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胰腺癌是一种发病隐匿且进展迅速的消化系统恶性肿瘤。胰腺癌发病与吸烟、肥胖、遗传、糖尿病和饮酒等多种危险因素相关,其首次确诊时通常已是晚期,预后极差[1]。尽管过去10年内全球癌症的生存率不断提高,但胰腺癌的发病率在不断提高的同时,其5年生存率在我国胰腺癌患者中却并未提高,仅为7%左右[2]。晚期胰腺癌的治疗主要以化疗为主,其中,FOLFIRINOX化疗方案与白蛋白结合型紫杉醇联合吉西他滨的治疗方案对胰腺癌患者的生存率有一定的提高[3]。尽管联合化疗方案对胰腺癌的治疗有一定效果,但化疗药物的毒副作用与肿瘤细胞的耐药性限制了其在胰腺癌治疗中的应用。
中医秉承整体调理与辨证论治的治疗理念,在胰腺癌的治疗方面具有独特的优势,如缓解临床症状,提高抗癌药物的临床效果,改善患者的生活质量以及延长患者的生命等[4]。研究表明,在中医治疗胰腺癌的用药规律中,白术-半夏-茯苓是最常用且置信度最高的药组。三药协同可产生健脾益气、燥湿化痰等功效[5]。中药白术是将菊科植物白术的根茎干燥后制得,具有健脾益气、除湿益燥的功效。其活性成分可以促进肿瘤细胞凋亡,抑制胰腺肿瘤细胞生长与转移,调节免疫代谢等[6]。中药半夏为天南星科植物半夏的干燥块茎,具有燥湿化痰、消痞散结的作用效果。半夏的化学成分包括生物碱类、 有机酸类、黄酮类和糖类等,具有抗肿瘤、 抗炎等药理活性[7]。中药茯苓是真菌茯苓的干燥菌核,其多糖与三萜类化合物成分可以通过增强机体免疫力、增加其他化疗药物的抗肿瘤效果以及降低其他化疗药物的毒副作用等机制发挥抗肿瘤效果[8]。尽管临床上白术-半夏-茯苓药组常用于胰腺癌的配伍治疗,但其包含的活性成分复杂,具体分子作用机制尚未系统阐述。
网络药理学可以利用药物疾病相关数据库以及生物系统分析方法来预测药物、疾病、靶点间的联系,从而了解药物成分对机体的潜在调控机制[9]。分子对接技术则利用计算机模拟配受体间的相互结合模式,通过一系列参数筛选出能相互结合的药物活性成分与靶点蛋白,从而提高药物开发与分子机制验证的成功率[10]。本研究利用网络药理学与分子对接技术分析验证白术-半夏-茯苓配伍治疗胰腺癌的潜在分子机制,以期为临床使用该药组提供理论参考依据。
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在TCMSP数据库中检索到白术55个化合物,半夏116个化合物,茯苓34个化合物。在OB≥30%,DL≥0.18的筛选条件下,共得到35 个活性成分化合物,其中,白术 7个, 半夏13个,茯苓15 个。通过 PharmMapper平台对 35个化合物的靶点进行预测,将得到的结果与TCMSP搜集的靶点汇总去重后,经 UniProt 数据库标准化后得到190个潜在作用靶点。通过检索OMIM和GeneCards数据库,筛选汇总后共获得1 566个胰腺癌相关靶点。通过绘制韦恩图,得到白术-半夏-茯苓配伍治疗胰腺癌的交集靶点共76个(图1)。
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在Cytoscape 3.9.1软件中导入白术-半夏-茯苓配伍治疗胰腺癌的活性成分与交集靶点数据,构建药物-成分-交集靶点的关系网络(图2),包括114个节点(3个中药节点、35个活性成分节点、76个交集靶点节点)和516条边。其中,用拼音首字母BZ、BX、FL分别代表白术、半夏、茯苓;首字母+数字代表每种药物的活性成分。从图2 可以看出,同一活性分子可作用多个靶点,而不同活性分子可作用同一靶点,说明该药组配伍治疗胰腺癌可能是通过多成分、多基因靶点发挥作用的。在网络中,节点越大,节点相连的线条数越多,则节点的度值越大,其在网络中发挥的作用也相对越大。所有活性成分中,白术活性成分度值排名前三的是12-千里光酰基-8-反式白术三醇(12-senecioyl-2E,8E,10E-atractylentriol,度值15),14-乙酰基-12-千里光酰基-8-反式白术三醇(14-acetyl-12-senecioyl-2E,8E,10E-atractylentriol,度值15),3β-乙酰氧基苍术酮(3β-acetoxyatractylone,度值9);半夏活性成分度值排名前三的是黄芩素(baicalein,度值31),12,13-环氧-9-羟基十九碳-7,10-二烯酸(12,13-epoxy-9-hydroxynonadeca-7,10-dienoic acid,度值23),β-谷甾醇(beta-sitosterol,度值22);茯苓活性成分度值排名前三的是茯苓酸A(poricoic acid A,度值22),茯苓酸B(poricoic acid B,度值22),3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸(3beta-Hydroxy-24-methylene-8-lanostene-21-oic acid,度值21)。度值排名前10的靶点分别为CA2(度值32),PIM1(度值27),ALB(度值25),MAPK1(度值21),AR(度值20),GSTP1(度值20),CASP3(度值19),PGR(度值17),BMP2(度值17),CASP7(度值15)。
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使用STRING 11.5平台对交集靶点进行分析,并将结果数据导入Cytoscape 3.9.1构建PPI网络(图3)。其中,节点颜色越深、直径越大代表它与其他靶点的相关性越大,在蛋白相互作用的网络中处于越中心的位置。对PPI网络进行拓扑分析,筛选得到网络中degree值>2倍中位数值的17个核心靶点蛋白基因(图4),degree值排名前十的靶点蛋白基因分别是SRC、TP53、MAPK1、ESR1、JUN、RELA、FOS、AKT1、HIF1A、MAPK14。在PPI网络中,靶点degree值越大,说明该靶点与其他靶点的相互作用越强。因此,上述10个靶点基因可能是白术-半夏-茯苓配伍治疗胰腺癌的核心靶点。
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将白术-半夏-茯苓与胰腺癌的交集靶点基因导入DAVID数据库进行GO富集分析,得到P<0.05的结果共计498个。其中,包含生物过程(BP)条目373个,细胞组成(CC)条目36个,分子功能(MF)条目89个。将生物过程、细胞组成和分子功能排名前十的分析结果绘制成柱状图(图5)。
通过KEGG分析筛选出P<0.05的通路共134条,前20条通路见图6。其中,胰腺癌信号通路的潜在靶点有AKT1,MAPK1,MAPK8,RELA,TP53,CASP9,TGFB1,BAX,EGFR,TGFBR1,TGFBR2,VEGFA。通过进一步分析,白术-半夏-茯苓配伍治疗胰腺癌是通过影响KRAS下游的PI3K-Akt信号通路、MAPK信号通路、ErbB信号通路、VEGF信号通路、p53信号通路以及TGF-β信号通路发挥作用的(图7)。
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将KEGG富集分析得到的前20条显著差异的信号通路与相应靶点导入 Cytoscape 3.9.1软件构建白术-半夏-茯苓治疗胰腺癌的靶点-通路网络(图8)。该网络中共75个节点,其中 55个菱形节点为药组治疗胰腺癌的潜在作用靶点,20个V形节点代表信号通路。度值排名前10的靶点为AKT1(度值19)、MAPK1(度值18)、MAPK8(度值16)、RELA(度值16)、JUN(度值16)、FOS(度值15)、BCL2(度值14)、TP53(度值13)、CASP3(度值13)、MAPK14(度值12)。
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一般而言,配受体间的结合能越小,结合越稳定。结合能≤−20.93 kJ/mol,表明配受体间有较好的结合活性[11]。为了能更好地阐述白术-半夏-茯苓配伍治疗胰腺癌的可能作用机制,选取靶点-通络网络中胰腺癌通路相关的关键靶点AKT1,MAPK1,MAPK8,RELA和TP53,并筛选出与胰腺癌通路关键靶点相关的位于药物-成分-交集靶点网络图中度值排名前三的核心活性成分进行分子对接验证,结果见表1。其中,茯苓酸A与靶点AKT1,3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸与靶点MAPK8,茯苓酸B与靶点MAPK8的结合能≤−20.93 kJ/mol,表明它们与靶点间有较好的结合活性。利用PyMOL软件将稳定结合排名前三的对接结果进行可视化处理(图9)。茯苓酸A与靶点AKT1的氨基酸残基ALA-230/GLU-228形成3个氢键,3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸与靶点MAPK8的氨基酸残基ILE-304形成1个氢键,茯苓酸B与靶点MAPK8的氨基酸残基LYS-288/ASN-322/GLN-317形成4个氢键,这些氢键可能是配体结合到相应靶蛋白位点的主要作用力。
表 1 白术-半夏-茯苓核心成分与胰腺癌关键靶点的结合能
序号 活性分子 靶点 结合能(kJ/mol) 1 黄芩素 AKT1 −26.87 2 3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸 MAPK8 −21.64 3 茯苓酸 B MAPK8 −21.39 4 黄芩素 RELA −19.46 5 黄芩素 TP53 −19.38 6 茯苓酸 A MAPK1 −19.30 7 3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸 MAPK1 −18.71 8 茯苓酸 A MAPK8 −18.59 9 14-乙酰基-12-千里光酰基-8-反式白术三醇 MAPK8 −13.44 10 茯苓酸 B MAPK1 −11.13 11 12-千里光酰基-8-反式白术三醇 MAPK8 −8.58 12 12,13-环氧-9-羟基十九碳-7,10-二烯酸 MAPK1 −8.33 13 12-千里光酰基-8-反式白术三醇 MAPK1 −6.15 14 12,13-环氧-9-羟基十九碳-7,10-二烯酸 MAPK8 −3.10
Research on the mechanism of Atractylodes-Pinellia-Poria in the treatment of pancreatic cancer based on network pharmacology and molecular docking
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摘要:
目的 采用网络药理学和分子对接技术预测白术-半夏-茯苓配伍治疗胰腺癌的作用靶点,探讨其治疗胰腺癌的潜在分子作用机制。 方法 通过中药系统药理学数据库和分析平台(TCMSP)、PharmMapper、OMIM、GeneCards、STRING、DAVID等在线数据库与Cytoscape软件构建一系列网络图、 筛选核心靶点并对靶基因进行GO分析和KEGG通路富集分析,最后通过AutoDock软件对关键活性成分与潜在作用靶点进行分子对接验证。 结果 共筛选得到药组活性成分35个,相关靶点190个,胰腺癌靶点1 566个以及白术-半夏-茯苓治疗胰腺癌的交集靶点76个。这些交集靶点主要参与基因表达的正调控,细胞因子介导的信号通路以及细胞凋亡调控等生物学过程,并与癌症通路、乙型肝炎、大肠癌、化学致癌受体激活、胰腺癌以及MAPK 信号通路等相关。分子对接结果显示,白术-半夏-茯苓的主要活性成分与胰腺癌的潜在作用靶点间具有一定的结合活性。 结论 白术-半夏-茯苓主要通过多成分、多靶点、多通路发挥对胰腺癌的治疗作用,为临床应用白术-半夏-茯苓配伍治疗胰腺癌提供一定的理论依据。 Abstract:Objective To predict the target of Atractylodes-Panxia-Poria in the treatment of pancreatic cancer, and to explore its potential molecular mechanism by using network pharmacology and molecular docking. Methods Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), PharmMapper, OMIM, GeneCards, STRING, DAVID and Cytoscape software were used to construct a series of network diagrams. The core targets and conduct GO analysis and KEGG pathway enrichment analyses of the target genes were selected. Finally, molecular docking verification of key active ingredients and potential targets were conducted by AutoDock software. Results A total of 35 active ingredients, 190 related targets, 1566 targets of pancreatic cancer and 76 intersection targets were screened for the treatment of pancreatic cancer with Atractylodes-Panxia-Poria. These intersection targets were mainly involved in several biological processes, including positive regulation of gene expression, cytokine-mediated signaling pathway and regulation of apoptotic process, etc, which were also related to pathways in cancer, hepatitis B, colorectal cancer, chemical carcinogenesis-receptor activation, pancreatic cancer, and MAPK signaling pathway, etc. Molecular docking results showed that the main active components of Atractylodes-Panxia-Poria had certain affinity with the potential targets of pancreatic cancer. Conclusion Atractylodes-Panxia-Poria mainly exerts a therapeutic effect on pancreatic cancer through multi-component, multi-target and multi-pathway, which provides a certain theoretical basis for the clinical application of Atractylodes -Panxia-Poria in the treatment of pancreatic cancer. -
表 1 白术-半夏-茯苓核心成分与胰腺癌关键靶点的结合能
序号 活性分子 靶点 结合能(kJ/mol) 1 黄芩素 AKT1 −26.87 2 3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸 MAPK8 −21.64 3 茯苓酸 B MAPK8 −21.39 4 黄芩素 RELA −19.46 5 黄芩素 TP53 −19.38 6 茯苓酸 A MAPK1 −19.30 7 3β-羟基-24-亚甲基-8-羊毛甾烯-21-酸 MAPK1 −18.71 8 茯苓酸 A MAPK8 −18.59 9 14-乙酰基-12-千里光酰基-8-反式白术三醇 MAPK8 −13.44 10 茯苓酸 B MAPK1 −11.13 11 12-千里光酰基-8-反式白术三醇 MAPK8 −8.58 12 12,13-环氧-9-羟基十九碳-7,10-二烯酸 MAPK1 −8.33 13 12-千里光酰基-8-反式白术三醇 MAPK1 −6.15 14 12,13-环氧-9-羟基十九碳-7,10-二烯酸 MAPK8 −3.10 -
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