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

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 apoptosis in Caco-2 cells(P<0.05).Conclusion STM inhibits intestinal epithelial cell apoptosis at least in part by suppressing activation of the PI3K/AKT pathway to ameliorate intestinal barrier dysfunction and colitis in mice.