有17.75个分子，然后利用Cytoscape 3.0中插件NetworkAnalyzer^[35]进一步对HDTN网络中分子与靶蛋白间的相互关系网的分析发现，此关系网的平均最短路径为17.827，远小于整个节点数（231），提示分子与靶标的关系网为典型的小世界网络（small world），而从分子与靶标关系网中各节点的度分布服从幂律分布［P(κ)＝28.96×κ^−0.73（r＝0.865）］来看，说明分子与靶标构成的关系网具有良好的稳定性^{[36, 37]}，提示桂枝茯苓胶囊中这些活性成分能纠正疾病状态下的机体平衡网络促使其形成新的机体平衡，进而可能起到调控机体的治疗作用^[38]。同时，在分子与药材的相互关系分析发现，46个分子来自于君药桂枝、46个分子来自于茯苓，而来自白芍和牡丹皮的分子各有27个，此外还有桃仁的28个分子，其中桃仁与桂枝之间的活性成分有较大的重叠，这也印证了桂枝茯苓胶囊组方的合理性（图 2）。

圆形代表从茯苓桂枝胶囊中筛选的化合物；菱形代表蛋白靶点；圆角矩形代表组方药材 Ellipse represents small molecules from GFC;Diamond represents target proteins; Round rectangle represents herbs图 1 药材-分子-靶点网络 Fig.1 Herb-drug-target network (HDTN)

图 2 桂枝茯苓胶囊中活性成分在药味归属中的分布 Fig.2 Distribution of activeconstituents from GFC in herbalattribution

2.2 主要活性化合物和潜在靶蛋白分析

在网络分析中，除了整体网络特征分析，还提出了度（degree）、介数（betweenness）、聚类系数（clustering coefficient）等一系列可定量刻画网络内部结构的度量^[39]，并已被应用到生物网络中来鉴别可药的靶点^[40]、发现药物新适应症^[15]、解析药物作用新机制以及活性化合物的发现^[14]等，尤其是度、介数常被用于研究分子网络中各节点的重要性。因此，在对HDTN网络的整体拓扑学特征分析的基础上，进一步对网络中各节点的度和介数进行了详细分析，以确定桂枝茯苓胶囊中主要活性化合物和潜在的靶蛋白。

从HDTN网络中分子节点的网络度和介数来看（表 3、4），67个分子（35个化合物）可与6个以上的靶蛋白相互作用，其中网络度和介数较高的分子多数为五环三萜类化合物和甾醇类化合物，如柠檬甾二烯醇（citrostadienol）、菜油甾醇（campesterol）、麦角甾醇（ergosterol）、去氢茯苓酸（dehydropachymic acid）、茯苓酸（pachymic acid）、去氢土莫酸（dehydrotumulosic acid）、ergosta-4,6,8 (14),22-tetraen-3-one、齐墩果酸（oleanolic acid），还有部分黄酮类化合物如槲皮素（quercetin）、芹菜素（apigenin）。而文献分析也发现，ergosta-4,6,8 (14),22-tetraen-3-one、oleanolic acid、polyporenicacid C、eburicoic acid 和dehydroeburicoic acid能明显抑制NO、IL-1β和TNF-α^{[41, 42, 43, 44]}，β-amyrin acetate具有镇痛和抗炎活性^{[45, 46]}，campesterol可抑制血管形成^[47]，与用于治疗子宫肌瘤的Progesterone受体激动剂和调节剂药物有类似的结构的甾醇类化合物也被报道治疗多种癌症^{[6, 8, 48]}，黄酮类化合物与用于子宫肌瘤治疗的genistein、isoliquiritigenin等化合物有类似的结构，且槲皮素已被作为有可能治疗子宫肌瘤的多酚类化合物被研究，儿茶素类化合物用于癌症的治疗^{[9, 49]}。

表 3(Table 3)

表 3 HDTN网络中部分节点（化合物）的网络特征 Table 3 Network features of nodes (compoundsfrom GFC) with high degree inHDTN 编码 名称 度 介数 编码 名称 度 介数 UNPD166501 α-amyrin 76 0.048 5 UNPD80675 citrostadienol 25 0.003 4 UNPD164160 α-amyrin 74 0.055 0 UNPD14867 polyporenic acid C 24 0.011 9 UNPD31327 α-amyrin 73 0.061 3 UNPD191345 hederagenin 17 0.004 0 UNPD15360 α-amyrin 72 0.043 9 UNPD129222 dehydroeburicoic acid 16 0.005 5 UNPD140430 ergosta-4,6,8(14),22-tetraen-3-one 65 0.061 0 UNPD105452 betulinic acid 15 0.001 4 UNPD118360 α-amyrin 62 0.030 0 UNPD115852 polyporenic acid C 14 0.002 7 UNPD25688 α-amyrin 60 0.023 6 UNPD148754 lactoflorin 14 0.001 9 UNPD194734 α-amyrin 59 0.029 6 UNPD184770 tumulosic acid 12 0.001 5 UNPD96215 α-amyrin 58 0.022 9 UNPD147757 15α-hydroxydehydrotumulosic acid 12 0.001 3 UNPD14414 α-amyrin 58 0.019 7 UNPD176049 taxifolin 11 0.001 2 UNPD73147 α-amyrin 57 0.029 6 UNPD45635 daucosterol 11 0.000 7 UNPD66565 β-amyrin acetate 57 0.024 7 UNPD48910 apigenin 10 0.004 1 UNPD137580 α-amyrin 55 0.022 9 UNPD162992 taxifolin 10 0.003 2 UNPD130468 α-amyrin 55 0.021 5 UNPD10084 3-epi-dehydrotumulosic acid 10 0.001 1 UNPD186476 α-amyrin 55 0.019 5 UNPD37924 eburicoic acid 9 0.002 5 UNPD13342 α-amyrin 53 0.020 6 UNPD167512 dehydropachymic acid 9 0.002 4 UNPD136822 α-amyrin 52 0.022 6 UNPD31682 dehydropachymic acid 9 0.000 8 UNPD102502 α-amyrin 43 0.012 0 UNPD105334 guanosine 9 0.000 6 UNPD182597 α-amyrin 41 0.024 7 UNPD65702 dehydroeburicoicacid 8 0.001 4 UNPD123516 citrostadienol 41 0.014 5 UNPD81316 pachymic acid 8 0.000 5 UNPD38191 campesterol 39 0.011 4 UNPD102661 3-epi-dehydropachymic acid 8 0.000 5 UNPD12116 oleanolic acid 38 0.033 3 UNPD187258 3β-p-hydroxybenzoyl-dehydrotumulos ic acid 8 0.000 5 UNPD79460 citrostadienol 38 0.008 0 GZFL_3 mudanpioside-E 8 0.000 4 UNPD11020 β-sitosterol 36 0.017 7 UNPD80387 catechins 7 0.001 3 UNPD52570 β-sitosterol 35 0.009 9 UNPD49205 quercetin 7 0.000 7 UNPD38563 citrostadienol 35 0.005 9 UNPD72055 dehydrotumulosic acid 7 0.000 5 UNPD46148 β-sitosterol 34 0.017 8 UNPD195952 sodium paeoniflorin sulfonate 7 0.000 4 UNPD6300 β-sitosterol 33 0.009 2 UNPD172347 taxifolin 7 0.000 3 UNPD152846 citrostadienol 31 0.007 1 UNPD161634 kaempferol 6 0.002 5 GZFL_1 3-hydroxy-tirucallon-(7)-oic acid 28 0.014 0 UNPD183645 3,3'-di-O-methyl ether ellagic acid 6 0.000 3 UNPD191562 lactoflorin 27 0.004 9 UNPD6707 daucosterol 6 0.000 2 UNPD143140 dehydroeburicoic acid 26 0.008 1 UNPD59312 5a,8a-peroxydehydrotumulosic acid 6 0.000 1 UNPD152742 citrostadienol 26 0.004 9 UNPD135747 lactoflorin 6 0.000 1 UNPD89095 ergosterol 25 0.010 2

表 3 HDTN网络中部分节点（化合物）的网络特征 Table 3 Network features of nodes (compoundsfrom GFC) with high degree inHDTN

表 4(Table 4)

表 4 HDTN网络中部分节点（靶蛋白）的网络特征 Table 4 Network features of nodes (target proteins) with high degree in HDTN Uniprot 名称 度 介数 P29474 nitric oxide synthase,endothelial 36 0.033 6 P14416 D(2) dopamine receptor 35 0.027 1 Q13557 calcium/calmodulin-dependent protein kinase type II subunit δ 32 0.017 2 P45452 collagenase 3 32 0.013 1 Q14914 prostaglandin reductase 1 32 0.009 4 P07550 beta-2 adrenergic receptor 32 0.009 0 P14061 estradiol 17-beta-dehydrogenase 1 31 0.015 3 P49841 glycogen synthase kinase-3 beta 31 0.010 9 O75469 nuclear receptor subfamily 1 group I member 2 30 0.020 0 P14555 phospholipase A2,membrane associated 30 0.018 5 Q02750 dual specificity mitogen-activated protein kinase kinase 1 29 0.010 6 P41743 protein kinase C iota type 29 0.007 6 P49888 estrogen sulfotransferase 28 0.013 0 P42345 serine/threonine-protein kinase mTOR 28 0.010 2 P35228 nitric oxide synthase,inducible 28 0.007 4 Q9UQM7 calcium/calmodulin-dependent protein kinase type II subunit α 27 0.019 7 Q16539 mitogen-activated protein kinase 14 27 0.018 0 P29466 caspase-1 27 0.008 4 Q13555 calcium/calmodulin-dependent protein kinase type II subunit γ 27 0.008 0 Q9HBL8 nmrA-like family domain-containing protein 1 25 0.017 5 P62826 GTP-binding nuclear protein Ran 25 0.017 1 Q96NX5 calcium/calmodulin-dependent protein kinase type 1G 25 0.013 8 Q99683 mitogen-activated protein kinase kinase kinase 5 25 0.007 4 P23921 ribonucleoside-diphosphate reductase large subunit 24 0.015 1 P41222 prostaglandin-H2 D-isomerase 24 0.013 8 O76054 SEC14-like protein 2 24 0.009 3 P53778 mitogen-activated protein kinase 12 24 0.006 8 Q16566 calcium/calmodulin-dependent protein kinase type IV 24 0.005 7 Q8IU85 calcium/calmodulin-dependent protein kinase type 1D 24 0.004 8 P27815 cAMP-specific 3',5'-cyclic phosphodiesterase 4A 24 0.003 5 O76083 high affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A 24 0.003 3 P00533 epidermal growth factor receptor 23 0.005 0 Q9NWZ3 interleukin-1 receptor-associated kinase 4 22 0.006 5 Q15759 mitogen-activated protein kinase 11 22 0.005 8 O60658 high affinity cAMP-specific and IBMX-insensitive 3',5'-cyclic phosphodiesterase 8A 22 0.004 7 P00374 dihydrofolate reductase 22 0.004 0 P61073 C-X-C chemokine receptor type 4 22 0.003 8 P05771 protein kinase C beta type 22 0.002 6 P14780 matrix metalloproteinase-9 21 0.018 5 P11362 fibroblast growth factor receptor 1 21 0.006 5 P48736 phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit γ 21 0.005 4 P03951 coagulation factor XI 21 0.004 5 P08069 insulin-like growth factor 1 receptor 21 0.003 3 P00742 coagulation factor X 20 0.007 8 P52564 dual specificity mitogen-activated protein kinase kinase 6 20 0.005 9 P01375 tumor necrosis factor 20 0.005 7 P53779 mitogen-activated protein kinase 10 20 0.005 1 P23219 prostaglandin G/H synthase 1 20 0.004 7 Q08499 cAMP-specific 3',5'-cyclic phosphodiesterase 4D 20 0.004 3 P09237 matrilysin 20 0.003 0 P45983 mitogen-activated protein kinase 8 19 0.025 5 P23458 tyrosine-protein kinase JAK1 19 0.012 9 O14684 prostaglandin E synthase 19 0.007 8 P45984 mitogen-activated protein kinase 9 19 0.007 1 P05093 steroid 17-alpha-hydroxylase/17,20 lyase 19 0.005 5 P22680 cholesterol 7-alpha-monooxygenase 19 0.003 2 P00746 complement factor D 19 0.002 5 P01008 antithrombin-III 18 0.013 2 Q07343 cAMP-specific 3',5'-cyclic phosphodiesterase 4B 18 0.006 1 P09917 arachidonate 5-lipoxygenase 18 0.005 1 Q13946 high affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A 18 0.003 4 Q04759 protein kinase C theta type 18 0.001 6 P80192 mitogen-activated protein kinase kinase kinase 9 17 0.010 5 P00740 coagulation factor IX 17 0.007 8 P17948 vascular endothelial growth factor receptor 1 17 0.007 4 P36897 TGF-beta receptor type-1 17 0.003 3 Q13554 calcium/calmodulin-dependent protein kinase type II subunit β 17 0.003 0 P27361 mitogen-activated protein kinase 3 17 0.002 3 P62158 calmodulin 17 0.002 1 P07333 macrophage colony-stimulating factor 1 receptor 16 0.007 7 P04626 receptor tyrosine-protein kinase erbB-2 16 0.007 4 Q8NBQ5 estradiol 17-beta-dehydrogenase 11 16 0.007 0 P35968 vascular endothelial growth factor receptor 2 15 0.002 8 Q8NEB9 phosphatidylinositol 3-kinase catalytic subunit type 3 15 0.001 2 Q9BVM4 gamma-glutamylaminecyclotransferase 14 0.007 7 P67775 serine/threonine-protein phosphatase 2A catalytic subunit α 14 0.004 0 P02751 fibronectin 14 0.002 6 O75051 plexin-A2 13 0.032 2 P62993 growth factor receptor-bound protein 2 13 0.008 2 P03372 estrogen receptor 13 0.004 6 Q00722 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase β2 13 0.004 5 P21802 fibroblast growth factor receptor 2 13 0.001 6 P62508 estrogen-related receptor gamma 13 0.000 8 P08709 coagulation factor VII 12 0.003 1 P04629 high affinity nerve growth factor receptor 12 0.002 5 Q92731 estrogen receptor beta 12 0.002 5 O60882 matrix metalloproteinase-20 12 0.002 1 P49137 MAP kinase-activated protein kinase 2 12 0.001 1 Q9BY41 histone deacetylase 8 12 0.000 7 P14174 macrophage migration inhibitory factor 11 0.036 8 P17252 protein kinase C alpha type 11 0.001 8 P24723 protein kinase C eta type 11 0.000 9 P22303 acetylcholinesterase 11 0.000 6 Q9NQU5 serine/threonine-protein kinase PAK 6 10 0.011 2 P51512 matrix metalloproteinase-16 10 0.004 6 P57735 ras-related protein Rab-25 10 0.004 5 Q96GD4 aurora kinase B 10 0.001 1 P36507 dual specificity mitogen-activated protein kinase kinase 2 9 0.003 1 P09960 leukotriene A-4 hydrolase 9 0.000 9 Q99558 mitogen-activated protein kinase kinase kinase 14 8 0.003 6 P12259 coagulation factor V 7 0.018 4 P08235 mineralocorticoid receptor 7 0.001 3 P19438 tumor necrosis factor receptor superfamily member 1A 7 0.001 3 P51575 P2X purinoceptor 1 7 0.000 7 O43318 mitogen-activated protein kinase kinase kinase 7 7 0.000 3 P42336 phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α 7 0.000 3 P05230 fibroblast growth factor 1 6 0.000 4 P28482 mitogen-activated protein kinase 1 6 0.000 4

表 4 HDTN网络中部分节点（靶蛋白）的网络特征 Table 4 Network features of nodes (target proteins) with high degree in HDTN

在对HDTN5网络中116个靶蛋白节点的网络特征分析发现（表 5），有109个靶蛋白可与6个以上的活性分子存在较强相互作用，其中NOS、collagenase 3、coagulation factor X、GSK、PLA₂、FGFR、MMP-9、EGFR、COX等50个靶点可与20个以上的活性分子存在较强相互作用，在这些靶蛋白中，即包括可与NO、PGE₂、TNF-α、leukotriene炎症因子的合成与释放炎症因子密切相关的NOS、COX、PLA2、PGES、5-LOX、caspase-1等关键酶、与凝血过程相关的antithrombin-III、coagulation factor XI、coagulation factor VII、cogulation factor X、cogulation factor IX、coagulation factor V、antithrombin-III酶以及与血管形成和激素作用密切相关的GFR、TGFR、FGR、VEGFR、estrogen receptor、P2X purinoceptor 1受体^[9]。同时桂枝茯苓胶囊中化合物还可与PI3K、GSK3、IRAK4、ERK、PKC、mTOR、MMP-9等靶蛋白相互作用来调控PI3K、PKC、MAPKs等多条信号通路和钙离子通道，进而来影响子宫平滑肌收缩和细胞增殖。

表 5(Table 5)

表 5 TPN中部分节点（生物通路）的网络特征 Table 5 Network features of nodes (pathwayfrom KEGG database) with high degree in TPN ID 名称 度 介数 ID 名称 度 介数 hsa05205 proteoglycans in cancer 30 0.039 4 hsa04730 long-term depression 10 0.007 9 hsa05200 pathways in cancer 29 0.034 6 hsa04666 Fc gamma R-mediated phagocytosis 10 0.005 6 hsa04010 MAPK signaling pathway 28 0.042 0 hsa05132 salmonella infection 10 0.004 4 hsa04722 neurotrophin signaling pathway 27 0.021 9 hsa04713 circadian entrainment 10 0.002 6 hsa04151 PI3K-Akt signaling pathway 23 0.023 0 hsa04930 type II diabetes mellitus 10 0.002 3 hsa05164 influenza A 23 0.021 3 hsa04726 serotonergic synapse 9 0.012 3 hsa04912 GnRH signaling pathway 23 0.020 0 hsa04070 phosphatidylinositol signaling system 9 0.008 9 hsa04380 osteoclast differentiation 23 0.016 4 hsa05203 viral carcinogenesis 9 0.005 8 hsa04012 ErbB signaling pathway 23 0.013 9 hsa04060 cytokine-cytokine receptor interaction 9 0.004 1 hsa04066 HIF-1 signaling pathway 21 0.017 2 hsa04150 mTOR signaling pathway 9 0.003 7 hsa05152 tuberculosis 20 0.025 0 hsa05162 measles 9 0.002 3 hsa04664 hsa05145 Fc epsilon RI signaling pathway toxoplasmosis 20 20 0.018 9 0.017 3 hsa05120 epithelial cell signaling in helicobacter pylori infection 9 0.001 7 hsa04510 focal adhesion 20 0.014 8 hsa04622 RIG-I-like receptor signaling pathway 9 0.001 0 hsa05142 chagas disease (American trypanosomiasis) 19 0.016 4 hsa05211 renal cell carcinoma 9 0.001 0 hsa05214 glioma 19 0.008 6 hsa05220 chronic myeloid leukemia 9 0.000 7 hsa01100 metabolic pathways 18 0.150 6 hsa05221 acute myeloid leukemia 9 0.000 6 hsa04660 T cell receptor signaling pathway 18 0.011 1 hsa04960 aldosterone-regulated sodium reabsorption 8 0.009 3 hsa05160 hepatitis C 18 0.008 4 hsa05034 alcoholism 8 0.007 8 hsa04620 toll-like receptor signaling pathway 18 0.006 1 hsa04064 NF-κB signaling pathway 8 0.004 6 hsa04728 dopaminergic synapse 17 0.018 7 hsa04210 apoptosis 8 0.002 4 hsa04370 VEGF signaling pathway 17 0.015 3 hsa05014 amyotrophic lateral sclerosis (ALS) 8 0.001 9 hsa05161 hepatitis B 17 0.009 4 hsa04520 adherens junction 8 0.001 4 hsa04020 calcium signaling pathway 16 0.022 8 hsa04971 gastric acid secretion 8 0.001 2 hsa05169 epstein-barr virus infection 16 0.019 0 hsa05168 herpes simplex infection 8 0.000 9 hsa04725 cholinergic synapse 15 0.013 4 hsa05131 shigellosis 8 0.000 4 hsa04910 insulin signaling pathway 15 0.008 3 hsa04610 complement and coagulation cascades 7 0.964 3 hsa05215 prostate cancer 15 0.004 2 hsa05032 morphine addiction 7 0.047 0 hsa05133 pertussis 14 0.009 8 hsa00230 purine metabolism 7 0.012 6 hsa05166 HTLV-I infection 13 0.014 6 hsa05010 alzheimer's disease 7 0.004 8 hsa04310 wnt signaling pathway 13 0.013 5 hsa00590 arachidonic acid metabolism 7 0.003 4 hsa04916 melanogenesis 13 0.005 3 hsa04920 adipocytokine signaling pathway 7 0.001 5 hsa04720 long-term potentiation 13 0.004 4 hsa04911 insulin secretion 7 0.000 8 hsa04810 regulation of actin cytoskeleton 13 0.003 9 hsa05219 bladder cancer 7 0.000 8 hsa05223 non-small cell lung cancer 13 0.003 5 hsa04913 ovarian steroidogenesis 6 0.010 7 hsa04914 progesterone-mediated oocyte maturation 13 0.002 2 hsa04360 axon guidance 6 0.009 9 hsa05212 pancreatic cancer 13 0.002 2 hsa04530 tight junction 6 0.004 6 hsa04270 vascular smooth muscle contraction 12 0.025 2 hsa04724 glutamatergic synapse 6 0.002 8 hsa05140 leishmaniasis 12 0.006 3 hsa04630 jak-STAT signaling pathway 6 0.000 4 hsa04650 natural killer cell mediated cytotoxicity 12 0.005 9 hsa00140 steroid hormone biosynthesis 5 0.016 8 hsa04540 gap junction 11 0.010 6 hsa05143 african trypanosomiasis 5 0.003 2 hsa04062 chemokine signaling pathway 11 0.007 3 hsa00562 inositol phosphate metabolism 5 0.003 2 hsa04670 leukocyte transendothelial migration 11 0.006 4 hsa04970 salivary secretion 5 0.002 9 hsa04144 endocytosis 11 0.006 3 hsa04350 TGF-beta signaling pathway 5 0.001 7 hsa04621 NOD-like receptor signaling pathway 11 0.003 8 hsa05222 small cell lung cancer 5 0.001 2 hsa04723 retrograde endocannabinoid signaling 11 0.003 6 hsa04390 hippo signaling pathway 5 0.001 0 hsa04114 oocyte meiosis 11 0.003 2 hsa04973 carbohydrate digestion and absorption 5 0.000 9 hsa04662 B cell receptor signaling pathway 11 0.003 0 hsa05202 transcriptional misregulation in cancer 5 0.000 5 hsa05218 melanoma 11 0.002 0 hsa05216 thyroid cancer 5 0.000 4 hsa05210 colorectal cancer 11 0.001 8 hsa04320 dorso-ventral axis formation 5 0.000 1 hsa05213 endometrial cancer 11 0.001 6 hsa04740 olfactory transduction 5 0.000 1 hsa05146 amoebiasis 10 0.009 6

表 5 TPN中部分节点（生物通路）的网络特征 Table 5 Network features of nodes (pathwayfrom KEGG database) with high degree in TPN

2.3 潜在生物通路分析

生物通路通过其构成的不同靶蛋白间的相互作用来执行特定的生物学功能，是理解疾病临床表现的生理学基础^{[50, 51]}。因此药物的作用不仅与靶蛋白有关，而且也受靶蛋白所在的生物通路的影响，多靶点的中药更是如此，故在上述靶蛋白分析的基础上，又进一步将116个潜在靶点投射到KEGG数据库^[52]，其中98个靶蛋白可投射到KEGG上的157条人源生物通路上（图 3）。进一步的网络分析发现（表 6），网络度最高的3条生物通路分别为proteoglycans in cancer、pathways in cancer和MAPK signaling pathway，均是与肿瘤、炎症等密切相关，介数最高的生物通路为complementand coagulation cascades，是与凝血系统密切相关。

图 3 靶蛋白-生物通路网络 Fig.3 Potentialtarget proteins -biological pathway network

表 6(Table 6)

表 6 TPN网络中与痛经、盆腔炎和子宫肌瘤密切相关生物通路的网络特征 Table 6 Network features of biological pathways related to dysmenorrhea,PID,and hysteromyoma in TPN ID 通路 度 介数 hsa05200 pathways in cancer 29 0.034 6 hsa04010 MAPK signaling pathway 28 0.042 0 hsa04151 PI3K-Akt signaling pathway 23 0.023 0 hsa04912 GnRH signaling pathway 23 0.020 0 hsa04370 VEGF signaling pathway 17 0.015 3 hsa04020 calcium signaling pathway 16 0.022 8 hsa04310 wnt signaling pathway 13 0.013 5 hsa04914 progesterone-mediated oocyte maturation 13 0.002 2 hsa04270 vascular smooth muscle contraction 12 0.025 2 hsa04064 NF-kappa B signaling pathway 8 0.004 6 hsa04210 apoptosis 8 0.002 4 hsa04610 complement and coagulation cascades 7 0.964 3 hsa00590 arachidonic acid metabolism 7 0.003 4 hsa00140 steroid hormone biosynthesis 5 0.016 8 hsa04350 TGF-beta signaling pathway 5 0.001 7

表 6 TPN网络中与痛经、盆腔炎和子宫肌瘤密切相关生物通路的网络特征 Table 6 Network features of biological pathways related to dysmenorrhea,PID,and hysteromyoma in TPN

为了更好地理解桂枝茯苓胶囊发挥治疗痛经、盆腔炎和子宫肌瘤的作用机制，从上述的靶点-通路网络中抽提了与痛经、盆腔炎、子宫肌瘤密切相关的15条生物通路（表 6）。在这15条生物通路中，GnRH signaling pathway和progesterone-mediated oocytematuration分别涉及到雌激素对子宫平滑肌细胞的生理作用、子宫内膜增生、月经出血^{[6, 8]}；VEGF signaling pathway和arachidonic acid metabolism分别涉及到子宫内膜内血管形成、PGE₂、PGF_2α、leukotriene B4生成以及子宫内膜内腺体增生等^{[28, 31, 53, 54, 55]}；TGF-beta signaling pathway涉及到调控细胞生长、纤维化以及炎症反应等^[9]；而complement and coagulation cascades和steroid hormone biosynthesis则分别与凝血系统以及类固醇激素合成密切相关^[56]；calcium signaling pathway和vascular smooth musclecontraction与子宫平滑肌的松弛有密切关系^[57]。而从target-pathway中节点网络特征分析来看，这15条生物通路多数均有较高的网络度和或较高的介数，且包含了98个潜在蛋白中77个，占78.57%（图 4）。因此，推测桂枝茯苓胶囊可能是通过多个成分对多条生物通路的作用来抑制子宫平滑肌的增殖、改善子宫内膜的血管形成和血液循环、降低/减弱雌激素和黄体酮的分泌、子宫平滑肌收缩以及前列腺素等炎症因子合成或释放，从而起到缓解痛经、盆腔炎和子宫肌瘤引起的疼痛、降低炎症反应、改善患者生活质量的作用。在上述分析结果的基础上，对前期的实验报道分析也发现桂枝茯苓胶囊的确能明显降低子宫肌瘤组织中progesterone receptor和大鼠血清中progesterone水平^{[58, 59]}，能下调盆腔炎大鼠血清中TGF-β1的水平^[60]。同时，整体动物实验也发现桂枝茯苓胶囊的醋酸乙酯部位能显著抑制缩宫素诱导的子宫收缩^[61]，能显著降低痛经大鼠血清中PGF_2α的量^[62]。

图 4 靶蛋白在与痛经、盆腔炎及子宫肌瘤密切相关的15条生物通路中的分布 Fig.4 Potentialtarget proteins in 15 biological pathway related to dysmenorrheal,PID,and mysteromyoma

3 讨论

通过分子对接、分子-靶蛋白网络特征分析以及生物通路的信息挖掘对桂枝茯苓胶囊治疗痛经、盆腔炎以及子宫肌瘤疾病的主要活性成分和可能的分子进行了分析，明确了67个分子（35化合物），主要为五环三萜类、甾醇类和黄酮类，进一步的药材归属发现这些分子在各单味中药中的分布与桂枝茯苓方的君臣佐使组方相一致，这也从侧面印证了该处方的合理性。生物通路分析发现桂枝茯苓胶囊中的活性化合物通过与arachidonic acid metabolism、complement and coagulation cascades、calcium signaling pathway、VEGF signaling pathway、GnRH signaling pathway等15条生物通路作用来抑制子宫平滑肌的增殖、改善子宫内膜的血管形成和血液循环、降低/减弱雌激素和黄体酮的分泌、子宫平滑肌收缩以及NO、PGE₂、PGF_2α、TNF-α、leukotriene等炎症因子合成或释放。本研究通过分子计算、网络分析和信息挖掘的组合应用，初步解析了桂枝茯苓胶囊治疗痛经、盆腔炎及子宫肌瘤的药效物质基础、可能的分子机制和相关的生物通路，这将为后续桂枝茯苓胶囊药效成分分析和作用机制探索等实验研究选择提供一定的参考方向。