南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (3): 474-482.doi: 10.12122/j.issn.1673-4254.2023.03.19

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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

杨 子，赵天豪，程 阳，周约青，李岳彤，王欣茹，张小凤，左芦根，葛思堂   

  1. 蚌埠医学院第一附属医院胃肠外科，消化内科，输血科，中心实验室，安徽 蚌埠 233004；蚌埠医学院，安徽 蚌埠 233030；蚌埠医学院组织移植重点实验室，安徽 蚌埠 233030
  • 出版日期:2023-03-20 发布日期:2023-03-20

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

YANG Zi, ZHAO Tianhao, CHENG Yang, ZHOU Yueqing, LI Yuetong, WANG Xinru, ZHANG Xiaofeng, ZUO Lugen, GE Sitang   

  1. Department of Gastrointestinal Surgery, Department of Gastroenterology, Department of Blood Transfusion, Central Laboratory, First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, China; Bengbu Medical College, Bengbu 233030, China; Key Laboratory of Tissue Transplantation, Bengbu Medical College, Bengbu 233030, China
  • Online:2023-03-20 Published:2023-03-20

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摘要： 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。

关键词: 克罗恩病；结肠炎；香叶木素；肠黏膜免疫

Abstract: 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.

Key words: Crohn's disease; colitis; diosmetin; intestinal mucosal immunity