Verbenalin ameliorates intestinal inflammation and colitis in a mouse model of Crohn's disease by inhibiting the PI3K-AKT pathway

doi:10.12122/j.issn.1673-4254.2026.02.20

Abstract

Abstract:

Objective To investigate the therapeutic effect of verbenalin (VE) on Crohn's disease (CD)‑like colitis and the underlying molecular mechanism. Methods Fifty C57BL/6 mice were randomly divided into control group, TNBS group, and low-, medium-, and high-dose VE treatment groups (n=10). Mouse models of CD-like colitis were established in all but the control group by enema with 25 mg/L TNBS dissolved in ethanol, and the mice in VE treatment groups received daily intraperitoneal injections of VE at 5, 10, or 20 mg/kg for 7 days. Cultured colon organoids derived from mouse crypts were exposed to 100 μg/mL lipopolysaccharide (LPS) for 24 h and treated with 5, 10, or 20 μmol/L VE. The therapeutic effects of VE in the mouse models were evaluated by assessing changes in disease activity index (DAI), histopathological scores, and spleen index. In both colonic mucosa of the mouse models and the colon organoids, the levels of inflammatory cytokines, expressions of tight junction proteins, and changes in PI3K-AKT pathway proteins were analyzed, and the regulatory mechanism of VE was verified using the PI3K-AKT agonist 740 Y-P. Results In TNBS-treated mice, VE treatment significantly reduced DAI, histopathological scores, and spleen index, and mitigated weight loss, colon shortening and bacterial translocation. VE obviously lowered the expression of pro-inflammatory cytokines in colonic mucosa of the mice and the colon organoids, upregulated ZO-1 and claudin-1 expressions, and reduced bacterial translocation. VE significantly downregulated p-PI3K and p-AKT protein expressions, which was reversed by treatment with 740 Y-P. Conclusion VE inhibits intestinal inflammation and protects intestinal barrier function in mice with CD-like colitis by modulating the PI3K-AKT signaling pathway.

Key words: Crohn's disease, inflammatory bowel disease, intestinal barrier, PI3K-AKT, verbenalin

Linlin HUANG, Wang ZHENG, Jianguo HU, Xue SONG, Lu TAO, Zhijun GENG, Jing LI, Lugen ZUO, Sitang GE. Verbenalin ameliorates intestinal inflammation and colitis in a mouse model of Crohn's disease by inhibiting the PI3K-AKT pathway[J]. Journal of Southern Medical University, 2026, 46(2): 423-433.

Figures/Tables 8

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence (5'to 3')
TNF-α	F: CACGCTCTTCTGTCTACTGAACTTC
TNF-α	R: CTTGGTGGTTTGTGAGTGTGAGG
IL-1β	F: AATCTCGCAGCAGCACATCAAC
IL-1β	R: AGGTCCACGGGAAAGACACAG
IL-6	F: GAGAGGAGACTTCACAGAGGATACC
IL-6	R: TCATTTCCACGATTTCCCAGAGAAC
GAPDH	F: AACTCCCACTCTTCCACCTTCG R: TCCACCACCCTGTTGCTGTAG

Fig.1 Effects of VE treatment on TNBS-induced colitis symptoms in mice. A: Body weight changes. B: Disease activity index (DAI) scores. C: Spleen index. D: Spleen size. E: Gross morphology of mouse colon. F: Quantitative analysis of colon length. G: Histological inflammation score. H: HE staining of the colon tissue (n=10). *P<0.05.

Fig.2 Effects of VE on intestinal barrier function and structure in mice. A-D: Bacterial culture and translocation rates in the lymph nodes, liver, and spleen. E: Immunofluorescence staining of ZO-1 and claudin-1 in colonic mucosa. F-G: Western blotting bands and quantitative analysis of ZO-1 and claudin-1 protein expression (n=10). *P<0.05.

Fig.3 Effects of VE on inflammatory mediator levels in colonic mucosa of TNBS-treated mice. A-C: ELISA detection of IL-6, IL-1β, and TNF-α protein levels. D-F: RT-qPCR analysis of IL-6, IL-1β, and TNF-α mRNA expression levels (n=10). *P<0.05.

Fig.4 Effects of VE on LPS-induced intestinal barrier injury in colonic organoids. A: Immunofluorescence staining of ZO-1 and claudin-1. B: Microscopic images of colonic organoids. C: Quantitative analysis of organoid area. D-F: Western blotting for analysis of ZO-1 and claudin-1 protein expression levels (n=3). *P<0.05.

Fig.5 Effects of VE on inflammatory mediator levels in LPS-induced colonic organoids. A-C: ELISA detection of IL-6, IL-1β, and TNF-α protein levels. D-F: RT-qPCR analysis of IL-6, IL-1β, and TNF-α mRNA expression levels (n=3). *P<0.05.

Fig.6 Effects of VE on PI3K-AKT signaling pathway activation. A, B: Western blotting for analysis of p-PI3K/PI3K and p-AKT/AKT protein expressions in colon tissues of TNBS-treated mice (n=10). C, D: Western blotting for analysis of p-PI3K/PI3K and p-AKT/AKT protein expressions in LPS-induced colonic organoids (n=3). *P<0.05.

Fig.7 VE ameliorates colitis in mice by inhibiting the PI3K-AKT signaling pathway. A: DAI scores following 740 Y-P intervention. B: Histological inflammation scores. C: HE staining of the colon tissue. D-E: mRNA and protein expression levels of IL-6, IL-1β, and TNF-α in the colonic mucosa. n=10, *P<0.05.

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