基于网络药理学和细胞分子生物学探讨鸡血藤治疗宫颈癌的作用机制

Research of Molecular Mechanism of Spatholobi Caulis in Treatment of Cervical Cancer Based on Network Pharmacology and Cell Molecular Biology

[目的]运用网络药理学和细胞分子生物学探讨鸡血藤治疗宫颈癌的分子作用机制。[方法]通过中药药理学系统分析平台(Traditional Chinese Medicine Database and Analysis Platform,TCMSP)筛选鸡血藤的有效成分;利用Drugbank数据库、基因组注释数据平台(genome annotation database platform,GeneCard)数据库预测和筛选鸡血藤有效成分和宫颈癌相关靶点;采用String数据库构建靶点蛋白相互作用(protein-protein interaction,PPI)网络;借助生物学信息注释数据库(the Database for Annotation,Visualization and Integrated Discovery,DAVID)对靶点进行基因本体(gene ontology,GO)分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析;利用KEGG数据库构建鸡血藤治疗宫颈癌的通路图。最后通过分子生物学实验验证分析鸡血藤抗宫颈癌的作用,通过3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]法检测细胞增殖情况,通过免疫印迹法检测凋亡相关蛋白的表达情况。[结果]筛选得出鸡血藤有21个有效成分,涉及100个作用靶点,鸡血藤抗宫颈癌的关键蛋白为蛋白激酶B1(protein kinase B1,AKT1)、丝裂原活化蛋白激酶1(mitogen-activated protein kinase 1,MAPK1)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、原癌基因JUN和MYC、胱天蛋白酶3(caspase 3,CASP3)、血管内皮生长因子A(vascular endothelial growth factor A,VEGFA)、白细胞介素-6(interleukin-6,IL-6)、信号传导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)。鸡血藤通过调节CASP3、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、CASP9、氨基末端转移酶(N-terminal kinase,JNK)和肿瘤抑制基因p53通路等信号通路来发挥抗癌作用。MTT结果表明,鸡血藤醇提物具有抑制宫颈癌细胞增殖的作用;免疫印迹结果显示,鸡血藤醇提物能够升高宫颈癌Hela细胞中CASP3和Bax蛋白的表达,降低Bcl-2蛋白的表达,并且调节Vimentin和E-cadherin的表达,诱导宫颈癌细胞凋亡,抑制细胞迁移。[结论]网络药理学和细胞分子生物学研究提示,鸡血藤治疗宫颈癌可能是通过多成分、多靶点和多通路交互发挥抗癌作用。

[Objective] To explore the molecular mechanism of Spatholobi Caulis in treatment of cervical cancer by network pharmacology and cell molecular biology. [Methods] The main active ingredients of Spatholobi Caulis were obtained through Traditional Chinese Medicine Database and Analysis Platform(TCMSP) database. Drugbank and genome annotation database platform(GeneCard) were used to predict and screen the active ingredients of Spatholobi Caulis and targets of cervical cancer. The protein-protein interaction(PPI) network was constructed by using the String database. The gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways involved in the targets were analyzed by the Database for Annotation,Visualization and Integrated Discovery(DAVID). The KEGG database was used to construct a pathway map of Spatholobi Caulis in treatment of cervical cancer. Finally,molecular biology verification was used to analyze the effect of Spatholobi Caulis on cervical cancer in vitro experiments on human cervical cancer cells. Cell proliferation was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) method,and the expressions of apoptosis-related protein were detected by using Western blot. [Results] The results showed that 21 active components and 100 targets of Spatholobi Caulis were involved. The key protein of Spatholobi Caulis anti-cervical cancer was protein kinase B1(AKT1),mitogen-activated protein kinase 1(MAPK1),epidermal growth factor receptor(EGFR),JUN,MYC,caspase 3(CASP3),vascular endothelial growth factor A(VEGFA),interleukin-6(IL-6),signal transducer and activator of transcription 3(STAT3). Spatholobi Caulis played an anti-cancer effect by regulating signaling pathways such as CASP3,B-cell lympnoma-2(Bcl-2),CASP9,N-terminal kinase(JNK) and p53 pathways. MTT results showed that ethanolic extract of Spatholobi Caulis had the inhibitory effect on Hela cells. Western blot results showed that ethanolic extract of Spatholobi Caulis could induce cell apoptosis and migration by increasing the protein expressions of CASP3 and Bax,decreasing the expression of Bcl-2,regulating the expression of Vimentin and Ecadherin in Hela cell. [Conclusion] Network pharmacology and cell molecular biology suggest that Spatholobi Caulis may play an anticancer role in the treatment of cervical cancer through multi-component,multi-target and multi-channel interaction.