连翘脂素通过抑制IL-17信号改善2,4,6-三硝基苯磺酸诱导的小鼠克罗恩病样结肠炎

Phillygenin alleviated 2,4,6-trinitrobenzene sulfonic acid-induced Crohn′s disease-like colitis by inhibiting IL-17 signaling

目的:探究连翘脂素(PHI)对2,4,6-三硝基苯磺酸(TNBS)诱导的克罗恩病(CD)样结肠炎的治疗效果及潜在的分子机制。方法:选取18只6~8周龄的野生型小鼠(C57BL/6J),并将其随机分为对照组(WT组)、模型组(TNBS组)和PHI干预组(PHI组),每组6只。其中TNBS组和PHI组小鼠均采用TNBS诱导CD样结肠炎模型,PHI组小鼠于造模成功后给予PHI干预(20 mg·kg^(-1)·d^(-1),灌胃),而WT组和TNBS组小鼠给予等量的0.9%氯化钠溶液。干预7 d后,采用疾病活动度指数(DAI)、体质量变化及结肠长度评估小鼠肠炎症状;采用HE染色和炎症评分观察小鼠结肠组织炎症程度;采用酶联免疫吸附实验检测小鼠结肠黏膜组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和干扰素-γ(IFN-γ)水平。使用网络药理学预测PHI作用于CD的可能机制,并进一步采用免疫印迹法进行验证。结果:PHI组小鼠DAI评分、结肠缩短程度均低于TNBS组(P<0.01),高于WT组(P<0.01);PHI小鼠体质量高于TMBS组(P<0.01),低于WT组(P<0.01);PHI组小鼠结肠组织学评分及肠黏膜炎症介质(TNF-α、IL-6和IFN-γ)水平均低于TNBS组(P<0.01),高于WT组(P<0.01)。网络药理学分析获得PHI作用于CD的潜在靶点共63个;生物信息学富集分析显示,PHI主要参与炎症反应的调控过程,且可能与白细胞介素-17(IL-17)信号通路有关。免疫印迹结果显示PHI组小鼠结肠黏膜组织中IL-17、白细胞介素-17受体A及核因子-κB激活剂1表达水平较TNBS组降低,但仍高于WT组(P<0.05~P<0.01)。结论:PHI可改善TNBS诱导的小鼠CD样结肠炎,这可能与其抑制IL-17信号通路有关。

Objective:To explore the effect and potential molecular mechanism of phillygenin(PHI)on Crohn′s disease(CD)-like colitis induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS).Methods:Eighteen wild-type mice(C57BL/6J)aged 6-8 weeks were randomly divided into control group(WT group),model group(TNBS group)and PHI administration group(PHI group),with 6 mice in each group.Mice in the TNBS group and PHI group were treated with TNBS-induced CD colitis model while mice in the PHI group were given PHI administration(20 mg·kg^(-1)·d^(-1),gavage)after modeling,while mice in the WT group and the TNBS group were given the same amount of 0.9%sodium chloride solution.After 7 days,the disease activity index(DAI),bodyweight changes and colon length were used to evaluate the symptoms of colitis.HE staining and histological inflammation scores were used to observe the injury degree of colon tissue.The levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interferon-γ(IFN-γ)in colonic mucosal tissues of mice were determined by enzyme-linked immunosorbent assay.Network pharmacology was used to predict the possible mechanism of PHI action on CD and further verified by Western blotting.Results:The DAI scores and colon shortening in the PHI group were significantly lower than those in the TNBS group(P<0.01),but still higher than those in the WT group(P<0.01).The body mass of mice in the PHI group was higher than that in the TNBS group and lower than that in the WT group(P<0.01).The scores of colon histology and the levels of inflammatory mediators(TNF-α,IL-6 and IFN-γ)in colonic mucosa in the PHI group were significantly lower than those in the TNBS group(P<0.01),but still higher than those in the WT group(P<0.01).Network pharmacology analysis identified a total of 63 potential targets of PHI on CD.Bioinformatics enrichment analysis showed that PHI was mainly involved in the regulation of inflammatory response,and might be related to interleukin-17(IL-17)signaling pathway.Finally,Western blotting showed that the expression levels of IL-17,interleukin-17RA and nuclear factorκB activator 1 in colonic mucosal tissue of mice in the PHI group were significantly lower than those in the TNBS group,but still higher than those in the WT group(P<0.05 to P<0.01).Conclusions:PHI can improve TNBS-induced CD-like colitis in mice,which may be related to the inhibition effect of IL-17 signaling pathway.