基于网络药理学及实验验证探讨连夏散结汤调控Wnt/β-catenin信号通路干预结直肠腺瘤的机制

The Mechanism of Lianxia Sanjie Decoction(连夏散结汤)Regulating Wnt/β-catenin Signaling Pathway to Intervene CRA Based on Network Pharmacology and Experimental Validation

目的:探讨连夏散结汤干预结直肠腺瘤(CRA)的效果,并探究其潜在作用机制。方法:利用中药系统药理学数据库和分析平台(TCMSP)筛选连夏散结汤的主要有效成分和作用靶点。通过人类基因数据库(GeneCards)、人类孟德尔遗传靶点数据库(OMIM)获取CRA相关的疾病靶点。通过韦恩图获得两者的共同靶标,使用蛋白相互作用数据库(String)构建蛋白相互作用(PPI)网络,并用Cytoscape 3.9.1建立连夏散结汤“药物-成分-疾病-靶点”网络。借助Metascape网站对交集基因进行基因本体(GO)及京都基因与基因百科组全书(KEGG)富集分析。采用连夏散结汤干预CRA小鼠模型,并对关键信号通路进行验证。结果:分别获取连夏散结汤活性成分235个,相关靶点4645个,收集连夏散结汤与CRA的共同靶点170个,PPI拓扑分析获得39个核心基因,通过GO及KEGG富集分析得到Wnt/β-catenin信号通路为连夏散结汤干预CRA的关键通路。动物实验结果表明,模型组小鼠结直肠长度低于空白组(P<0.01),结直肠质量、肠道腺瘤数量高于空白组(P<0.01);模型组小鼠4、6、8周体质量均低于空白组,DAI评分高于空白组(P<0.05);模型组小鼠结肠组织中β-catenin mRNA、c-myc mRNA、cyclinD1m RNA相对表达量及β-catenin、c-myc、cyclinD1蛋白相对表达量均高于空白组(P<0.01);连夏散结汤中、高剂量组小鼠结直肠长度高于模型组(P<0.01);阿司匹林组及连夏散结汤低、中、高剂量组小鼠体质量高于模型组,DAI评分低于模型组,结直肠质量、肠道腺瘤数量、结肠组织中β-catenin m RNA、c-myc mRNA、cyclinD1 mRNA相对表达量及β-catenin、c-myc、cyclinD1蛋白相对表达量均低于模型组(P<0.05或P<0.01)。结论:连夏散结汤可能通过抑制Wnt/β-catenin信号通路的异常表达干预CRA的形成。

Objective:To investigate the effect of Lianxia Sanjie decoction on colorectal adenoma(CRA)and to explore its potential mechanism of action.Methods:The Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform(TCMSP)was utilized to screen the main active ingredients and action targets of Lianxia Sanjie decoction.The disease targets related to CRA were obtained through the human gene database(GeneCards)and the human Mendelian genetic target database(OMIM).The common targets of both were obtained through the Wayne diagram,and the protein interaction(PPI)network was constructed using the protein interaction database(String).The drug-ingredient-disease-target(DIT)network of Lianxia Sanjie Decoction was established using Cytoscape 3.9.1.Metascape website was used to analyze the GO and KEGG pathway enrichment of the intersecting genes.Lianxia Sanjie decoction was used to intervene the CRA mouse model,and the key signaling pathways were verified.Results:Totally 235 active ingredients and 4645 related targets of Lianxia Sanjie decoction were obtained respectively.A total of 170 common targets of Lianxia Sanjie decoction and CRA were collected,and 39 core genes were obtained by PPI topology analysis.Wnt/β-catenin signaling pathway was obtained as the key pathway of Lianxia Sanjie decoction intervening CRA by GO and KEGG enrichment analysis.The results of animal experiments showed that the model group showed lower colorectal length than blank group(P<0.01),while higher colorectal weight and number of intestinal adenomas than blank group(P<0.01).The model group showed lower body mass than blank group at 4,6 and 8 weeks,while higher DAI scores than blank group(P<0.05).The model group showed higher levels ofβ-catenin mRNA,c-myc mRNA,c-myc mRNA,cyclinD1 mRNA relative expression andβ-catenin,c-myc,cyclinD1 protein relative expression in colonic tissues,than blank group(P<0.01).The medium-dose group of Lianxia Sanjie decoction(medium-dose group)and high-dose group of Lianxia Sanjie decoction(high-dose group)showed higher colorectal length than model group(P<0.01).The aspirin group,low-dose group of Lianxia Sanjie decoction(low-dose group),medium-dose group and high-dose group showed higher body mass than model group,while lower DAI score than model group.The aspirin group,low-dose group,medium-dose group and high-dose group showed lower colorectal weight,number of intestinal adenomas,relative expression ofβ-catenin mRNA,c-myc mRNA,cyclinD1 mRNA in colonic tissues,and relative expression ofβ-catenin,c-myc,and cyclinD1 proteins in colonic tissues,than model group(P<0.05 or P<0.01).Conclusion:Lianxia Sanjie decoction may intervene the formation of CRA by inhibiting the aberrant expression of Wnt/β-catenin signaling pathway.