异长叶烯通过减少肠上皮细胞凋亡改善小鼠克罗恩病样结肠炎  

Isongifolene Improves Crohn's Disease-Like Colitis in Mice by Reducing Apoptosis of Intestinal Epithelial Cells

作　　者：段婷 耿志军 杨晶晶 殷丽霞 孙明熙 王舜印 张小凤 李静 胡建国 陆国玉 DUAN Ting;GENG Zhijun;YANG Jingjing;YIN Lixia;SUN Mingxi;WANG Shunyin;ZHANG Xiaofeng;LI Jing;HU Jianguo;LU Guoyu(Department of Emergency,The First Affiliated Hospital of Bengbu Medical University,Bengbu 233000,China;Central Laboratory,The First Affiliated Hospital of Bengbu Medical University,Bengbu 233000,China;Anhui Province Key Laboratory of Basic and Translational Research of Inflammation-Related Diseases,Bengbu 233000,China;Department of Gastrointestinal Surgery,The First Affiliated Hospital of Bengbu Medical University,Bengbu 233000,China;Clinical Laboratory,The First Affiliated Hospital of Bengbu Medical University,Bengbu 233000,China)

机构地区：[1]蚌埠医科大学第一附属医院急诊内科,蚌埠233000 [2]蚌埠医科大学第一附属医院中心实验室,蚌埠233000 [3]炎症相关性疾病基础与转化研究安徽省重点实验室,蚌埠233000 [4]蚌埠医科大学第一附属医院胃肠外科,蚌埠233000 [5]蚌埠医科大学第一附属医院检验科,蚌埠233000

出　　处：《四川大学学报（医学版）》2024年第5期1175-1185,共11页Journal of Sichuan University(Medical Sciences)

基　　金：安徽省高校优秀科研创新团队项目(No.2023AH010067);蚌埠医学院重大科技项目孵育计划(No.2020byfy003)资助。

摘　　要：目的探究异长叶烯(isolongifolene,ISO)对肠上皮细胞凋亡和2,4,6-三硝基苯磺酸(TNBS)诱导的小鼠克罗恩病(Crohn's disease,CD)样结肠炎的作用及分子机制。方法动物实验:将小鼠随机分为Wild type(WT)组、2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)组和TNBS+ISO组,每组8只。TNBS组及TNBS+ISO组小鼠经直肠灌注TNBS构建结肠炎模型,TNBS+ISO组小鼠在造模后予ISO灌胃(10 mg/kg)干预,其余两组予等量生理盐水灌胃,第7天处死小鼠。检测小鼠体质量变化、疾病活动评分(disease activity index,DAI)、结肠长度,进行结肠组织跨上皮电阻(TEER)检测,根据HE染色计算结肠炎症程度评分,RT-PCR和ELISA法检测肠黏膜炎症因子[肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素(IL)-1β和IL-6]水平,TUNEL染色检测小鼠结肠组织细胞凋亡,Western blot和免疫荧光检测其凋亡蛋白(cleaved caspase-3/caspase-3和Bax)、抗凋亡蛋白(Bcl-2)和紧密连接蛋白(ZO-1和claudin-1)表达。细胞实验:TNF-α诱导肠上皮细胞Caco-2凋亡模型,进行ISO治疗,再加入AMPK抑制剂Compound C干预。TUNEL染色、Western blot和免疫荧光检测同前。基因功能注释(Gene Ontology,GO)富集预测ISO的生物学功能。然后用小鼠和Caco-2细胞进行机制验证:Western blot检测各组小鼠结肠组织及Caco-2细胞中p-AMPK/AMPK和p-PGC1α表达水平,TUNEL染色检测细胞凋亡。结果动物实验结果显示,ISO能缓解实验性结肠炎和肠屏障功能障碍,表现为小鼠体质量降低(P<0.05)、结肠长度缩短(P<0.05),DAI评分(P<0.05)、炎症程度评分(P<0.05)、TEER值(P<0.05)改善,结肠组织中促炎因子(TNF-α、IFN-γ、IL-1β和IL-6)(P<0.05)、紧密连接蛋白[ZO-1(P<0.05)和claudin-1(P<0.05)]的表达改善。细胞实验中,在TNF-α诱导的肠上皮细胞模型中也发现ISO能保护肠屏障受损。ISO减少肠上皮细胞的凋亡率(P<0.05)、cleaved caspase-3/caspase-3(P<0.05)和Bax(P<0.05)的表达,且�Objective To investigate the effect and molecular mechanism of isolongifolene(ISO)on the apoptosis of intestinal epithelial cells and 2,4,6-trinitrobenzenesulfonic acid(TNBS)-induced Crohn's disease(CD)-like colitis in mice.Methods In the animal experiments,mice were randomly assigned to the wild type(WT)group,TNBS group and TNBS+ISO group,with 8 mice in each group.Colitis models of mice were established in the TNBS group and the TNBS+ISO group by rectal injection of TNBS.After modeling,the mice in the TNBS+ISO group were given ISO intervention via intragastric gavage(10 mg/kg),and the other two groups were given the same amount of normal saline via intragastric gavage.The mice were sacrificed on the 7th day.The changes in body mass,disease activity scores(DAI),and the colon length of mice were measured,and transepithelial electrical resistance(TEER)of the colon tissues was determined.The score of colon inflammation was calculated according to HE staining.The levels of intestinal mucosal inflammatory factors,including tumor necrosis factor alpha(TNF-α),interferon(IFN)-γ,interleukin(IL)-1β,and IL-6,were measured by RT-PCR and ELISA.The apoptosis of colon tissue cells was determined by TUNEL assay.The expressions of apoptotic proteins(cleaved caspase-3/caspase-3 and Bax),an anti-apoptotic protein(Bcl-2),and tight junction proteins(ZO-1 and claudin-1)were detected by Western blot and immunofluorescence.In the cell experiment,TNF-αwas used to induce intestinal epithelial cell Caco-2 apoptosis model,which was treated with ISO.Then,intervention with the AMPK inhibitor Compound C was given.TUNEL assay,Western blot assay,and immunofluorescence assay were performed to measure apoptosis and the expression of apoptosis proteins in the Caco-2 cells.Gene Ontology(GO)enrichment analysis was performed to predict the biological function of ISO.Then,the mechanism involved was verified by examination of the mice and Caco-2 cells.Western blot was performed to determine the expression levels of p-AMPK/AMPK and p-PGC1αin the col

关 键 词：克罗恩病 异长叶烯 肠上皮细胞凋亡 肠屏障 AMPK/PGC1α 

分 类 号：R574.62[医药卫生—消化系统]