溃结灌肠液Ⅱ改善溃疡性结肠炎菌群结构和炎症机制的网络药理学筛查及实验验证

Network Pharmacology Screening and Experimental Validation of Kuijie EnemaⅡ(溃结灌肠液Ⅱ)on Improvement of the Microbiota Structure and Inflammatory Mechanism of Ulcerative Colitis

目的:借助网络药理学筛查溃结灌肠液Ⅱ改善溃疡性结肠炎(UC)菌群结构及炎症的主要成分和靶点,并通过动物实验探究溃结灌肠液Ⅱ对葡聚糖硫酸钠盐(DSS)造模小鼠肠道菌群结构和炎症因子的调节作用,从菌群平衡角度阐明其治疗UC的机制及现代生物学内涵。方法:通过TCMSP数据库和相关文献搜集溃结灌肠液Ⅱ的主要化学成分,通过PubChem数据库获得其2D分子结构,利用Swiss Target Prediction平台预测溃结灌肠液Ⅱ主要化学成分作用的靶点。通过GeneCards数据库获取UC和肠道菌群的基因表达数据;利用String平台对交集差异表达基因进行蛋白质相互作用分析,构建PPI网络;通过R软件进行GO分析和KEGG分析可视化;最后通过Cytoscape3.7.1软件制作“活性成分-靶点-富集通路”网络图。随机选10只小鼠作为空白对照组,26只小鼠自由饮用2.5%DSS溶液诱导构建小鼠UC疾病模型,将25只造模成功小鼠随机分为模型对照组、给药组。监测小鼠体质量,处死各组小鼠后测量结肠长度,观察小鼠结肠组织病理学变化。16S rDNA测序分析小鼠粪便菌群,采用PCR检测小鼠结肠组织TNF-αmRNA、IL-1βmRNA表达情况。结果:共获得溃结灌肠液Ⅱ主要活性成分127个,关键靶点23个,其中主要活性成分为槲皮素、山奈酚、木犀草素、β-谷甾醇、豆甾醇。通过功能和通路分析发现肠道菌群失衡是UC免疫应答异常的关键因素。溃结灌肠液Ⅱ干预能改善DSS诱导UC模型小鼠相对体质量减轻情况,增加结肠长度,修复结肠黏膜损伤,减轻炎症细胞浸润。小鼠肠道菌群在种水平阿克曼菌(Akkermansia muciniphila)、约氏乳杆菌(Lactobacillus_johnsonii)、罗伊氏乳杆菌(Lactobacillus_reuteri)、uncultured_bacterium_g__norank_f__Muribaculaceae等益生菌丰度变化与TNF-αmRNA、IL-1βmRNA表达呈负相关;大肠杆菌(Escherichia_coli)、溃疡梭杆菌(Fusobacterium_ulcerans)等致病菌丰度变化与TNF-αmRNA、IL-1βmRNA表达呈正相关。结论:溃结灌肠液Ⅱ治疗UC疗效显著,可能通过增加益生菌阿克曼菌(Akkermansia muciniphila)、约氏乳杆菌(Lactobacillus_johnsonii)、罗伊氏乳杆菌(Lactobacillus_reuteri)、uncultured_bacterium_g__norank_f__Muribaculaceae丰度,减少致病菌大肠杆菌(Escherichia_coli)、溃疡梭杆菌(Fusobacterium_ulcerans)丰度,并抑制TNF-αmRNA、IL-1βmRNA表达来降低肠道炎症反应。

Objective:To screen the main components and targets of Kuijie enemaⅡin improving the microbiota structure and inflammation of ulcerative colitis(UC)with the help of network pharmacology,and to explore the regulatory effect of Kuijie enemaⅡon the intestinal microbiota structure and inflammatory factors of dextran sodium sulfate(DSS)model mice through animal experiments,so as to clarify its mechanism of treating UC and its modern biological connotation from the perspective of bacterial balance.Methods:The main chemical components of Kuijie enemaⅡwere collected through TCMSP database and relevant literature,and the 2D molecular structure was obtained through PubChem database.The target of the main chemical components of Kuijie enemaⅡwas predicted by Swiss Target Prediction platform.The gene expression data of UC and intestinal flora were obtained through the GeneCards database.The protein-protein interaction analysis of cross differentially expressed genes was performed by using the String platform to construct PPI network.The Go analysis and KEGG analysis were visualized by R software.Finally,the network diagram of"active ingredient-target-enrichment pathway"was made by Cytoscape 3.7.1 software.10 mice were randomly selected as the blank control group,and the remaining 26 mice were free to drink 2.5%DSS solution to induce the construction of a mouse UC disease model,and 25 successful mice were randomly divided into model control group and drug administration group.The weight of mice were monitored,the length of colon was measured after the mice in each group were executed,and the histopathological changes of colon were observed.16S rDNA sequencing was used to analyze the fecal flora of mice,and PCR was used to detect the expression of TNF-αand IL-1βmRNA in the colon of mice.Results:127 main active components and 23 key targets of Kuijie enemaⅡwere obtained,and the main active components were quercetin,kaempferol,luteolinβ-Sitosterol and stigmasterol.Through functional and pathway analysis,it was found that the imbalance of intestinal flora was the key factor of abnormal immune response in UC.Kuijie enemaⅡintervention can improve the relative body weight reduction induced by DSS in UC model mice,increase the length of colon,repair the damage of colon mucosa,and reduce the infiltration of inflammatory cells.The abundance of probiotics such as Akkermansia muciniphila,Lactobacillus_johnsonii,Lactobacillus_reuteri and uncultured_bacterium_g__norank_f__Muribaculaceae in the intestinal flora of mice at the species level was negatively correlated with the expression of TNF-αmRNA and IL-1βmRNA.The abundance of Escherichia_coli,Fusobacterium_ulcerans and other pathogenic bacteria were positively correlated with the expression of TNF-αmRNA and IL-1βmRNA.Conclusion:Kuijie enemaⅡwas effective in the treatment of UC,which may reduce intestinal inflammatory reaction by increasing the abundance of probiotics Akkermansia muciniphila,Lactobacillus_johnsonii,Lactobacillus_reuteri and uncultured_bacterium_g__norank_f__Muribaculaceae,reducing the abundance of pathogenic bacteria Escherichia_coli and Fusobacterium_ulcerans,and inhibiting the expression of TNF-αmRNA and IL-1βmRNA.