长春胺调控TLR4/NF-κB信号改善2,4,6-三硝基苯磺酸诱导的小鼠结肠炎

Vincamine improves 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice through regulating TLR4/NF-κB signal

目的:探讨长春胺(VC)对2,4,6-三硝基苯磺酸(TNBS)诱导的克罗恩病(CD)样小鼠结肠炎的作用及可能机制。方法:选取6~8周龄Wild-type(WT)小鼠(C57BL/6J,雄性),将其随机分为对照组(WT组)、模型组(TNBS组)和VC干预组(VC组),每组8只;其中VC组经TNBS造模成功后给予VC干预(40 mg·kg^(-1)·d^(-1),溶于1%吐温80,灌胃),WT组和TNBS组接受等量的1%吐温80;通过分析各组小鼠体质量改变、肠炎疾病活动度(DAI)评分、结肠长度、组织学炎症评分及肠黏膜炎症介质水平[(肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)]评估VC对TNBS模型小鼠CD样肠炎的作用效果;采用SuperPred和DisGeNet数据库预测VC作用于CD的潜在靶点;DAVID数据库进行基因本体论(GO)及京都基因与基因组百科全书(KEGG)富集分析,并利用Cytoscape软件对结果进行可视化;采用分子对接技术分析VC与PTGS3、NFKB1及TLR4的结合情况,并于动物实验进一步验证VC对TLR4/NF-κB信号通路的调控作用。结果:VC组小鼠体质量降低幅度、DAI评分及结肠缩短程度均低于TNBS组小鼠(P<0.05),但仍高于WT组(P<0.05);VC组小鼠肠道组织学炎症评分低于TNBS组(P<0.05);VC组结肠黏膜炎症介质水平(TNF-α、IL-1β、IL-6)均低于TNBS组(P<0.05),但仍高于WT组(P<0.05)。网络药理学及分子对接结果显示VC对TLR4/NF-κB具有调控作用,且进一步的Western blotting结果显示,VC组小鼠结肠黏膜组织中TLR4和p-p65水平较TNBS组均降低(P<0.05),但仍高于WT组(P<0.05)。结论:VC可能通过抑制TLR4/NF-κB信号改善TNBS诱导的小鼠结肠炎。

Objective:To investigate the effect and possible mechanism of vincamine(VC)on 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced Crohn′s disease(CD)-like colitis in mice.Methods:Wild-type(WT)mice aged 6-8 weeks old(C57BL/6J,male)were randomly divided into control group(WT group),model group(TNBS group)and VC-treated group(VC group),with 8 mice in each group.VC group was treatment with VC(40 mg·kg^(-1)·d^(-1),soluble in 1%Tween 80,gavage)after successful modeling,while WT group and TNBS group received the same amount of 1%Tween 80.The body weight changes,disease activity index(DAI)scores,colon lengths,histological scores and the level of inflammatory mediators in colon mucosa(TNF-α,IL-1β,IL-6)was used to evaluate the effect of VC on CD-like colitis in TNBS model mice.The potential target of VC acting on CD was predicted by SuperPred and DisGeNet database.The DAVID database was used for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,and the results were visualized using the Cytoscape software.The combination of VC with PTGS3,NFKB1 and TLR4 were analyzed by the molecular docking technology and the effect of VC on TLR4/NF-κB signal were further verified by animal experiments.Results:The weight loss,DAI scores and colonic shortening in the VC group were lower than that in the TNBS group(P<0.05),but still higher than the WT group(P<0.05);the histological scores in VC group was significantly lower than that in TNBS group(P<0.05);inflammatory mediators(TNF-α、IL-1β、IL-6)level in colon mucosa of VC group were lower than those in TNBS group(P<0.05),but still higher than those in WT group(P<0.05).Network pharmacology and molecular docking showed that VC could regulate TLR4/NF-κB signal,and further Western blotting results showed that the level of TLR4 and p-p65 in colon mucosa of VC group were significantly lower than those in TNBS group(P<0.05),but still higher than that in WT group(P<0.05).Conclusions:VC can improve TNBS-induced colitis in mice probably through inhibiting TLR4/NF-κB signal.