基于网络药理学的黄瑞香抗胃癌作用机制探讨及实验验证

目的 采用网络药理学方法预测黄瑞香发挥抗胃癌作用的潜在机制，并通过体外细胞实验进行初步验证。方法 利用中药系统药理学数据库和分析平台（TCMSP）筛选黄瑞香的有效成分，通过Swiss Target Prediction数据库和PharmMapper数据库筛选获得黄瑞香活性成分的相关靶点。通过GeneCards及DisGeNET数据库检索获得胃癌疾病相关靶点。利用Cytoscape软件构建黄瑞香活性成分-靶点网络；通过STRING、Metascape数据库对成分和胃癌疾病交集靶点进行蛋白质相互作用（PPI）网络和基因本体（GO）注释及京都基因与基因组百科全书（KEGG）通路富集分析。采用MTT实验考察黄瑞香异戊烯基黄酮成分构树黄酮醇F（broussoflavonol F）、daphnegiravone D、daphgiflavone C体外对胃癌MGC803细胞生长的影响，采用实时荧光定量PCR方法检测这3种异戊烯基黄酮类化合物作用48h后胃癌MGC803细胞表皮生长因子受体（EGFR）、磷脂酰肌醇3-激酶（PI3K）和蛋白激酶B（Akt）基因的表达。结果 筛选得到黄瑞香70个活性成分，黄瑞香作用于胃癌的42个相关靶点，其中表皮生长因子受体（EGFR）、鼠肉瘤病毒癌基因同源物B1（BRAF）、Ⅰ类磷脂酰肌醇-3激酶催化亚基α（PIK3CA）、酪氨酸激酶（MET）以及丝裂原活化蛋白激酶1（MAPK1）等为核心靶点。KEGG富集分析发现黄瑞香作用于胃癌主要富集在癌症、PI3K/Akt以及丝裂原活化蛋白激酶（MAPK）等通路中。活性测试结果发现，异戊烯基黄酮成分构树黄酮醇F、daphnegiravone D、daphgiflavone C对胃癌MGC803细胞生长具有较好的抑制作用。同时，与对照组比较，构树黄酮醇F、daphnegiravone D、daphgiflavone C可显著抑制胃癌MGC803细胞EGFR、PI3K和Akt基因的表达（P＜0.05、0.01、0.001）。结论 黄瑞香可能以异戊烯基黄酮为主要的抗胃癌活性成分，以EGFR、BRAF、PIKC3A、MET等蛋白为核心作用靶点，主要经I3K/Akt以及MAPK等相关信号通路发挥治疗胃癌的作用。

Study on mechanism of Daphne giraldii against gastric cancer based on network pharmacology and experimental verification

Objective To investigate the mechanism of Daphne giraldii in the treatment of gastric cancer by using the method of network pharmacology and cellular experiments. Methods Systematic Pharmacology Database and TCMSP database were used to obtain the chemical components and action targets of D.giraldii. Then, from the Swiss target prediction, PharmMapper, GeneCards and DisGeNET databases, the targets of chemical constituents from D.giraldii and gastric cancer related target information were searched. The active ingredient-target network of D.giraldii was further constructed by Cytoscape. Afterwards, STRING and Metascape databases were used for protein-protein interaction, GO and KEGG pathway enrichment analysis. MTT assay was used to detect the anti-tumor activity of broussoflavonol F, daphnegiravone D, daphgiflavone C. The expression of mRNA level of EGFR, PI3K and Akt of gastric cancer MGC803 cells were examined by using real-time fluorescence quantitative PCR (qRT-PCR) analysis after 48h treated by these three prenylated flavonoids. Results Totally 70 potential active ingredients of D.giraldii were obtained. And through screening, 42 targets of D.giraldii on gastric cancer were obtained. Among them, EGFR, BRAF, PIK3CA, MET and MAPK1 were the core targets, which were mainly enriched in cancer, PI3K/Akt and MAPK signaling pathways. MTT assay showed that three prenylated flavones (broussoflavonol F, daphnegiravone D, daphgiflavone C) had better activity against gastric cancer MGC803 cells growth, and they also significantly decreased the mRNA level of EGFR, PI3K and Akt. Conclusion The main antigastric cancer active component of D.giraldii may be prenylated flavonoids, with EGFR, BRAF, PIKC3A, MET and other proteins as core targets, and it mainly plays a role in the treatment of gastric cancer through PI3K/Akt and MAPK signaling pathways.