Objectives: To investigate the effects of asperosaponin VI (AVI) on intestinal epithelial cell apoptosis and intestinal barrier function in a mouse model of Crohn's disease (CD)-like colitis and explore its mechanisms.
Methods: Male C57BL/6 mice with TNBS-induced CD-like colitis were treated with saline or AVI (daily dose 150 mg/kg) by gavage for 6 days. The changes in body weight, colon length, DAI scores, and colon pathologies of the mice were observed, and the expressions of inflammatory factors and tight injunction proteins were detected using ELISA and RT-qPCR. The effects of AVI on barrier function and apoptosis of mouse intestinal epithelial cells and TNF‑α‑treated Caco-2 cells were analyzed using immunofluorescence staining, TUNEL assay, and Western blotting. Network pharmacology, TUNEL assay, and Western blotting were performed to explore and validate the therapeutic mechanisms of AVI for CD.
Results: In the mouse models of CD-like colitis, AVI significantly improved body weight loss, colon shortening and DAI and tissue inflammation scores, alleviated intestinal villi and goblet cell injuries, and lowered the expressions of inflammatory factors. AVI treatment significantly reduced the loss of tight junction proteins and apoptosis in both mouse intestinal epithelial cells and TNF‑α-stimulated Caco-2 cells. KEGG enrichment pathway analysis suggested that the therapeutic effect of AVI on CD was associated with inhibition of PI3K/AKT/NF-κB pathway activation, which was confirmed by lowered expressions of p-PI3K, p-AKT, and p-p65 in AVI-treated mouse models and Caco-2 cells. In Caco-2 cells, Recilisib significantly blocked the inhibitory effect of AVI on the PI3K/AKT/NF-κB pathway and TNF-α-induced apoptosis, and AKT1 knockdown experiment confirmed the role of the PI3K/AKT pathway for mediating the activation of downstream NF-κB signaling.
Conclusions: AVI can improve TNBS-induced CD-like colitis in mice by reducing intestinal epithelial cell apoptosis and intestinal barrier damage via inhibiting the PI3K/AKT/NF-κB signaling pathway.
目的: 探讨川续断皂苷VI（AVI）对小鼠克罗恩病（CD）样结肠炎的肠上皮细胞凋亡和肠屏障的影响及其作用机制。方法: 将30只雄性C57BL/6小鼠随机分成对照组（WT组）、2，4，6-三硝基苯磺酸诱导模型组（TNBS组）、AVI药物治疗组（AVI组，150 mg/kg），每组10只。通过监测小鼠体质量、测量结肠长度、疾病活动度（DAI）评分、HE染色、AB-PAS染色、组织炎症评分、ELISA和RT-qPCR实验，验证AVI对小鼠结肠炎的缓解作用。采用TNF-α诱导Caco-2细胞建立体外凋亡模型，分为Control组、TNF-α组、AVI组（250 μmol/L）。CCK-8实验检测AVI对Caco-2细胞活力的影响。采用免疫荧光、TUNEL实验和Western blotting检测AVI对小鼠肠上皮细胞和Caco-2细胞屏障损伤与凋亡的改善情况。利用网络药理学预测AVI干预CD的分子机制可能与PI3K/AKT/NF-κB通路有关，Western blotting检测体内外通路的蛋白表达，以及经PI3K/AKT通路激活剂（Recilisib）和AKT1 siRNA转染干预细胞后，通过TUNEL实验和Western blotting验证其对细胞凋亡的调控作用。结果: AVI能明显地缓解小鼠体质量降低、结肠缩短、DAI与组织炎症评分的增加、肠绒毛和杯状细胞的损伤，以及炎症因子的高表达（P<0.05）。AVI浓度在0~250 μmol/L时对Caco-2细胞活力无影响。体内外实验证实，AVI可阻断紧密连接蛋白的缺失，减少肠上皮细胞的凋亡（P<0.05）。KEGG富集通路分析发现AVI干预CD可能与抑制PI3K/AKT/NF-κB通路活化有关，在体内外模型证实AVI干预后p-PI3K、p-AKT和p-p65的表达降低（P<0.05）。此外，Recilisib干预后可逆转AVI对通路的抑制作用和抗凋亡作用（P<0.05），AKT1 siRNA转染细胞后证实PI3K/AKT通路可介导下游NF-κB信号的活化（P<0.05）。结论: AVI可通过拮抗肠上皮细胞的凋亡和减轻肠屏障损伤，达到改善TNBS诱导的CD小鼠结肠炎的目的，其机制可能与AVI负向调控PI3K/AKT/NF-κB有关。.
Keywords: Crohn's disease; PI3K/AKT/NF-κB signaling pathway; apoptosis; asperosaponin VI; intestinal barrier.