Hypaphorine alleviates Crohn's disease-like colitis in mice by inhibiting intestinal epithelial inflammatory response and protecting intestinal barrier function

doi:10.12122/j.issn.1673-4254.2025.11.18

Abstract

Abstract:

Objective To investigate the effect of hypaphorine (HYP) on Crohn's disease (CD)‑like colitis in mice and its molecular mechanism. Methods Thirty male C57BL/6J mice were equally randomized into WT, TNBS, and HYP groups, and in the latter two groups, mouse models of CD-like colitis were established using TNBS with daily gavage of 15 mg/kg HYP or an equivalent volume of saline. The treatment efficacy was evaluated by assessing the disease activity index (DAI), body weight changes, colon length and histopathology. The effect of HYP was also tested in a LPS-stimulated Caco-2 cell model mimicking intestinal inflammation by evaluating inflammatory responses and barrier function of the cells using qRT-PCR and immunofluorescence staining. GO and KEGG analyses were conducted to explore the therapeutic mechanism of HYP, which was validated in both the cell and mouse models using Western blotting. Results In the mouse models of CD-like colitis, HYP intervention obviously alleviated colitis as shown by significantly reduced body weight loss, colon shortening, DAI and inflammation scores, and expressions of pro-inflammatory factors in the colon tissues. HYP treatment also significantly increased the TEER values, reduced bacterial translocation to the mesenteric lymph nodes, liver, and spleen, lowered serum levels of I-FABP and FITC-dextran, increased the number of colonic tissue cup cells, and upregulated colonic expressions of MUC2 and tight junction proteins (claudin-1 and ZO-1) in the mouse models. In LPS-stimulated Caco-2 cells, HYP treatment significantly inhibited the expressions of pro-inflammatory factors and increased the expressions of tight junction proteins. Western blotting showed that HYP downregulated the expressions of the key proteins in the TLR4/MyD88 signaling pathway in both the in vitro and in vivo models. Conclusion HYP alleviates CD-like colitis in mice possibly by suppressing intestinal epithelial inflammation and improving gut barrier function.

Key words: Crohn's disease, colitis, hypaphorine, intestinal barrier, intestinal epithelial cells

Qingqing HUANG, Jingjing YANG, Xuening JIANG, Wenjing ZHANG, Yu WANG, Lugen ZUO, Lian WANG, Yueyue WANG, Xiaofeng ZHANG, Xue SONG, Jianguo HU. Hypaphorine alleviates Crohn's disease-like colitis in mice by inhibiting intestinal epithelial inflammatory response and protecting intestinal barrier function[J]. Journal of Southern Medical University, 2025, 45(11): 2456-2465.

Figures/Tables 8

Tab.1 Primer sequences for qRT-PCR in this study

Gene name	Primer sequences
TNF-α	Forward:5'-CAGGCGGTGCCTATGTCTC-3'
TNF-α	Reverse:5'-CGATCACCCCGAAGTTCAGTAG-3'
IL-6	Forward:5'-TCTATACCACTTCACAAGTCGGA-3'
IL-6	Reverse:5'-GAATTGCCATTGCACAACTCTTT-3'
IL-1β	Forward:5'-GAAATGCCACCTTTTGACAGTG-3'
IL-1β	Reverse:5'-TGGATGCTCTCATCAGGACAG-3'
GAPDH	Forward:5'-AGGTCGGTGTGAACGGATTTG-3'
GAPDH	Reverse:5'-GGGGTCGTTGATGGCAACA-3'

Fig.1 HYP treatment alleviates CD-like colitis in TNBS-treated mice. A: Body weight changes. B: DAI score. C: Gross observation of the dissected colon from each groups. D: Colon length in each group. E: HE staining of colonic sections of the mice from each group (scale bar=1 mm or 100 μm). F: Colonic inflammation scores in each group. *P<0.05 vs WT, #P<0.05 vs TNBS.

Fig.2 HYP treatment reduces the levels of inflammatory mediators in the colonic mucosa of TNBS-treated mice. A-C: Expression levels of TNF-α, IL-6 and IL-1β mRNAs in mouse colonic mucosal tissue. D-F: Expression levels of TNF-α, IL-6 and IL-1β proteins in mouse colonic mucosal tissue. *P<0.05 vs WT, #P<0.05 vs TNBS.

Fig.3 HYP ameliorates intestinal barrier damage in TNBS-treated mice. A: TEER values of mouse colon tissues in each group. B-D: Bacterial translocation rate in the mesenteric lymph nodes, liver, and spleen. E, F: Serum levels of I-FABP and FD4 in each group. G: AB-PAS and immunohistochemical staining of colonic sections (scale bar=100 μm). H, I: Number of goblet cells per crypt and MUC2 expression level in mouse colon tissue in the 3 groups. J, L: Immunofluorescence staining of colonic sections from each group (scale bar=100 μm). K, M: Expression levels of claudin-1 and ZO-1 in mouse colon tissues in each group. *P<0.05 vs WT, #P<0.05 vs TNBS.

Fig.4 HYP treatment reduces inflammatory mediator levels in LPS-induced Caco-2 cells. A-C: Expression levels of TNF-α, IL-6 and IL-1β mRNAs in Caco-2 cells with different treatments. D-F: Protein levels of TNF-α, IL-6 and IL-1β in Caco-2 cells with different treatments. *P<0.05 vs Control, #P<0.05 vs LPS.

Fig.5 HYP treatment ameliorates LPS-induced barrier damage in Caco-2 cells. A: TEER value of Caco-2 cells. B: Immunofluorescence staining of Caco-2 cells with different treatments (scale bar=100 μm). C-E: Expression levels of claudin-1 and ZO-1 in Caco-2 cells with different treatments. *P<0.05 vs Control, #P<0.05 vs LPS.

Fig.6 Therapeutic mechanism of HYP for CD may involve inflammatory response and TLR signaling pathway. A, B: GO and KEGG enrichment analyses.

Fig.7 HYP downregulates the TLR4/MyD88 signaling pathway in both the mouse and cell models. A-C: Protein levels of TLR4 and MyD88 in mouse colonic mucosa tissues. D-F: Protein levels of TLR4 and MyD88 in Caco-2 cells with different treatments. *P<0.05 vs WT or Control, #P<0.05 vs TNBS or LPS.

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