[Isongifolene Improves Crohn's Disease-Like Colitis in Mice by Reducing Apoptosis of Intestinal Epithelial Cells]

Abstract

Objective: To investigate the effect and molecular mechanism of isolongifolene (ISO) on the apoptosis of intestinal epithelial cells and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced Crohn's disease (CD)-like colitis in mice.

Methods: In the animal experiments, mice were randomly assigned to the wild type (WT) group, TNBS group and TNBS+ISO group, with 8 mice in each group. Colitis models of mice were established in the TNBS group and the TNBS+ISO group by rectal injection of TNBS. After modeling, the mice in the TNBS+ISO group were given ISO intervention via intragastric gavage (10 mg/kg), and the other two groups were given the same amount of normal saline via intragastric gavage. The mice were sacrificed on the 7th day. The changes in body mass, disease activity scores (DAI), and the colon length of mice were measured, and transepithelial electrical resistance (TEER) of the colon tissues was determined. The score of colon inflammation was calculated according to HE staining. The levels of intestinal mucosal inflammatory factors, including tumor necrosis factor alpha (TNF-α), interferon (IFN)-γ, interleukin (IL)-1β, and IL-6, were measured by RT-PCR and ELISA. The apoptosis of colon tissue cells was determined by TUNEL assay. The expressions of apoptotic proteins (cleaved caspase-3/caspase-3 and Bax), an anti-apoptotic protein (Bcl-2), and tight junction proteins (ZO-1 and claudin-1) were detected by Western blot and immunofluorescence. In the cell experiment, TNF-α was used to induce intestinal epithelial cell Caco-2 apoptosis model, which was treated with ISO. Then, intervention with the AMPK inhibitor Compound C was given. TUNEL assay, Western blot assay, and immunofluorescence assay were performed to measure apoptosis and the expression of apoptosis proteins in the Caco-2 cells. Gene Ontology (GO) enrichment analysis was performed to predict the biological function of ISO. Then, the mechanism involved was verified by examination of the mice and Caco-2 cells. Western blot was performed to determine the expression levels of p-AMPK/AMPK and p-PGC1α in the colon tissues from the mice of different groups and Caco-2 cells. The apoptosis of the cells was determined by TUNEL assay.

Results: According to the results of the animal experiment, ISO could alleviate experimental colitis and intestinal barrier dysfunction, leading to improvements in body mass loss, colon length shortening, DAI score, inflammatory rating, and TEER values (all P<0.05) in mice. Furthermore, ISO decreased the expression of pro-inflammatory factors TNF-α, IFN-γ, IL-1β, and IL-6 and increased the expression of the tight junction proteins ZO-1 and claudin-1 (all P<0.05). In the cell experiment, in a TNF-α-induced intestinal epithelial cell model, ISO was also found to protect intestinal barrier against damage. ISO reduced the proportion of apoptotic intestinal epithelial cells, reduced the expression of cleaved-caspase-3/caspase-3 and Bax, and upregulated the level of Bcl-2 (all P<0.05). GO enrichment predictive analysis showed that the role of ISO in improving CD-like enteritis might be associated with the negative regulation of apoptosis. Verification of the mechanism showed that the expression of p-AMPK and p-PGC1α in the mice colon tissue was significantly upregulated after ISO intervention (P<0.05). In contrast, the AMPK inhibitor Compound C increased the apoptosis rate of ISO-treated Caco-2 cells and decreased the relative expression levels of ZO-1 and claudin-1 (P<0.05).

Conclusion: ISO reduces intestinal epithelial cell apoptosis at least in part by activating AMPK/PGC1α signaling pathway, thereby alleviating TNBS-induced intestinal barrier dysfunction and CD-like colitis in mice.

Keywords: AMPK/PGC1α; Crohn's disease; Intestinal barrier; Intestinal epithelial cell apoptosis; Isolongifolene.

图 1

Effect of ISO on enteritis symptoms in TNBS mice ISO对TNBS小鼠肠炎症状的影响

图 2

ISO treatment improves intestinal barrier injury in TNBS model mice ISO治疗改善TNBS模型小鼠肠屏障损伤

图 3

ISO treatment reduces the apoptosis of intestinal epithelial cells in the colonic tissue of TNBS model mice ISO治疗减少TNBS模型小鼠结肠组织中肠上皮细胞凋亡

图 4

Effect of ISO on tight junction proteins in Caco-2 cells ISO对Caco-2细胞紧密连接蛋白的作用

图 5

Effect of ISO intervention on the apoptosis of Caco-2 cells ISO干预对Caco-2细胞凋亡的影响

图 6

Predictive analysis of the possible biological function and effect of ISO in CD was made by GO enrichment 生物信息学预测ISO在CD中的功能与作用

图 7

Western blot was conducted to assess the effect of ISO intervention on the AMPK/PGC1α pathway in vivo and in vitro Western blot检测ISO对AMPK/PGC1α信号通路的作用

图 8

The effect of ISO on Caco-2 cell apoptosis and intestinal barrier by blocking AMPK/PGC1α signaling pathway ISO干预后阻断AMPK/PGC1α信号通路对Caco-2的细胞凋亡及肠屏障的作用