基于网络药理学和分子对接研究丹参-赤芍药对治疗结直肠癌的作用机制

Mechanism of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair in treatment of colorectal cancer based on network pharmacology and molecular docking

目的 基于网络药理学和分子对接技术探究丹参-赤芍药对治疗结直肠癌(colorectal cancer,CRC)的作用机制,并进一步运用体外细胞实验进行验证。方法 通过TCMSP数据库筛选出丹参-赤芍药对活性成分及其对应的靶点,通过检索GeneCards、OMIM、PharmGkb、TTD、DrugBank数据库检索获得CRC相关靶点,将丹参-赤芍活性成分靶点与CRC靶点取交集,采用Cytoscape 3.8.2软件构建“药物-成分-疾病-靶点”网络,运用STRING数据库构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络,运用R语言进行基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析;选取关键靶点及核心活性成分,使用AutoDock软件进行分子对接。通过体外细胞实验验证网络药理学结果,分别采用MTT及划痕实验检测丹参-赤芍药对对HCT116细胞增殖及迁移能力的影响,采用qRT-PCR及Western blotting检测丹参-赤芍药对对网络药理学核心靶点表达的影响。结果 网络药理学预测显示,丹参-赤芍药对活性成分206个,共有352个对应的靶基因,CRC疾病相关靶点9143个,得到交集靶点283个。通过PPI网络筛选得到丹参-赤芍药对治疗CRC的关键治疗靶点3个:雌激素受体1(estrogen receptor 1,ESR1)、黏着连接蛋白β1(catenin β1,CTNNB1)和视网膜母细胞瘤基因1(retinoblastoma 1,RB1)。GO功能和KEGG通路分析显示,关键靶点涉及磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)-蛋白激酶B(protein kinase B,Akt)信号通路、肿瘤坏死因子(tumor necrosis factor,TNF)信号通路、p53信号通路、缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)、细胞凋亡等。分子对接结果显示关键靶点与重要活性成分结合构象稳定,筛选出丹参-赤芍药对治疗CRC的核心活性成分主要是黄芩苷、芍药内酯苷、苯甲酰芍药苷等。MTT及划痕实验表明,丹参-赤芍药对对HCT116细胞增殖和迁移能力具有明显的抑制作用(P<0.05、0.01);丹参-赤芍药对显著抑制ESR1和CTNNB1 m RNA表达水平(P<0.05、0.01),显著上调RB1m RNA表达水平(P<0.05);显著下调ESR1、β-catenin、半胱氨酸天冬氨酸蛋白酶-3(cystein-asparate protease-3,Caspase-3)和聚腺苷酸二磷酸核糖转移酶(poly-ADP-ribose polymerase,PARP)蛋白表达水平(P<0.05、0.01),显著上调RB1蛋白表达水平(P<0.05、0.01)。结论 丹参-赤芍药对可能作用于ESR1、CTNNB1和RB1等靶点,通过PI3K-Akt信号通路、TNF信号通路、p53信号通路、HIF-1信号通路等相关通路,从而抑制CRC细胞的增殖和迁移能力。

Objective To explore the mechanism of Danshen(Salviae Miltiorrhizae Radix et Rhizoma)-Chishao(Paeoniae Radix Rubra) drug pair in treatment of colorectal cancer(CRC), and further verified by in vitro cell experiments. Methods Active ingredients of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair and corresponding targets were screened out through TCMSP,targets related to CRC were obtained by GeneCards, OMIM, PharmGkb, TTD and DrugBank databases, CRC-related targets were obtained by searching GeneCards, OMIM, PharmGkb, TTD and DrugBank databases. Active ingredients targets of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair were intersected with CRC targets, Cytoscape 3.8.2 software was used to construct “drug-active ingredient-disease-target” network, STRING database was used to construct protein-protein interaction(PPI) network, R language was used for gene ontology(GO) function and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis;Key targets and core active components were selected, and AutoDock software was performed for molecular docking. The results of network pharmacology were verified by in vitro cell experiments. MTT and scratch experiments were used to detect the effects of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair on proliferation and migration of HCT116 cells. qRT-PCR and Western blotting were used to detect the effect of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair on network pharmacology core targets expression. Results Network pharmacology prediction showed 206 active ingredients of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair, with a total of 352 corresponding target and 9143 CRC-related targets,and 283 intersecting targets were obtained. Three key therapeutic targets of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair in treatment of CRC were identified by PPI network screening: estrogen receptor 1(ESR1), catenin β1(CTNNB1) and retinoblastoma 1(RB1). GO function and KEGG pathway analysis showed that key targets involved phosphatidylinositol-3-kinase(PI3K)-protein kinase B(Akt) signaling pathway, tumor necrosis factor(TNF) signaling pathway, p53 signaling pathway, hypoxiainducible factor-1(HIF-1), apoptosis, etc. Molecular docking results showed that key targets and important active components were bound in a stable conformation. The core active components of Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair in treatment of CRC were mainly baicalin, paeoniflorin and benzoylpaeoniflorin. MTT and scratch experiments showed that Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair significantly inhibited the proliferation and migration of HCT116 cells(P < 0.05, 0.01), significantly inhibited m RNA expression levels of ESR1 and CTNNB1(P < 0.05, 0.01), up-regulated RB1 m RNA expression level(P < 0.05), down-regulated ESR1, β-catenin, cysteine-aspartate protease-3(Caspase-3) and poly-ADP-ribose polymerase(PARP) protein expressions(P < 0.05, 0.01), up-regulated RB1 protein expression level(P < 0.05, 0.01). Conclusion Salviae Miltiorrhizae Radix et Rhizoma-Paeoniae Radix Rubra drug pair may act on ESR1, CTNNB, RB1 and other targets, and inhibit the proliferation and migration of CRC cells through PI3K-Akt signaling pathway, TNF signaling pathway, p53 signaling pathway, HIF-1 signaling pathway and other related pathways, thereby inhibiting the proliferation and migration ability of CRC cells.