珠子草素通过拮抗肠上皮细胞凋亡与调控肠道Th1/Th2免疫平衡改善克罗恩病样小鼠结肠炎

doi:10.12122/j.issn.1673-4254.2026.03.19

摘要/Abstract

摘要：

目的探究珠子草素（Nir）对克罗恩病样结肠炎的作用与机制。方法建立2，4，6-三硝基苯磺酸（TNBS，2.5%）诱导的结肠炎小鼠模型，通过疾病活动度活动（DAI）评分、体质量改变、结肠长度、病理学检测评估结肠炎症状改变，AB-PAS、免疫荧光和免疫印迹评估肠屏障功能，TUNEL染色和免疫印迹评估细胞凋亡，流式细胞术分析肠系膜淋巴结中T淋巴细胞亚群（Th1/Th2）变化；ELISA检测TNF-α和IL-10表达水平变化。体外实验采用脂多糖诱导小鼠结肠类器官炎症模型，检测类器官出芽数及屏障蛋白表达。通过网络药理学和体内实验探索验证Nir拮抗结肠炎的潜在机制。结果体内实验显示，Nir可改善TNBS诱导结肠炎小鼠体质量下降、DAI升高、结直肠缩短等表型，降低组织炎症评分，减少杯状细胞丢失（P<0.05）及肠上皮凋亡细胞数量。体外实验表明，Nir能增加结肠类器官出芽数目（P<0.05）。免疫荧光和免疫印迹证实，Nir可恢复体内外实验结肠组织紧密连接蛋白（ZO-1、claudin-1）水平，下调促凋亡蛋白C-caspase3和BAX表达，上调抗凋亡蛋白Bcl-2（P<0.05）；流式细胞术显示，Nir可降低小鼠肠系膜淋巴结Th1比例，升高Th2比例，改善Th1/Th2失衡，同时下调结肠组织TNF-α、上调IL-10水平（P<0.05）；网络药理学预测Nir作用与PI3K/AKT通路相关；免疫印迹发现，Nir可下调p-PI3K、p-AKT和p-p65表达（P<0.05）。并且PI3K/AKT通路激活剂Recilisib可逆转Nir拮抗肠上皮细胞凋亡和调节Th1/Th2平衡的作用。结论 Nir通过拮抗肠上皮细胞凋亡与调控Th1/Th2平衡来改善克罗恩病样结肠炎，其作用机制与抑制PI3K/AKT通路活性，进而调控下游凋亡信号和炎症免疫分子表达有关。

关键词: 克罗恩病, 珠子草素, 肠屏障, 凋亡, Th1/Th2, PI3K/AKT

Abstract:

Objective To investigate the effect of niranthin (Nir) in mice with Crohn's disease (CD)‑like colitis and its therapeutic mechanism. Methods In a mouse model of CD-like colitis induced using 2,4,6-trinitrobenzene sulfonic acid, the effects of niranthin on colitis symptoms were evaluated by measuring changes in disease activity index (DAI) score, body weight, colon length, and colonic pathologies. Intestinal barrier function and cell apoptosis were evaluated using AB-PAS staining, immunofluorescence staining, Western blotting, and TUNEL staining. The changes in Th1 and Th2 cells in the mesenteric lymph nodes and colonic TNF-α and IL-10 expression levels were determined with flow cytometry and ELISA. In lipopolysaccharide (LPS)-induced mouse colon organoids, the effects of niranthin on organoid budding number and barrier protein expressions were observed. Network pharmacology and in vivo experiments were employed to explore and verify the therapeutic mechanism of niranthin on colitis. Results In the mouse models of CD-like colitis, niranthin treatment obviously improved weight loss, DAI scores, and colorectal shortening and significantly reduced tissue inflammation scores, goblet cell loss, and intestinal epithelial cell apoptosis. Niranthin significantly increased the budding number in LPS-induced mouse colon organoids. In both mouse colon tissues and LPS-induced mouse colon organoids, niranthin obviously increased the expressions of ZO-1 and claudin-1, downregulated the expressions of cleaved caspase-3 and BAX, and upregulated Bcl‑2 expression. The niranthin-treated mouse models showed significantly ameliorated Th1/Th2 imbalance in the mesenteric lymph nodes, downregulated TNF‑α and upregulated IL‑10 levels in the colon tissues. Network pharmacology predicted that the therapeutic mechanism of niranthin for CD-like colitis involved the PI3K/AKT pathway, which was validated in the mouse models treated with niranthin and the PI3K/AKT pathway activator Recilisib. Conclusion Niranthin ameliorates colitis in mice by antagonizing epithelial apoptosis and regulating Th1/Th2 balance via inhibiting the PI3K/AKT pathway.

Key words: Crohn's disease, niranthin, intestinal barrier, apoptosis, Th1/Th2, PI3K/AKT

温贺新, 林洁, 左芦根, 刘牧林. 珠子草素通过拮抗肠上皮细胞凋亡与调控肠道Th1/Th2免疫平衡改善克罗恩病样小鼠结肠炎[J]. 南方医科大学学报, 2026, 46(3): 655-665.

Hexin WEN, Jie LIN, Lugen ZUO, Mulin LIU. Niranthin ameliorates Crohn's disease-like colitis in mice via antagonizing intestinal epithelial cell apoptosis and regulating intestinal Th1/Th2 immune homeostasis[J]. Journal of Southern Medical University, 2026, 46(3): 655-665.

图/表 8

图1 Nir干预对TNBS诱导小鼠结肠炎的作用

Fig.1 Effects of niranthin (Nir) intervention in mice with TNBS-induced colitis. A: Body weight changes. B: DAI scores. C, D: Colon length. E, G: Inflammation scores and HE staining of mouse colon tissues. F, H: Number of goblet cells and AB-PAS staining. n=6, *P<0.05 vs WT; #P<0.05 vs TNBS.

图2 Nir干预对TNBS诱导结肠炎小鼠肠屏障功能的影响

Fig.2 Effects of Nir intervention on intestinal barrier function in TNBS-induced colitis mice. A: Immunofluorescence assay of ZO-1 and claudin-1 expression. B, C: Western blotting for claudin-1 and ZO-1 expressions. n=6, *P<0.05 vs WT; #P<0.05 vs TNBS.

图3 Nir干预对TNBS诱导结肠炎小鼠结肠组织细胞凋亡的影响

Fig.3 The effect of Nir intervention on colonic cell apoptosis in TNBS-induced colitis mice. A: TUNEL staining. B, C: Western blotting for C-caspase-3, Bax and Bcl-2 expressions. n=6, *P<0.05 vs WT; #P<0.05 vs TNBS.

图4 Nir干预对LPS诱导结肠类器官肠上皮细胞凋亡的作用

Fig.4 Effect of Nir on LPS-induced intestinal epithelial cell apoptosis in mouse colon organoids. A: Representative images of colon organoids. B: Number of organoids budding. C, D: Western blotting and bar chart for C-caspase-3, Bax and Bcl-2 expressions. n=6, *P<0.05 vs Con; #P<0.05 vs LPS.

图5 Nir干预对LPS诱导结肠类器官肠屏障功能的影响

Fig.5 Effects of Nir intervention on intestinal barrier function in LPS-induced colonic organoids. A, B: Immunofluorescence assay of ZO-1 and claudin-1 expression. C, D: Western blotting for claudin-1 and ZO-1 expressions. n=6, *P<0.05 vs Con; #P<0.05 vs LPS.

图6 Nir干预对TNBS诱导结肠炎小鼠肠系膜淋巴结Th1/Th2平衡和结肠组织炎症因子水平的影响

Fig.6 Effects of Nir intervention on Th1/Th2 balance in mesenteric lymph nodes and inflammatory factor levels in colonic tissues of TNBS-induced colitis mice. A, B: Proportion of Th1 cells in the mesenteric lymph nodes. C, D: Proportion of Th2 cells in the mesenteric lymph nodes. E: Th1/Th2 ratio. F, G: Expression levels of intestinal mucosal inflammatory mediators. n=6, *P<0.05 vs WT; #P<0.05 vs TNBS.

图7 Nir对肠炎的改善作用可能与PI3K/AKT通路相关

Fig.7 Ameliorative effect of Nir on colitis is possibly associated with the PI3K/AKT pathway. A: Venn diagram. B: KEGG enrichment analysis. C: GO enrichment analysis. D, F: Western blotting of p-PI3K, PI3K, p-AKT and AKT expressions in the colon tissue. E, G: Western blotting of p-PI3K, PI3K, p-AKT and AKT expressions in the colonic organoids. n=6, *P<0.05 vs WT; #P<0.05 vs TNBS.

图8 Nir可以通过负调控PI3K/AKT通路恢复免疫稳态与屏障完整性

Fig.8 Nir can restore immune homeostasis and barrier integrity by negatively regulating the PI3K/AKT pathway. A, C: Western blotting of p-PI3K, PI3K, p-AKT and AKT expressions. B, D: Western blotting for C-caspase-3, Bax and Bcl-2 expressions. E, F: Proportion of Th1 cells in the mesenteric lymph nodes. G, H: Proportion of Th2 cells in the mesenteric lymph nodes. I: Th1/Th2 ratio. J, K: Western blotting for p-p65 and p65 expressions. n=6, *P<0.05 vs TNBS; #P<0.05 vs Nir.

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