Swertiamarin通过抑制肠上皮细胞细胞凋亡改善TNBS诱导的实验性结肠炎  

Swertiamarin ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice by inhibiting intestinal epithelial cell apoptosis

作　　者：刘硕 李静 吴兴旺[4] LIU Shuo;LI Jing;WU Xingwang(First Clinical Medical College,Anhui Medical University,Hefei 230000,China;Clinical Laboratory,,First Affiliated Hospital of Bengbu Medical University,Bengbu 233003,China;Anhui Provincial Key Laboratory of Basic and Translational Research of Inflammation-related Diseases,First Affiliated Hospital of Bengbu Medical University,Bengbu 233003,China;Department of Radiology,First Affiliated Hospital of Anhui Medical University,Hefei 230000,China)

机构地区：[1]安徽医科大学第一临床医学院,安徽合肥230000 [2]蚌埠医科大学第一附属医院检验科,安徽蚌埠233003 [3]蚌埠医科大学第一附属医院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233003 [4]安徽医科大学第一附属医院放射科,安徽合肥230000

出　　处：《南方医科大学学报》2024年第8期1545-1552,共8页Journal of Southern Medical University

基　　金：安徽省高校优秀青年基金(2022AH030138);安徽省自然科学基金(2308085MH241);安徽省学术和技术带头人及后备人选科研活动经费资助项目(2021D299)。

摘　　要：目的本研究旨在探讨Swertiamarin(STM)通过拮抗肠上皮细胞凋亡改善CD样结肠炎的作用和机制。方法体外建立TNF-α刺激的Caco-2细胞凋亡模型,分为3组:对照组(Con)、TNF-α刺激组(TNF-α)和STM干预组(STM),通过Tunel染色、免疫印迹、免疫荧光和上皮电阻检测等方法,评估STM对细胞凋亡和屏障功能的影响。体内建立TNBS诱导的CD样结肠炎小鼠模型,分为3组:WT、TNBS和STM组,利用小鼠体质量变化、疾病活动指数评分、炎症评分和黏膜组织中炎症因子含量分析STM对结肠炎的作用;通过通透性、细菌移位率和紧密连接蛋白表达与定位观察STM对肠屏障功能的影响;使用Tunel染色和免疫印迹检测凋亡相关蛋白水平评估STM对上皮细胞凋亡的作用。体内外研究验证PI3K/AKT通路在STM抗肠上皮细胞凋亡中的调控作用。结果体外研究中TUNEL染色结果显示,STM显著得减少TUNEL着色的Caco-2细胞的比例(P<0.05);免疫印迹数据显示,STM组中cleavedcaspase3和Bax的表达低于TNF-α组(P<0.05),而Bcl2的水平则增高(P<0.05);肠屏障完整性和功能检测显示,STM恢复了TEER值(P<0.05)、促进了紧密连接蛋白(ZO1和claudin 1)的定位正常化和表达水平的上调(P<0.05),以及抑制了促炎因子(IL-6和CCL3)的表达(P<0.05)。体内研究显示STM能缓解结肠炎和肠屏障功能障碍,具体表现为体重下降、疾病活动指数(DAI)评分、炎症评分和促炎因子(IL-6和CCL3)释放以及肠屏障通透性、结肠TEER、细菌移位和紧密连接蛋白(ZO1和Claudin-1)定位与表达均得到了改善(P<0.05)。机制上,STM在体和体外均抑制了p-PI3K和p-AKT的表达(P<0.05),且PI3K/AKT通路的激活剂(740YP)阻遏了STM抗TNF-α诱导的Caco-2凋亡作用(P<0.05)。结论STM至少部分是通过抑制PI3K/AKT通路的激活,拮抗肠上皮细胞细胞的凋亡,进而改善肠屏障功能障碍和实验性结肠炎。Objective To investigate the mechanism by which swertiamarin(STM)ameliorates CD-like colitis in mice.Methods A Caco-2 cell model of TNF‑α‑stimulated apoptosis was established and divided into three groups:Con,TNF-αand STM,and the effects of STM on apoptosis and barrier function were assessed by Tunel staining,western blotting,immunofluorescence,and transepithelial electric resistance(TEER).A mouse model of 2,4,6-trinitrobenzenesulfonic acid(TNBS)-induced CD-like colitis was established to assess the effects of STM on colitis,intestinal barrier function and epithelial cell apoptosis.The regulatory role of the PI3K/AKT pathway in STM-induced resistance to intestinal epithelial cell apoptosis was investigated in both the cell model and mouse models.Results TUNEL staining showed that in Caco-2 cells with TNF‑αstimulation,STM treatment significantly reduced the percentage of TUNEL-stained cells(P<0.05).STM obviously reduced TNF‑α‑induced enhancement of cleaved-caspase 3 and Bax expressions(P<0.05),increased Bcl-2 expression(P<0.05),protected intestinal barrier integrity and function by restoring transepithelial electrical resistance(TEER)of the cells,promoted normal localization and expressions of the tight junction proteins(ZO1 and claudin 1)(P<0.05),and inhibited the expression of pro-inflammatory factors(IL-6 and CCL3)(P<0.05)in TNF‑α-stimulated Caco-2 cells.In the mouse models,STM significantly alleviated TNBS�induced CD-like colitis and intestinal barrier dysfunction(P<0.05)as shown by improved weight loss,lowered Disease Activity Index(DAI)score and inflammation score,reduction of IL-6 and CCL3 release,and restoration of intestinal barrier permeability,colonic TEER,bacterial translocation,and localization and expressions of the tight junction proteins.Mechanistically,STM inhibited the expressions of p-PI3K and p-AKT in both the cell model and mouse model(P<0.05),and treatment with 740Y-P(a PI3K/AKT pathway activator)significantly attenuated the inhibitory effect of STM on TNF‑α‑induced ap

关 键 词：克罗恩病 肠屏障 肠上皮细胞凋亡 SWERTIAMARIN PI3K/AKT通路 

分 类 号：R574.62[医药卫生—消化系统]