Network Pharmacology Analysis and Experiments Validation of the Inhibitory Effect of JianPi Fu Recipe on Colorectal Cancer LoVo Cells Metastasis and Growth
Network Pharmacology Analysis and Experiments Validation of the Inhibitory Effect of JianPi Fu Recipe on Colorectal Cancer LoVo Cells Metastasis and Growth
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DOI：
10.1155/2020/4517483
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发表时间：
2020-07-26
影响因子：
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通讯作者：
Ren, Jianlin
中科院分区：
文献类型：
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作者：
Lu, Xinyi;Wu, Xingli;Ren, Jianlin
目标。以网络药理学和生物信息学为基础，对健脾附方的活性成分、潜在靶点和疾病进行分析，验证健脾附方体外的潜在生物学功能和作用机制。利用TCMSP、Tcm-PTD、TCMID、DrugBank等网络药理数据库筛选JPFR的活性成分和潜在的药物靶点。应用Cytoscape3.7软件构建活性化合物与潜在靶点之间的相互作用网络。根据GO功能和KEGG通路丰富分析的结果，进行David在线数据库分析，以研究潜在的有效疾病和涉及的信号通路。为确保JPFR的标准化和批间可靠性，采用高效液相色谱(HPLC)建立“化学指纹图谱”。为了验证JPFR的生物学功能，采用CCK-8法和Transwell和创伤愈合实验观察了JPFR对体外培养的结直肠癌细胞增殖和迁移的影响，并通过建立裸鼠肺转移模型和活体成像检测了JPFR对结直肠癌细胞在体内生长和转移的影响。通过实时荧光定量聚合酶链式反应、免疫印迹和免疫组织化学方法检测MALAT1、PTBP-2和β-连环蛋白在结直肠癌细胞和移植瘤中的表达。根据口服生物利用度(OB>；30%)和类药物度(DL>；0.18)规则，筛选出244个有效成分和132个潜在的药物靶点。经David数据库分析，所有这些关键靶点都与前列腺癌、结直肠癌、膀胱癌、小细胞肺癌、胰腺癌、肝细胞癌等癌症密切相关。此外，多条信号通路与JPFR密切相关，包括P53、Wnt、PI3K-Akt、IL-17、HIF-1、p38-MAPK、NF-kappa B、PD-L1表达和PD-1检查点通路、VEGF、JAK-STAT和HIPPO。系统分析表明，JPFR的多种活性成分与Wnt/β-catenin、EGFR、HIF-1、转化生长因子β/Smads和IL-6-STAT3信号通路密切相关，包括山奈酚、异鼠李素、毛蕊异黄素、栎素、木犀草素和异三叶醇。在体外实验中，JPFR提取物以剂量依赖的方式抑制人结直肠癌细胞的迁移和生长，并以剂量依赖的方式下调MALAT1、PTBP-2、β-catenin、MMP7、c-Myc和Cyclin D1的表达。在体内转移实验中，JPFR提取物呈剂量依赖性地减少肺转移数目，并呈剂量依赖性地下调肺转移相关基因MALAT1、PTBP-2、β-catenin和MMP7的表达。在原位移植瘤实验中，JPFR提取物能抑制原位移植瘤的生长，下调c-Myc和Cyclin D1的表达，并呈剂量依赖关系。此外，JPFR提取物还能剂量依赖性地延长荷瘤小鼠的存活时间。通过有效的网络药理分析，我们发现JPFR含有许多可能直接靶向肿瘤相关信号通路的有效化合物。体内外实验进一步证实，JPFR通过调节β-连环蛋白信号相关基因或蛋白来抑制大肠癌细胞的生长和转移。
Objective. To analyze the active compounds, potential targets, and diseases of JianPi Fu Recipe (JPFR) based on network pharmacology and bioinformatics and verify the potential biological function and mechanism of JPFRin vitroandin vivo.Methods. Network pharmacology databases including TCMSP, TCM-PTD, TCMID, and DrugBank were used to screen the active compounds and potential drug targets of JPFR. Cytoscape 3.7 software was applied to construct the interaction network between active compounds and potential targets. The DAVID online database analysis was performed to investigate the potential effective diseases and involved signaling pathways according to the results of the GO function and KEGG pathways enrichment analysis. To ensure standardization and maintain interbatch reliability of JPFR, High Performance Liquid Chromatography (HPLC) was used to establish a "chemical fingerprint." For biological function validation, the effect of JPFR on the proliferation and migration of CRC cellsin vitrowas investigated by CCK-8 and transwell and wound healing assay, and the effect of JPFR on the growth and metastasis of CRC cellsin vivowas detected by building a lung metastasis model in nude mice andin vivoimaging. For the potential mechanism validation, the expressions of MALAT1, PTBP-2, and beta-catenin in CRC cells and transplanted CRC tumors were detected by real-time PCR, western blot, and immunohistochemical staining analysis.Results. According to the rules of oral bioavailability (OB) > 30% and drug-likeness (DL) > 0.18, 244 effective compounds in JPFR were screened out, as well as the corresponding 132 potential drug targets. By the analysis of DAVID database, all these key targets were associated closely with the cancer diseases such as prostate cancer, colorectal cancer, bladder cancer, small cell lung cancer, pancreatic cancer, and hepatocellular carcinoma. In addition, multiple signaling pathways were closely related to JPFR, including p53, Wnt, PI3K-Akt, IL-17, HIF-1, p38-MAPK, NF-kappa B, PD-L1 expression and PD-1 checkpoint pathway, VEGF, JAK-STAT, and Hippo. The systematical analysis showed that various active compounds of JPFR were closely connected with Wnt/beta-catenin, EGFR, HIF-1, TGF beta/Smads, and IL6-STAT3 signaling pathway, including kaempferol, isorhamnetin, calycosin, quercetin, medicarpin, phaseol, spinasterol, hederagenin, beta-sitosterol, wighteone, luteolin, and isotrifoliol. Forin vitroexperiments, the migration and growth of human CRC cells were inhibited by the JPFR extract in a dose-dependent way, and the expression of MALAT1, PTBP-2,beta-catenin, MMP7, c-Myc, and Cyclin D1 in CRC cells were downregulated by the JPFR extract in a dose-dependent way. Forin vivometastasis experiments, the numbers of lung metastasis were found to be decreased by the JPFR extract in a dose-dependent manner, and the expressions of metastasis-associated genes including MALAT1, PTBP-2, beta-catenin, and MMP7 in the lung metastases were downregulated dose dependently by the JPFR extract. For the orthotopic transplanted tumor experiments, the JPFR extract could inhibit the growth of orthotopic transplanted tumors and downregulate the expression of c-Myc and Cyclin D1 in a dose-dependent manner. Moreover, the JPFR extract could prolong the survival time of tumor-bearing mice in a dose-dependent manner.Conclusions. Through effective network pharmacology analysis, we found that JPFR contains many effective compounds which may directly target cancer-associated signaling pathways.The in vitro and in vivoexperiments further confirmed that JPFR could inhibit the growth and metastasis of CRC cells by regulating beta-catenin signaling-associated genes or proteins.