Ethyl caffeate alleviates dextran sulfate sodium-induced colitis in mice by restoring intestinal epithelial tight junctions via activating the Wnt/β-Catenin pathway

doi:10.12122/j.issn.1673-4254.2026.09.18

Abstract

Abstract:

Objective To observe the effect of ethyl caffeate (EC) on intestinal barrier function in mice with dextran sulfate sodium (DSS)-induced murine colitis and explore the possible mechanism. Methods Forty male C57BL/6 mice were randomized into control group, DSS model group, and 3 EC groups treated with EC at 5, 10, or 20 mg/kg. Mouse models of colitis were established bytreatment with 2.5% DSS for 7 days, and EC treatment was administered daily by gavage. Colitis phenotypes were assessed by changes in body weight, disease activity index (DAI), colon length and histology. Immunofluorescence staining and PAS staining were used to examine the changes in expressions of the tight junction proteins ZO-1 and claudin-1 and goblet cells, respectively. The intestinal levels of inflammatory factors were detected by ELISA, RT-qPCR and immunohistochemistry. Network pharmacology combined with Western blotting and Wnt inhibitor (IWR-1) was used to verify the mechanism of EC for ameliorating colitis. Results Treatment with EC at 10 mg/kg significantly alleviated DSS-induced colitis in the mouse models, reduced intestinal pro-inflammatory cytokines and MPO levels, increased goblet cell counts, and restored ZO-1 and claudin-1 expressions and their membrane localization. Network pharmacology identified Wnt/β‑catenin and tight junction pathways as the core targets of EC, which activated the Wnt/β‑catenin pathway by upregulating WNT3A/β‑catenin, and this effect was blocked by treatment with IWR-1. Conclusion EC ameliorates DSS-induced colitis in mice by activating Wnt/β‑catenin signaling to restore tight junction protein expressions and localization, thereby improving intestinal barrier function, suggesting the value of EC as a novel candidate for treatment of inflammatory bowel disease.

Key words: Ethyl Caffeate, inflammatory bowel disease, Wnt/β-Catenin pathway, intestinal epithelial tight junctions, intestinal mucosal barrier, goblet cells

Keni ZHANG, Longtao ZHANG, Yu ZHANG, Ju HUANG, Qingqing LI, Zhijun GENG, Jianguo HU, Jing LI. Ethyl caffeate alleviates dextran sulfate sodium-induced colitis in mice by restoring intestinal epithelial tight junctions via activating the Wnt/β-Catenin pathway[J]. Journal of Southern Medical University, 2026, 46(9): 2196-2205.

Figures/Tables 10

Tab.1 Primer sequences for RT-qPCR

Gene name	Gene ID	Primer sequences (5'-3')
IL-6	16193	F:TCTATACCACTTCACAAGTCGGA
		R:GAATTGCCATTGCACAACTCTTT
TNF-α	21926	F:CAGGCGGTGCCTATGTCTC
		R:CGATCACCCCGAAGTTCAGTAG
ZO-1	21872	F: TTCACAGAGCAGCGACAACT
		R: TCTGCTGGAGTAGGCTACGA
Claudin-1	100771737	F: AAAGCACCGGGCAGATACAA
		R: TCATGCCAATGGTGGACACA
GAPDH	14433	F:TGGCCTTCCGTGTTCCTAC
		R:GAGTTGCTGTTGAAGTCGCA

Fig.1 Ethyl caffeate (EC) alleviates dextran sulfate sodium (DSS)-induced colitis symptoms in mice. A: Body weight alterations in mice from the Control, DSS model, EC-5 mg/kg, EC-10 mg/kg, and EC-20 mg/kg groups. B: Disease activity index (DAI) scores of the 5 groups. C: Representative colonic images of the mice. D: Mouse colon length measurements (cm). *P<0.05 vs Control; #P<0.05 vs DSS.

Fig.2 EC attenuates colonic pathology and inflammation in mice with DSS-induced colitis. A: Histological analysis and inflammation scoring of mouse colons using HE staining. B: MPO immunohistochemistry and the average optical density (AOD) in the 3 groups. C: TNF‑α and IL‑6 protein concentrations in colonic mucosa quantified using ELISA. D: Relative mRNA expression of TNF‑α and IL‑6 in colonic mucosa measured by RT‑qPCR. *P<0.05 vs Control; #P<0.05 vs DSS.

Fig.3 EC improves DSS-induced intestinal barrier dysfunction. A: AB-PAS staining of mouse colon and goblet cell counts. B: Claudin-1 expression (immunohistochemical staining) and the average optical density (AOD). *P<0.05 vs Control; #P<0.05 vs DSS.

Fig.4 EC upregulates expression of intestinal mucosal tight junction proteins. A: Western blotting of ZO-1 and claudin-1 expression in colonic mucosal tissues. B: Relative mRNA expression levels of ZO-1 and claudin-1 in colonic mucosa determined by RT-qPCR. *P<0.05 vs Control; #P<0.05 vs DSS.

Fig.5 Network pharmacology of EC. A: Venn map. B: PPI network C: KEGG pathway enrichment. D: GO functional annotation (BP, CC, MF).

Fig.6 EC exhibits no obvious cytotoxicity in Caco-2 cells. *P<0.05 vs EC=0 μmol/L.

Fig.7 EC improves DSS-induced barrier structure disorder in Caco-2 cells. A: Relative mRNA expression levels of ZO-1 and claudin-1 in Caco-2 cells detected by RT-qPCR. B: Western blotting of ZO-1 and claudin-1 protein expressions in Caco-2 cells. C: Immunofluorescence staining of ZO-1 (red) and claudin-1 (green) localization and fluorescence intensity in Caco-2 cells. *P<0.05 vs Control; #P<0.05 vs DSS.

Fig.8 EC activates the Wnt/β-catenin signaling pathway in DSS-induced Caco-2 cells. *P<0.05 vs Control; #P<0.05 vs DSS.

Fig.9 EC depend on the Wnt/β-catenin pathway to promote repair of intestinal epithelial barrier function. *P<0.05 vs DSS+EC.

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