乙酰紫堇灵通过抑制肠上皮细胞凋亡改善三硝基苯磺酸诱导的小鼠克罗恩病样结肠炎  

Acetylcorynoline relieves 2,4,6-trinitrobenesulfonic acid-induced Crohn's disease-like colitis in mice by regulating intestinal epithelial cell apoptosis

作　　者：李晴晴 黄菊 孙洋 徐赟辉 王炼 张小凤 王月月 耿志军 宋雪 左芦根[4,5] 李静 胡建国 LI Qingqing;HUANG Ju;SUN Yang;XU Yunhui;WANG Lian;ZHANG Xiaofeng;WANG Yueyue;GENG Zhijun;SONG Xue;ZUO Lugen;LI Jing;HU Jianguo(Clinical Laboratory,First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Department of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Central Laboratory,First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Department of Gastrointestinal Surgery,First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Anhui Province Key Laboratory of Basic and Translational Research of Inflammation-related Diseases,Bengbu 233004,China)

机构地区：[1]蚌埠医学院第一附属医院检验科,安徽蚌埠233004 [2]蚌埠医学院检验医学院,安徽蚌埠233030 [3]蚌埠医学院第一附属医院中心实验室,安徽蚌埠233004 [4]蚌埠医学院第一附属医院胃肠外科,安徽蚌埠233004 [5]炎症相关性疾病基础与转化研究安徽省重点实验室,安徽蚌埠233004

出　　处：《南方医科大学学报》2023年第8期1306-1314,共9页Journal of Southern Medical University

基　　金：安徽省教育厅优秀青年基金(2022AH030138);蚌埠医学院重大科技项目孵育计划(2020byfy003);蚌埠医学院研究生科研创新计划项目(Byycx22038)。

摘　　要：目的探究乙酰紫堇灵(Ace)对2,4,6-三硝基苯磺酸(TNBS)诱导的小鼠克罗恩病(CD)样结肠炎的作用及机制。方法将40只雄性C57BL/6小鼠随机分为5组:WT组、TNBS组和Ace干预组(Ace-10组、Ace-20组和Ace-40组),每组8只。TNBS组和Ace干预组小鼠均采用TNBS诱导建立结肠炎模型,Ace干预组分别给予10、20、40 mg/kg的Ace灌胃治疗,而TNBS组均给予等量生理盐水。通过监测体质量变化、疾病活动评分(DAI)、结肠长度测量、HE染色和组织学评分评估Ace对TNBS结肠炎小鼠的保护作用。小鼠结肠组织经免疫荧光检测Claudin-1、Western blot检测紧密连接蛋白(Claudin-1和ZO-1)和凋亡相关蛋白(C-caspase3、Bax和Bcl-2)以及Tunel染色检测肠上皮细胞凋亡的数量。采用结肠类器官经脂多糖诱导建立凋亡模型,分为Control组、脂多糖组及Ace组,采用免疫荧光检测Claudin-1以及Western blot检测Claudin-1、ZO-1、C-caspase3、Bax和Bcl-2的表达。利用网络药理学预测Ace抑制肠上皮细胞凋亡的分子机制可能与PI3K-AKT通路相关,采用Western blot验证小鼠结肠组织和结肠类器官中pPI3K和p-AKT的表达。体外凋亡模型中加入PI3K-AKT信号通路激活剂(740Y-P),Western blot验证结肠类器官中p-PI3K、pAKT、C-caspase3、Bax及Bcl-2的表达。结果与TNBS组相比,Ace治疗可显著缓解TNBS小鼠的体质量下降、结肠长度缩短、DAI评分和炎症评分升高(P<0.05)。在Ace组小鼠结肠组织中,免疫荧光结果显示:Claudin-1的表达上调(P<0.05);Western blot结果显示:Claudin-1、ZO-1和Bcl-2的蛋白水平升高,而C-caspase3和Bax的表达降低(P<0.05);Tunel染色结果显示:肠上皮细胞凋亡数量减少(P<0.05)。在体外凋亡模型中,经Ace干预后,免疫荧光和Western blot结果显示,结肠类器官组织中ZO-1和Claudin-1的表达升高(P<0.05)、C-caspase3和Bax表达降低,而Bcl-2表达升高(P<0.05)。KEGG通路富集预测发现PI3K-AKT信号通路与Ace治疗CD具有相关性,且WesternObjective To explore the effect of acetylcorynoline for relieving 2,4,6-trinitrobenzenesulfonic acid(TNBS)-induced Crohn's disease(CD)-like colitis in mice and explore the underlying mechanism.Methods Male C57BL/6 mice were subjected to TNBS treatment to establish models of TNBS-induced CD-like colitis,followed by treatment with saline or 10,20,or 40 mg/kg acetylcorynoline by gavage.The protective effect of acetylcorynoline against colitis was evaluated by monitoring body weight changes,measurement of DAI and colon length,and histological examination.The colon tissues and cultured colon organoids treated with LPS and acetylcorynoline were examined for expressions of tight junction proteins and apoptosis-related proteins using immunofluorescence assay,Western blotting,and TUNEL staining.The mechanism of acetylcorynoline-induced inhibition of intestinal epithelial cell apoptosis was predicted by network pharmacology and verified by Western blotting.Results Acetylcorynoline treatment significantly alleviated weight loss and colon length shortening and reduced DAI score and inflammation score in TNBS mice(P<0.05).Claudin-1 was significantly upregulated in the colon tissue of acetylcorynoline treated mice(P<0.05),where the protein levels of claudin 1,ZO-1,and Bcl 2 were increased and C-caspase3 and Bax were reduced(P<0.05)and the number of apoptotic intestinal epithelial cells decreased(P<0.05).In cultured colon organoids,acetylcorynoline significantly increased ZO-1,claudin-1 and Bcl-2 expressions and decreased C-caspase3 and Bax expressions(P<0.05).KEGG pathway enrichment analysis suggested that the PI3K-AKT signaling pathway was correlated with acetylcorynoline treatment for CD,and the expressions of p-AKT and p-PI3K decreased significantly after the treatment in both the in vivo and in vitro models(P<0.05).The PI3K-AKT activator(740Y-P)significantly promoted the expressions of p PI3K,p-AKT,C-caspase3 and Bax and inhibited Bcl-2 in the colon organoids(P<0.05).Conclusion Acetylcorynoline protects against TNBS-induce

关 键 词：克罗恩病 乙酰紫堇灵 肠上皮细胞凋亡 PI3K/AKT 

分 类 号：R285.5[医药卫生—中药学]