[Asperosaponin VI alleviates TNBS-induced Crohn's disease-like colitis in mice by reducing intestinal epithelial cell apoptosis via inhibiting the PI3K/AKT/NF-κB signaling pathway]

Abstract

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.

Keywords: Crohn's disease; PI3K/AKT/NF-κB signaling pathway; apoptosis; asperosaponin VI; intestinal barrier.

图1

AVI干预对TNBS模型小鼠疾病状态的影响 Fig.1 Effect of AVI intervention on body weight and disease activity in mice with TNBS-induced CD-like colitis. A: Changes of body weight. B: Changes of DAI scores. WT: Wild type group; TNBS: TNBS-induced model group; AVI: AVI treatment group. *P<0.05 vs WT group. ^# P<0.05 vs TNBS group.

图2

AVI干预对TNBS模型小鼠肠道炎症的影响 Fig.2 Effect of AVI on intestinal inflammation in the mouse models. A, B: Comparison of colon lengths among the groups. C: Histopathological score of the colon. D: HE staining of colon tissue in different groups. E: AB-PAS staining of colon tissue in different groups. *P<0.05 vs WT group. ^#P<0.05 vs TNBS group. Scale bar=50 μm.

图3

AVI干预对TNBS模型小鼠肠道炎症因子的影响 Fig.3 Effect of AVI on intestinal inflammatory factors in the mouse models. A, B: ELISA results of TNF‑α and IL-1β in the intestinal mucosa of the mice. C, D: TNF-α and IL-1β mRNA expression in the intestinal mucosa of the mice detected by RT-qPCR. *P<0.05 vs WT group. ^# P<0.05 vs TNBS group.

图4

不同浓度的AVI作用24 h对Caco-2细胞活力的影响 Fig.4 Changes in viability of Caco-2 cells after treatment with different concentrations of AVI for 24 h. *P<0.05 vs 0 μmol/L.

图5

AVI干预对TNBS模型小鼠结肠炎肠屏障的影响 Fig.5 Effect of AVI on intestinal barrier function in mice with TNBS-induced colitis. A: Immunofluorescence staining for detecting ZO-1, claudin-1 and MUC2 in the mouse colon (Scale bar=50 μm). B, C: Relative expression levels of ZO-1, Claudin-1 and MUC2 proteins in intestinal mucosa detected by Western blotting. *P<0.05 vs WT group. ^# P<0.05 vs TNBS group.

图6

AVI干预对TNF-α诱导的Caco-2细胞屏障损伤的影响 Fig.6 Effect of AVI on the barrier damage of Caco-2 cells induced by TNF-α. A: Immunofluorescence staining for detecting expressions of ZO-1 and claudin-1 in Caco-2 cells (Scale bar=50 μm). B, C: Relative expression levels of ZO-1 and claudin-1 proteins in Caco-2 cells detected by Western blotting. *P<0.05 vs Control group. ^# P<0.05 vs TNF-α group.

图7

AVI干预对TNBS模型小鼠肠上皮细胞凋亡的影响 Fig.7 Effect of AVI on intestinal epithelial cell apoptosis in mice with TNBS-induced colitis. A: TUNEL staining of the colon tissue. B: Apoptosis rate of the intestinal epithelial cells (Scale bar=50 μm). C, D: Western blotting for detecting relative expression levels of Bcl-2, Bax and C-caspase3 in colonic mucosa. *P<0.05 vs WT group. ^# P<0.05 vs TNBS group.

图8

AVI干预对TNF-α诱导的凋亡模型中Caco-2细胞凋亡的影响 Fig.8 Effect of AVI on apoptosis in TNF-α-induced Caco-2 cells. A: TUNEL staining of Caco-2 cells (Scale bar=50 μm). B: Apoptosis rate of Caco-2 cells. C, D: Relative expression levels of Bcl-2, Bax and C-caspase-3 in Caco-2 cells detected by Western blotting.*P<0.05 vs Control group. ^# P<0.05 vs TNF-α group.

图9

网络药理学分析结果 Fig.9 Network pharmacology analysis results. A: Venn diagram of the intersection between CD genes and AVI genes. B, C: PPI network diagram. D: Results of KEGG pathway enrichment analysis for the intersection genes between AVI and CD.

图10

AVI干预对体内和体外PI3K/AKT/NF-κB通路的影响 Fig.10 Effect of AVI on the PI3K/AKT/NF-κB pathway in the mouse models and in Caco-2 cells. A, B: Relative expression levels of PI3K, p-PI3K, AKT, p-AKT, p65 and p-p65 proteins in mouse colon tissue detected by Western blotting (*P<0.05 vs WT group; ^# P<0.05 vs TNBS group). C, D: Relative expression levels of PI3K, p-PI3K, AKT, p-AKT, p65 and p-p65 proteins in Caco-2 cells detected by Western blotting (*P<0.05 vs Control group. ^# P<0.05 vs TNF-α group).

图11

Recilisib对AVI治疗肠上皮细胞凋亡及AKT1 siRNA对体外凋亡模型的影响 Fig.11 Effect of Recilisib and AKT1 siRNA on apoptosis of AVI-treated Caco-2 cells. A, B: Relative expression levels of PI3K, p-PI3K, AKT, p-AKT, p65 and p-p65 proteins detected by Western blotting. C: TUNEL staining of Caco-2 cells (Scale bar=50 μm). D: Apoptosis rate of Caco-2 cells. E, F: Relative expression levels of Bcl-2, Bax, and C-caspase3 proteins detected by Western blotting. *P<0.05 vs AVI group, ^# P<0.05 vs Recilisib group.