[Niranthin ameliorates Crohn's disease-like enteritis in mice by inhibiting intestinal epithelial cell apoptosis and protecting intestinal barrier via modulating p38/JNK signaling]

Abstract

Objectives: To investigate the therapeutic effect of the natural compound niranthin on Crohn's disease-like colitis in mice and explore the underlying molecular mechanisms.

Methods: In a mouse model of colitis induced by 2,4,6-trinitro-benzenesulfonic acid (TNBS), the therapeutic effect of niranthin was evaluated by observing the changes in body weight, disease activity index (DAI), and colon length of the mice. The levels of inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-17A and IL-10) in the intestinal mucosal tissue were detected using ELISA and quantitative real-time PCR (qRT-PCR). TUNEL staining and Western blotting were used to assess intestinal epithelial cell apoptosis and the expressions of Bcl-2 and Bax. The expression levels of tight junction proteins (ZO-1 and claudin-1) and the activation of the p38/JNK signaling pathway were investigated using Western blotting, and diprovocim intervention experiments were conducted to explore the molecular regulatory mechanism of niranthin.

Results: Niranthin treatment significantly increased body weight of TNBS-treated mice, lowered the DAI and histological inflammation scores, and increased colon length of the mice. The niranthin-treated mouse models showed obviously reduced protein and mRNA levels of IL-6, IL-1β, IL-17A, and TNF-α and upregulated expression of IL-10 in the colon tissue. TUNEL staining and Western blotting demonstrated that niranthin significantly inhibited intestinal epithelial cell apoptosis and activated the anti-apoptotic pathway in the mouse models. Niranthin treatment obviously upregulated the expression levels of ZO-1 and claudin-1 and downregulated the phosphorylation levels of p38 and JNK in the colon tissues of the mice. Diprovocim intervention obviously attenuated the inactivation of the p38/JNK signaling pathway induced by niranthin in the mouse models.

Conclusions: Niranthin ameliorates TNBS-induced Crohn's disease-like colitis in mice by inhibiting intestinal epithelial cell apoptosis and protecting the integrity of the intestinal barrier via regulating the activation of the p38/JNK signaling pathway.

Keywords: Crohn's disease; Niranthin; inflammatory bowel disease; intestinal epithelial cell apoptosis; p38/JNK.

图1

NIR干预对TNBS诱导小鼠结肠炎症状的影响 Fig.1 Effect of niranthin (NIR) treatment on symptoms of TNBS-induced colitis in mice. A: Body weight changes of the mice. B: DAI score. C: Colon length. D: Comparison of colon length of the mice among the 4 groups. E: Colon inflammation score of the mice. F: HE staining of the colon tissues of the mice in the 4 groups.*P<0.05.

图2

NIR干预对TNBS诱导小鼠结肠炎炎症因子水平的影响 Fig.2 Effect of NIR treatment on inflammatory factor levels in mice with TNBS-induced colitis. A-E: Results of ELISA for detecting the levels of IL-6, IL-1β, IL-17A, TNF-α and IL-10 in the colon tissue of TNBS-induced mice. F-J: qRT-PCR for detecting IL-6, IL-1β, IL-17A, TNF‑α and IL-10 mRNA expressions in the colon tissues of TNBS-induced mice. *P<0.05.

图3

NIR干预对TNBS诱导小鼠结肠炎肠上皮细胞凋亡的影响 Fig.3 Effect of NIR treatment on TNBS-induced apoptosis of intestinal epithelial cells in the mouse models of colitis. A, B: TUNEL staining for detecting apoptosis in TNBS-induced mice with NIR treatment. C: Western blotting for detecting the expression of Bcl-2 and Bax proteins in TNBS-induced mice with NIR treatment. D: Immunofluorescence staining for detecting Bcl-2 expression in TNBS-induced mice with NIR treatment. *P<0.05.

图4

NIR干预对TNBS诱导小鼠结肠炎肠屏障的影响 Fig.4 Effect of NIR treatment on intestinal barrier function in mice with TNBS-induced colitis. A: Immunofluorescence staining for detecting ZO-1 and Claudin-1 expression in TNBS-induced mice with NIR treatment. B: Western blotting for detecting ZO-1 and claudin-1 protein expressions in TNBS-induced mice with NIR treatment. *P<0.05.

图5

NIR干预对LPS诱导Caco-2细胞凋亡的影响 Fig.5 Effect of NIR treatment on LPS-induced apoptosis in Caco-2 cells. A, B: TUNEL staining for detecting LPS-induced apoptosis of Caco-2 cells with NIR treatment. C: Western blotting for detecting the expression of Bcl-2 and Bax proteins in LPS-induced Caco-2 cells with NIR treatment. D: Immunofluorescence staining for detecting Bcl-2 expression in LPS-induced Caco-2 cells with NIR treatment. *P<0.05.

图6

NIR干预对LPS诱导Caco-2细胞TJ蛋白表达的影响 Fig. 6 Effect of NIR treatment on expressions of tight junction proteins in LPS-induced Caco-2 cells. A: Immunofluorescence staining showing ZO-1 and claudin-1 expressions in LPS-induced Caco-2 cells with NIR treatment. B: Western blotting for detecting ZO-1 and claudin-1 protein expressions in LPS-induced Caco-2 cells with NIR treatment. *P<0.05.

图7

NIR干预可调控p38/JNK信号 Fig. 7 NIR treatment modulates p38/JNK signaling. A: Western blotting for detecting p-p38 and p-JNK protein expressions in TNBS-induced mice with NIR treatment. B: Western blotting for detecting p-p38 and p-JNK protein expressions in LPS-induced Caco-2 cells with NIR treatment. C: Western blotting for detecting p-p38 and p-JNK protein expressions in the colon tissues of the mice with NIR and diprovocim treatment. *P<0.05.

图8

Diprovocim干预对NIR治疗Caco-2细胞中凋亡和TJ蛋白表达的影响 Fig. 8 Effects of diprovocim intervention on apoptosis and expressions of tight junction proteins in NIR-treated Caco-2 cells. A: TUNEL staining for detecting apoptosis in NIR-treated Caco-2 cells by Diprovocim treatment. B: Western blotting for detecting the expression of Bcl-2 and Bax proteins in NIR-treated Caco-2 cells with diprovocim treatment. C, D: Immunofluorescence staining for detecting Bcl-2, ZO-1 and Claudin-1 expression in NIR-treated Caco-2 cells with diprovocim treatment. *P<0.05.