基于网络药理学、分子对接和动物实验探讨菟丝子醇提物抗乳腺癌作用机制

目的 基于网络药理学、分子对接及体内实验验证探讨菟丝子醇提物抗乳腺癌的疗效及作用机制。方法 借助SwissADME平台筛选课题组前期通过UPLC/Q-TOF-MS分析得到的46个菟丝子醇提物候选化合物，利用TCMSP、ETCM、BATMAN-TCM数据库预测其潜在靶点； GeneCards、OMIM、DrugBank数据库收集疾病靶点。取交集构建“成分-靶点”和蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，利用DAVID数据库进行基因本体（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，并建立“成分-靶点-通路”网络，采用AutoDock等软件对关键活性成分和核心靶点进行分子对接验证。建立4T1荷瘤小鼠模型，设置模型组、环磷酰胺（20 mg/kg）组和菟丝子醇提物低、中、高剂量（3.12、6.24、12.48 g/kg）组，连续给药14 d，观察菟丝子醇提物对小鼠体质量、脾脏和胸腺指数的影响；小动物活体成像检测肿瘤细胞光子数；苏木素-伊红（hematoxylin-eosin，HE）染色检测瘤组织病理变化； Western blotting检测肿瘤组织磷脂酰肌醇-3-激酶/蛋白激酶B（phosphatidylinositol-3-kinase/protein kinaseB，PI3K/Akt）信号通路及凋亡相关蛋白表达。结果 网络药理学筛选得到17种菟丝子活性成分及靶点428个，乳腺癌疾病靶点1 534个，交集靶点184个，其中AKT1、雌激素受体1（estrogen receptor 1，ESR1）、表皮生长因子受体（epidermalgrowth factor receptor，EGFR）、肿瘤蛋白p53（tumor protein p53，TP53）等9个为核心靶点，与槲皮素、芹菜素等7个核心成分分子对接结果良好。通过KEGG和GO分析发现，PI3K/Akt信号转导通路可能是菟丝子抗乳腺癌的重要途径。动物实验结果证实成功构建4T1荷瘤小鼠模型，与模型组比较，菟丝子醇提物组小鼠体质量、胸腺指数显著升高（P＜0.05、0.01），脾脏指数及肿瘤细胞光子数显著降低（P＜0.05、0.01），同时瘤组织可见部分坏死区域，细胞核呈溶解现象。菟丝子醇提物组显著下调p-PI3K、p-Akt、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）蛋白表达（P＜0.05、0.01），上调Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）表达（P＜0.05、0.01），升高Bax/Bcl-2值（P＜0.05、0.01）。结论 菟丝子醇提物能够明显抑制荷瘤小鼠肿瘤的生长，其作用机制可能与调控PI3K/Akt通路、促进细胞凋亡有关。

Mechanism of alcohol extract of Cuscuta chinensis against breast cancer based on network pharmacology, molecular docking and animal experiments

Objective To explore the therapeutic effect and mechanism of alcohol extract of Cuscuta chinensis on breast cancer based on network pharmacology, molecular docking and in vivo experimental verification. Methods A total of 46 candidate compounds were identified from alcohol extract of C. chinensis using UPLC/Q-TOF-MS analysis in preliminary study. These components were analyzed on SwissADME platform to predict potential targets, which were further identified using TCMSP, ETCM and BATMANTCM databases. GeneCards, OMIM and DrugBank databases were used to collect disease targets. The intersections were used to construct the “component-target” and protein-protein interaction (PPI) network. Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed using DAVID database, and “component-target-pathway” network was constructed. Molecular docking verification of key active ingredients and core targets was conducted using AutoDock and other software. A 4T1 tumor-bearing model in mice was established, model group, cyclophosphamide (20 mg/kg) group and alcohol extract of C. chinensis low-, medium- and high-dose (3.12, 6.24, 12.48 g/kg) groups were set up. Drugs were administered continuously for 14 d, the effect of alcohol extract of C. chinensis on body weight, spleen and thymus indexes were monitored; Tumor cell photon counts were measured using live animal imager; Hematoxylin-eosin (HE) staining was employed to observe pathological changes in tumor tissue; Western blotting was used to detect phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling pathway and apoptosis-related protein expressions in tumor tissue. Results A total of 17 active ingredients and 428 potential targets of C. chinensis, along with 1 534 targets of breast cancer, and 184 intersection targets, were identified using network pharmacology. Molecular docking demonstrated favorable interactions for nine core targets, including AKT1, estrogen receptor 1 (ESR1), epidermal growth factor receptor (EGFR) and tumor protein p53 (TP53), etc, with seven core components such as quercetin and apigenin. KEGG and GO analysis indicated that PI3K/Akt signaling pathway may be a crucial pathway for C. chinensis to prevent breast cancer. The animal experiments confirmed the successful establishment of the mouse 4T1 tumor-bearing mouse model. Compared with model group, the body weight and thymus index of mice in alcohol extract of C. chinensis group were significantly increased (P < 0.05, 0.01), while the spleen index and photon number of tumor cells were significantly decreased (P < 0.05, 0.01). At the same time, some necrotic areas were visible in tumor tissue, and the nucleus showed dissolution. The alcohol extract of C. chinensis group significantly down-regulated the expressions of p-PI3K, p-Akt and B-cell lymphoma-2 (Bcl-2) protein (P < 0.05, 0.01), up-regulated the expression of Bcl-2 associated X protein (Bax) (P < 0.05, 0.01), and increased Bax/Bcl-2 value (P < 0.05, 0.01). Conclusion Alcohol extract of C. chinensis can significantly inhibit the growth of tumor in tumor-bearing mice, and its mechanism may be related to regulating PI3K/Akt pathway and promoting cell apoptosis.