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

doi:10.12122/j.issn.1673-4254.2024.08.13

摘要/Abstract

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。

关键词: 克罗恩病, 肠屏障, 肠上皮细胞凋亡, Swertiamarin, PI3K/AKT通路

Abstract:

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-α and 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‑α stimulation, 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.

Key words: Crohn's disease, intestinal barrier, intestinal epithelial cell apoptosis, swertiamarin, PI3K/AKT pathway

刘硕, 李静, 吴兴旺. 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[J]. 南方医科大学学报, 2024, 44(8): 1545-1552.

Shuo LIU, Jing LI, Xingwang WU. Swertiamarin ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice by inhibiting intestinal epithelial cell apoptosis[J]. Journal of Southern Medical University, 2024, 44(8): 1545-1552.

图/表 8

表1 引物序列

Tab.1 Primers Sequence

Primers	Forward (5'-3')	Reverse (5'-3')
IL-6	TCTATACCACTTCACAAGTCGGA	GAATTGCCATTGCACAACTCTTT
CCL3	CTCCCAGCCAGGTGTCATTTT	CTTGGACCCAGGTCTCTTTGG
GAPDH	TGGCCTTCCGTGTTCCTAC	GAGTTGCTGTTGAAGTCGCA

图1 STM对TNF-α诱导的Caco-2细胞凋亡的影响

Fig.1 Effect of STM on TNF-α-induced apoptosis in Caco-2 cells. A, B: Representative images of TUNEL staining and statistical analysis of the positively stained cells. C-F: Levels of c-caspase 3, Bax and Bcl2 detected by Western blotting. *P<0.05 vs Control group/WT group. #P<0.05 vs TNF-α group.

图2 STM对TNF-α诱导的Caco-2细胞的屏障和炎症反应的影响

Fig.2 Effect of STM on TNF-α-induced barrier dysfunction and inflammatory responses in Caco-2 cells. A: Detection of TEER values. B: Immunofluorescence staining of ZO1 and claudin 1. C-E: Levels of ZO1 and claudin 1 detected by Western blotting. F, G: qRT-PCR for detecting expressions of IL-6 and CCL3. H, I: Levels of IL-6 and CCL3 detected by ELISA. *P<0.05 vs Control group, WT group. #P<0.05 vs TNF-α group.

图3 STM对TNBS诱导小鼠的实验性结肠炎影响

Fig.3 Effect of STM on TNBS-induced CD-like colitis in mice. A: Body weight changes of the mice. B: DAI score. C, D: HE staining and inflammation score of the colonic tissue. E, F: qRT-PCR for detecting mRNA expressions of IL-6 and CCL3. G, H: Levels of IL-6 and CCL3 detected by ELISA. *P<0.05 vs WT group. #P<0.05 vs TNBS group.

图4 STM对TNBS诱导小鼠肠屏障的影响

Fig.4 Effect of STM on intestinal barrier in TNBS-induced mice. A: Intestinal barrier permeability assay. B: TEER analysis of the colonic tissues. C-E: Assessment of the percentage of bacterial translocation in the intestinal lymph nodes, spleen and liver. F: Immunofluorescence staining of ZO1 and claudin 1. G-I: Levels of ZO1 and claudin 1 detected by Western blotting. *P<0.05 vs WT group. #P<0.05 vs TNBS group.

图5 STM对TNBS诱导小鼠结肠组织中肠上皮细胞凋亡的影响

Fig. 5 Effect of STM on apoptosis of intestinal epithelial cells in TNBS-induced mice. A, B: Representative image of TUNEL staining and statistical analysis of the positively stained intestinal epithelial cells. C-F: Levels of c-caspase 3, Bax and Bcl2 detected by Western blotting. *P<0.05 vs WT group. #P<0.05 vs TNBS group.

图6 体内外验证STM对PI3K/AKT通路的影响

Fig. 6 In vitro and in vivo experiments for validating the effect of STM on the PI3K/AKT pathway. A-C: Levels of p-PI3K, PI3K, p-AKT and AKT detected in TNF-α-induced Caco-2 cells by Western blotting. D-F: Western blotting for detecting expression levels of p-PI3K, PI3K, p-AKT and AKT in the intestinal mucosal tissues of TNBS-induced mice. *P<0.05 vs WT or Con group. #P<0.05 vs TNBS or TNF-α group.

图7 PI3K/AKT通路参与STM抗肠上皮细胞凋亡

Fig.7 The PI3K/AKT pathway is involved in STM-mediated inhibition of intestinal epithelial cell apoptosis. A, B: TUNEL staining and statistical analysis of the positively stained intestinal epithelial cells. C-F: Levels of c-caspase 3, Bax and Bcl2 detected by Western blotting. *P<0.05 vs STM group.

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