南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (6): 935-942.doi: 10.12122/j.issn.1673-4254.2023.06.08

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茯苓酸缓解小鼠克罗恩病：基于抑制PI3K/AKT信号通路拮抗肠上皮细胞凋亡

邵荣瑢，杨 子，张文静，张 诺，赵雅静，张小凤，左芦根，葛思堂   

  1. 蚌埠医学院第一附属医院心电图室，胃肠外科，检验科，中心实验室，安徽 蚌埠 233004；蚌埠医学院临床医学院，组织移植安徽省重点实验室，安徽 蚌埠 233030
  • 出版日期:2023-06-20 发布日期:2023-07-06

Pachymic acid protects against Crohn's disease-like intestinal barrier injury and colitis in mice by suppressing intestinal epithelial cell apoptosisviainhibiting PI3K/AKT signaling

SHAO Rongrong, YANG Zi, ZHANG Wenjing, ZHANG Nuo, ZHAO Yajing, ZHANG Xiaofeng, ZUO Lugen, GE Sitang   

  1. Cardiogram Room, Department of Gastrointestinal Surgery, Department of Clinical Laboratory, Central Laboratory, First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, China; Clinical Medical School, Anhui Provincial Key Laboratory of Tissue Transplantation, Bengbu 233000, China
  • Online:2023-06-20 Published:2023-07-06

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摘要： 目的 明确茯苓酸（PA）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及可能机制。方法 将24只野生型小鼠随机分为对照组（WT组）、模型组（TNBS组）和PA干预组（PA组），每组8只。TNBS组和PA组采用TNBS诱导CD样结肠炎；PA组在TNBS造模的同时给予PA干预[5 mg/（kg·d），腹腔注射]，而WT组和TNBS组则每日给予等量的生理盐水腹腔注射。1周后处死小鼠，采用疾病活动度（DAI）评分、体质量改变、结肠长度、组织学炎症评分和小鼠结肠黏膜肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）水平（ELISA检测）评估肠炎程度；采用异硫氰酸荧光素-葡聚糖（FITC-dextran）、肠型脂肪酸结合蛋白（I-FABP）及跨上皮电阻抗值（TEER）评估小鼠肠屏障功能；TUNEL染色检测各组小鼠肠上皮细胞凋亡情况；通过网络药理学预测PA治疗CD可能的作用靶点；采用String平台和Cytoscape 3.7.2软件进行蛋白互作网络构建；采用David数据库对预测靶点进行GO功能及KEGG通路富集分析；应用AutoDock 4.2.6进行PA与关键靶点的分子对接；采用Western blot检测各组小鼠结肠黏膜组织中p-PI3K和p-AKT的蛋白表达水平以验证KEGG富集分析结果。结果 PA组小鼠DAI评分、组织学炎症评分、体质量降低幅度和结肠黏膜TNF-α、IL-6、IL-1β水平显著低于TNBS组（P<0.05），但仍高于WT组（P<0.05）；PA组小鼠结肠长度显著长于TNBS组（P<0.05），但仍短于WT组（P<0.05）；PA组小鼠血清中FITC-dextran和I-FABP水平较TNBS组显著降低（P<0.05），但仍高于WT组（P<0.05），而肠组织TEER水平较TNBS组显著升高（P<0.05），但仍低于WT组（P<0.05）；PA组小鼠肠上皮细胞凋亡率显著低于TNBS组（P<0.05），但仍高于WT组（P<0.05）。通过数据库共筛选到PA和CD交集靶点248个；构建PPI网络得到PA作用于CD的核心靶点分别为EGFR、HRAS、SRC、MMP9、STAT3、AKT1、CASP3、ALB、HSP90AA1、HIF1A；GO功能富集分析显示PA对细胞凋亡进程具有负向调控作用，KEGG富集分析显示PA治疗CD可能与PI3K/AKT信号通路密切相关；分子对接显示PA与AKT1、ALB、EGFR、HSP90AA1、SRC及STAT3等6个靶点有较强的结合力；同时，Western blot检测结果显示PA组小鼠结肠黏膜组织中p-PI3K和p-AKT水平较TNBS组显著降低（P<0.05），但仍高于WT组（P<0.05）。结论 PA可能通过调控PI3K/AKT信号通路拮抗肠上皮细胞凋亡保护肠屏障从而缓解TNBS小鼠CD样肠炎。

关键词: 炎症性肠病, 克罗恩病, 茯苓酸, 肠屏障, 凋亡

Abstract: Objective To investigate the effect of pachymic acid (PA) against TNBS-induced Crohn's disease (CD)-like colitis in mice and explore the possible mechanism. Methods Twenty-four C57BL/6J mice were randomized equally into control group, TNBS-induced colitis model group and PA treatment group. PA treatment was administered via intraperitoneal injection at the daily dose of 5 mg/kg for 7 days, and the mice in the control and model groups were treated with saline. After the treatments, the mice were euthanized for examination of the disease activity index (DAI) of colitis, body weight changes, colon length, intestinal inflammation, intestinal barrier function and apoptosis of intestinal epithelial cells, and the expressions of TNF-α, IL-6 and IL-1β in the colonic mucosa were detected using ELISA. The possible treatment targets of PA in CD were predicted by network pharmacology. String platform and Cytoscape 3.7.2 software were used to construct the protein-protein interaction (PPI) network. David database was used to analyze the GO function and KEGG pathway; The phosphorylation of PI3K/AKT in the colonic mucosal was detected with Western blotting. Results PA significantly alleviated colitis in TNBS-treated mice as shown by improvements in the DAI, body weight loss, colon length, and histological inflammation score and lowered levels of TNF-α, IL-6 and IL-1β. PA treatment also significantly improved FITC-dextran permeability, serum I-FABP level and colonic transepithelial electrical resistance, and inhibited apoptosis of the intestinal epithelial cells in TNBS- treated mice. A total of 248 intersection targets were identified between PA and CD, and the core targets included EGFR, HRAS, SRC, MMP9, STAT3, AKT1, CASP3, ALB, HSP90AA1 and HIF1A. GO and KEGG analysis showed that PA negatively regulated apoptosis in close relation with PI3K/AKT signaling. Molecular docking showed that PA had a strong binding ability with AKT1, ALB, EGFR, HSP90AA1, SRC and STAT3. In TNBS-treated mice, PA significantly decreased p-PI3K and p-AKT expressions in the colonic mucosa. Conclusion PA ameliorates TNBS-induced intestinal barrier injury in mice by antagonizing apoptosis of intestinal epithelial cells possibly by inhibiting PI3K/AKT signaling.

Key words: inflammatory bowel disease, Crohn's disease, pachymic acid, intestinal barrier, apoptosis