基于网络药理学白花蛇舌草治疗胰腺神经内分泌肿瘤作用机制探讨及体外实验验证

Mechanism of Hedyotis diffusa in treatment of pancreatic neuroendocrine tumor based on network pharmacology and in vitro experimental verification

目的采用网络药理学方法探讨白花蛇舌草治疗分化良好的胰腺神经内分泌肿瘤(pNET)的作用机制,并通过体外细胞实验进行验证。方法应用中药系统药理学数据库与分析平台(TCMSP)获取白花蛇舌草的活性成分和作用靶点,通过GEO、GeneCards、OMIM、PharmGkb、Drugbank数据库获取pNET的疾病相关靶点,通过R软件对共同靶点进行基因本体(GO)和京都基因和基因组百科全书(KEGG)通路富集分析。以CCK-8法和Western blotting体外实验观察白花蛇舌草提取物对人胰腺神经内分泌肿瘤BON-1、QGP-1细胞的增殖抑制作用及对细胞凋亡相关蛋白Bcl-2表达的影响。结果筛选得到白花蛇舌草活性成分7个,成分相关靶点158个;获得pNET疾病相关靶点2870个,成分与疾病共同相关靶点119个。共同靶点的GO功能富集分析共得到2099个条目,KEGG富集分析共得到162条信号通路,包括细胞凋亡、PI3K/Akt、P53等信号通路。不同质量浓度[0(对照)、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5 mg·mL^(-1)]白花蛇舌草提取物干预BON-1、QGP-1细胞48 h后,细胞增殖明显受到抑制,且抑制作用具有明显的浓度相关性。与对照组比较,随着白花蛇舌草提取物质量浓度增加,BON-1、QGP-1细胞的增殖受到明显抑制,漂浮死亡细胞数增加;与对照组比较,高质量浓度白花蛇舌草提取物可显著降低BON-1、QGP-1细胞Bcl-2蛋白表达(P<0.05)。结论白花蛇舌草提取物能够有效抑制人胰腺神经内分泌肿瘤BON-1、QGP-1细胞增殖,通过抑制Bcl-2蛋白表达促进细胞凋亡;白花蛇舌草能够通过多成分、多靶点、多通路的途径治疗胰腺神经内分泌肿瘤。

Objective To investigate the mechanism of Hedyotis diffusa in treatment of well differentiated pancreatic neuroendocrine tumor(pNET)via network pharmacology and in vitro cytologic experiments.Methods The active constituents of H.diffusa and drug targets were obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),and the disease-related targets of pNET were retrieved from GEO,GeneCards,OMIM,PharmGkb,and Drugbank databases.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis for common targets of drugs and disease was performed using the R software.CCK-8 assay were used to verify the inhibiton of H.diffusa extract on proliferation of human pancreatic neuroendocrine tumor of BON-1 and QGP-1 cells and apoptosis related proteins of the influence of the Bcl-2expression..Western blotting was used to observe effect of H.diffusa extract on apoptosis of BON-1 and QGP-1 cells.Results A total of 158 drug targets of seven active ingredients of H.diffusa,and 2870 disease-related targets orelated to pNET were obtained resulting in 119 common targets.A total of 2099 items were obtained by GO functional enrichment analysis.A total of 162 signaling pathways were obtained by KEGG enrichment analysis,including apoptosis,PI3K-Akt,and P53 signaling pathways,etc(P<0.05).CCK-8 assay showed that the extract of H.diffusa had a concentration-dependent inhibitory effect on proliferation of BON-1 and QGP-1cells.Western blotting showed that the protein expression of Bcl-2 in the high-concentration extract of H.diffusa group was significantly decreased compared with the control group(P<0.05).Conclusion The extract of H.diffusa can effectively inhibit the proliferation of human pancreatic neuroendocrine tumors of BON-1 and QGP-1 cells and promote apoptosis by inhibiting the expression of Bcl-2protein.H.diffusa can treat pancreatic neuroendocrine tumors through multi-component,multi-target and multi-pathway approaches.