香叶木素通过调节小鼠的肠道免疫平衡减轻克罗恩病样结肠炎:基于抑制PI3K/AKT通路

Diosmetin regulates intestinal immune balance by inhibiting PI3K/AKT signaling to relieve 2,4,6-trinitrobenzene sulfonic acid-induced Crohn's disease-like colitis in mice

目的明确香叶木素对2,4,6-三硝基苯磺酸(TNBS)诱导的小鼠克罗恩病(CD)样结肠炎的作用效果和机制。方法将野生型小鼠(WT)随机分为3组:WT组、TNBS诱导的CD样结肠炎组(TNBS组)和香叶木素干预的结肠炎组(50 mg·kg^(-1)·d-1;Dios组),8只/组;采用肠炎疾病活动(DAI)评分、体质量改变、肠炎组织学评分、结肠长度和ELISA法检测的肠黏膜炎症介质水平(TNF-α、IFN-γ、IL-17A)等评估肠炎程度;流式细胞术分析肠系膜淋巴结中T淋巴细胞亚群(Th1/Th2、Th17/Treg)变化;通过网络药理学和分子对接技术分析香叶木素调控PI3K/AKT通路的作用,并在体验证香叶木素改善TNBS小鼠CD样肠炎与抑制PI3K/AKT信号的关系。结果动物实验显示,Dios组小鼠DAI评分、肠炎组织学评分、体质量降低幅度及结肠黏膜炎症介质水平(TNF-α、IFN-γ、IL-17A)均低于TNBS组小鼠(P<0.05),但仍然高于WT组(P<0.05);Dios组小鼠结肠长度大于TNBS组(P<0.05),小于WT组(P<0.05);Dios组小鼠肠系膜淋巴结中Th1/Th17细胞比例低于TNBS组(P<0.05),高于WT组(P<0.05);Th2/Treg细胞比例高于TNBS组(P<0.05)和WT组(P<0.05)。网络药理学分析获得香叶木素和CD交集靶点46个,其中AKT1、EGFR、SRC、ESR1、MMP9、PTGS2为排名前6位的关键靶点;GO和KEGG分析显示,香叶木素改善CD样结肠炎作用与磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)等信号通路密切相关;分子对接显示香叶木素与关键靶点结合较好。Western blot检测结果显示,Dios组小鼠结肠黏膜p-PI3K和p-AKT水平低于TNBS组(P<0.05),高于WT组(P<0.05)。结论香叶木素可能通过抑制PI3K/AKT信号调控Th1/Th2和Th17/Treg平衡,改善肠黏膜免疫紊乱,从而缓解TNBS小鼠CD样肠炎。

Objective To investigate the therapeutic mechanism of diosmetin on 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced Crohn's disease(CD)-like colitis in mice.Methods Wild-type C57BL/6 mice were randomized into control group,TNBS-induced CD-like colitis group(TNBS group)and 50 mg·kg-1·d-1 diosmetin-treated group(n=8).Disease activity(DAI)scores,body weight changes,histological scores,colon lengths and colon mucosal levels of TNF-α,IFN-γ,and IL-17A were measured to evaluate the severity of colitis.The changes of T lymphocyte subsets(Th1/Th2 and Th17/Treg)in the mesenteric lymph nodes were analyzed by flow cytometry.Network pharmacology and molecular docking were used to analyze the effect of diosmetin on PI3K/AKT pathway.Results Compared with TNBS group,diosmetin treatment significantly lowered DAI scores,histological scores,body weight loss and colon mucosal levels of TNF-α,IFN-γ,and IL-17A(P<0.05)and increased the colon length of the rat models,but these improvements did not reach the control levels(P<0.05).Diosmetin significantly lowered the percentages of Th1/Th17 cells in the mesenteric lymph nodes in TNBS-treated mice,which remained higher than the control levels(P<0.05);The percentages of Th2/Treg cells were significantly higher in diosmetin group than in TNBS group(P<0.05)and the control group(P<0.05).Network pharmacologic analysis identified 46 intersection targets of diosmetin and CD,and among them AKT1,EGFR,SRC,ESR1,MMP9 and PTGS2 were the top 6 core targets.GO and KEGG analyses showed that the PI3K/AKT signaling pathway was closely related with the therapeutic effect of diosmetin on CD-like colitis.Molecular docking suggested strong binding of diosmetin to the key core targets.Diosmetin significantly reduced the levels of p-PI3K and p-AKT in the colon mucosa in TNBS-treated mice(P<0.05),but their levels remained higher than those in the control group(P<0.05).Conclusion Diosmetin ameliorates TNBS-induced CDPlike colitis in mice possibly by regulating Th1/Th2 and Th17/Treg balance to improve intestinal immune disorder through inhibition of PI3K/AKT signaling.