基于网络药理学探究黄芩苷联合京尼平苷治疗结直肠癌的作用机制

Exploring the mechanism of baicalin combined with geniposide in the treatment of colorectal cancer based on network pharmacology

目的 通过网络药理学的方法探究黄芩苷联合京尼平苷治疗结直肠癌的作用机制。方法 通过Swiss Target Prediction、中药系统药理数据库及分析平台(TCMSP)和STITCH数据库获取黄芩苷和京尼平苷的靶点,通过GeneCards、TTD和DrugBank数据库获取结直肠癌相关疾病靶点。靶点相互映射后经STRING构建蛋白质-蛋白质相互作用(PPI)网络图,经DAVID平台进行基因本体论(GO)分析和京都基因与基因组百科全书(KEGG)分析,随后将核心靶点与基因表达总括数据库(GEO)芯片数据比对。将直肠癌细胞系SW480细胞分为对照组、黄芩苷组、京尼平苷组、黄芩苷联合京尼平苷组,采用qRT-PCR检测核心靶点mRNA的表达情况。结果 从Swiss Target Prediction、TCMSP和STITCH数据库中获取黄芩苷相关靶点27个,京尼平苷相关靶点54个,合并、去重后共获得药物组合的靶点77个。使用“colorectal cancer”为关键词,在TTD数据库检索得到相关靶点19个,在GeneCards数据库检索得到靶点173个,在DrugBank获取相关靶点42个;将数据整合、去重共计获得结直肠癌相关靶点223个。将药物靶点和疾病靶点相互映射,黄芩苷联合京尼平苷治疗结直肠癌靶点PPI关系网络有17个节点,71条边,平均度值为8.353,大于平均度值的肿瘤坏死因子(TNF)、血管内皮生长因子A(VEGFA)、白细胞介素-6(interleukin-6,IL-6)、c-Myc原癌基因(MYC)、半胱氨酸蛋白酶3(CASP3)、基质金属蛋白酶2(MMP2)、表皮生长因子受体(EGFR)、人类RAS原癌基因家族成员HRAS、Toll样受体4(TLR4)、前列腺素内过氧化物合酶2(PTGS2)、TLR2是网络的核心节点。通过DAVID平台,将黄芩苷联合京尼平苷治疗结直肠癌靶点进行GO分析与KEGG分析,GO分析主要与蛋白结合、完全相同的蛋白质结合、酶结合、蛋白质异二聚活性、蛋白质同源二聚活性等分子功能有关;与胞质、质膜、质膜的整体成分、膜、细胞表面等细胞组分有关;与RNA聚合酶Ⅱ启动子对转录的正调控、基因表达的正向调控、对凋亡过程的负调控、ERK1和ERK2级联的正向调节、对药物的反应等生物过程相关;KEGG通路富集分析与磷脂酰肌醇3-激酶-蛋白激酶B信号通路(PI3K-Akt)、丝裂原活化蛋白激酶(MAPK)、低氧诱导因子-1(HIF-1)等多条癌症相关通路高度相关。芯片数据显示HRAS、TLR4、TNF差异显著,可能是黄芩苷联合京尼平苷直接抗癌的靶点。qRT-PCR检测结果显示,结直肠癌细胞系SW480在经过黄芩苷、京尼平苷处理后mRNA的表达量均有所改变,其中,京尼平苷组、黄芩苷联合京尼平苷组HRAS的mRNA表达量(0.73±0.11)、(0.60±0.06),低于对照组的(1.00±0.02),差异均有统计学意义(P均<0.05);黄芩苷组、黄芩苷联合京尼平苷组TLR4的mRNA表达量(0.71±0.11)、(0.66±0.04),低于对照组的(1.00±0.03),差异均有统计学意义(P均<0.05);京尼平苷组、黄芩苷联合京尼平苷组TNF-α的mRNA表达量(1.34±0.12)、(1.66±0.18),高于对照组的(1.00±0.02),差异均有统计学意义(P均<0.05)。结论 黄芩苷联合京尼平苷治疗结直肠癌,能显著影响核心靶点HRAS、TLR4、TNF,为该组合进一步开发提供了理论基础。

Objective To investigate the mechanism of action of baicalin combined with geniposide in the treatment of colorectal cancer through network pharmacology.Methods The targets of baicalin and geniposide were obtained from Swiss Target Prediction,Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and STITCH databases.The targets of colorectal cancer-related diseases were obtained from GeneCards,TTD,and DrugBank databases.The protein-protein interaction(PPI)network was constructed by mapping the targets,and gene ontology(GO)analysis and kyoto encyclopedia of genes and genomes(KEGG) analysis were performed using the DAVID platform.Subsequently,the core targets were compared with Gnen Expression Omnibus(GEO) chip data.The SW480 cells of colorectal cancer cell line were divided into control group,baicalin group,geniposide group,and baicalin combined with geniposide group,and the expression of core targets was detected by quantitative real-time reverse transcription PCR(qRT-PCR).Results A total of 27 targets of baicalin and 54 targets of geniposide were obtained from Swiss Target Prediction,TCMSP and STITCH databases.After merging and removing duplicates,a total of 77 drug combination targets were obtained.Using“colorectal cancer”as the keyword,19 related targets were retrieved from TTD database,173 targets from GeneCards database,and 42 targets from DrugBank;after integrating and removing duplicates,a total of 223 colorectal cancer-related targets were obtained.The PPI relationship network of drug targets and disease targets was mapped,and there were 17 nodes and 71 edges in the PPI network of baicalin combined with geniposide in the treatment of colorectal cancer,with an average degree value of 8.353.Tumor necrosis factor(TNF),vascular endothelial growth factor A(VEGFA),interleukin-6(IL-6),v-myc avian myelocytomatosis viral oncogene homolog(MYC),caspase 3(CASP3),matrix metalloproteinase 2(MMP2),epidermal growth factor receptor(EGFR),human RAS proto-oncogene family member HRAS,Toll-like receptor 4(TLR4),prostaglandin-endoperoxide synthetase 2(PTGS2),TLR2 were the core nodes with an average degree value greater than that of other nodes.GO analysis and KEGG analysis were performed on the targets of baicalin combined with geniposide in the treatment of colorectal cancer using the DAVID platform.GO analysis was mainly related to protein binding,identical protein binding,enzyme binding,protein homodimer activity,protein heterodimer activity,etc.in terms of molecular function;it was also related to cytoplasm,plasma membrane,integral component of plasma membrane,membrane,cell surface,etc.in terms of cell component;it was also related to positive regulation of transcription from RNA polymerase II promoter,positive regulation of gene expression,negative regulation of apoptotic process,positive regulation of ERK1 and ERK2 cascade,response to drugs,etc.in terms of biological process;KEGG pathway enrichment analysis was highly related to phosphatidylinositol 3-kinase-protein kinase B signaling pathway(PI3K-Akt),mitogen-activated protein kinase(MAPK),hypoxia inducible factor-1(HIF-1)and other cancer-related pathways.Chip data showed that HRAS,TLR4,TNF were significantly different and might be the targets of baicalin combined with geniposide to directly fight against cancer.The qRT-PCR results showed that the mRNA expression levels of SW480 cells of colorectal cancer cell line were changed after treatment with baicalin and geniposide.The mRNA expression levels of HRAS in geniposide group and baicalin combined with geniposide group were(0.73±0.11)and(0.60±0.06),respectively,which were lower than that in control group(1.00±0.02),and the differences were statistically significant(all P<0.05);the mRNA expression levels of TLR4 in baicalin group and baicalin combined with geniposide group were(0.71±0.11)and(0.66±0.04),respectively,which were lower than that in control group(1.00±0.03),and the differences were statistically significant(all P<0.05);the mRNA expression levels of TNF-α in geniposide group and baicalin combined with geniposide group were(1.34±0.12)and(1.66±0.18),respectively,which were higher than that in control group(1.00±0.02),and the differences were statistically significant(all P<0.05).Conclusions The action targets of baicalin combined with geniposide in the treatment of colorectal cancer at the molecular level.The combined action of baicalin and geniposide might target on HRAS,TLR4 and TNF-α,providing a theoretical basis for further development of this combination treatment.