Cimifugin ameliorates Crohn's disease-like colitis in mice by modulating Th-cell immune balance via inhibiting the MAPK pathway

doi:10.12122/j.issn.1673-4254.2025.03.17

Abstract

Abstract:

Objective To investigate the therapeutic effects of cimifugin on Crohn's disease (CD)-like colitis in mice and its possible mechanism. Methods Thirty adult male C57BL/6 mice were randomized equally into control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis model group, and cimifugin treatment (daily gavage at 12.5 mg/kg) group. The therapeutic effect of cimifugin was evaluated by observing changes in body weight, disease activity index (DAI) scores, colon length, histopathological inflammation scores, and inflammatory cytokine levels in the colonic mucosa. Intestinal barrier integrity in the mice was assessed using immunofluorescence assay and Western blotting for claudin-1 and ZO-1; T-helper (Th) cell subset ratios in the mesenteric lymph nodes were analyzed with flow cytometry. Network pharmacology, KEGG enrichment analysis and molecular docking were used to predict the targets of cimifugin and analyze the key pathways and cimifugin-MAPK protein interactions, which were validated by Western blotting in the mouse models. Results In mice with TNBS-induced colitis, cimifugin treatment significantly attenuated body weight loss and colon shortening, lowered DAI and histopathological scores, decreased IFN-γ and IL-17 levels, and increased IL-4 and IL-10 levels in the colonic mucosa. Cimifugin treatment also significantly improved TNBS-induced claudin-1 dislocation and reduction of goblet cells, upregulated claudin-1 and ZO-1 expressions, reduced Th1 and Th17 cell percentages, and increased Th2 and Treg cell percentages in the colonic mucosa of the mice. KEGG analysis suggested a possible connection between the effect of cimifugin and MAPK signaling, and molecular docking showed strong binding affinity between cimifugin and MAPK core proteins. Western blotting demonstrated significantly decreased phosphorylation levels of JNK, ERK, and p38 in the colonic mucosa of cimifugin-treated mouse models. Conclusion Cimifugin alleviates TNBS-induced CD-like colitis by repairing intestinal barrier damage and restoring Th1/Th2 and Th17/Treg balance via suppressing MAPK pathway activation.

Key words: Crohn's disease, cimifugin, MAPK, helper T cells, immune homeostasis, intestinal barrier

Lixia YIN, Minzhu NIU, Keni ZHANG, Zhijun GENG, Jianguo HU, Jiangyan LI, Jing LI. Cimifugin ameliorates Crohn's disease-like colitis in mice by modulating Th-cell immune balance via inhibiting the MAPK pathway[J]. Journal of Southern Medical University, 2025, 45(3): 595-602.

Figures/Tables 10

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequences (5'-3')
IL-17A	F:GGCCCTCAGACTACCTCAAC
IL-17A	R:TCTCGACCCTGAAAGTGAAGG
IL-4	F:CCCCAGCTAGTTGTCATCCTG
IL-4	R:CAAGTGATTTTTGTCGCATCCG
IL-10	F:GCTGGACAACATACTGCTAACC
IL-10	R:ATTTCCGATAAGGCTTGGCAA
IFN-γ	F:ACAGCAAGGCGAAAAAGGATG
IFN-γ	R:TGGTGGACCACTCGGATGA
GAPDH	F:TGACCTCAACTACATGGTCTACA
GAPDH	R:CTTCCCATTCTCGGCCTTG

Fig.1 Effect of cimifugin (CIM) on symptoms of TNBS-induced colitis in mice. A: Daily body weight changes of the mice in each group. B: Changes in DAI scores of the mice in each group. C: Gross observation of the mouse colon in each group. D: Comparison of colonic lengths of the mice among the 3 groups. *P<0.05 vs WT; #P<0.05 vs TNBS. WT: Type; TNBS: 2,4,6-trinitrobenzenesulfonic acid.

Fig.2 Effect of CIM on intestinal tissue damage in mice with TNBS-induced colitis. A: HE staining of mouse colon tissues. B: Inflammation scores of mouse colon tissue in each group. *P<0.05 vs WT; #P<0.05 vs TNBS.

Fig.3 Effect of CIM on intestinal mucosa inflammation of the mice in each group. A: Levels of inflammatory factors in the colonic mucosa detected by ELISA. B: mRNA expressions of inflammatory factors in the colonic mucosa detected by PCR. *P<0.05 vs WT; #P<0.05 vs TNBS.

Fig.4 Effect of CIM on intestinal barrier damage in mice with TNBS-induced colitis. A: Immunofluorescence staining of claudin-1 in the colon of the mice in each group. B: AB-PAS staining of mouse colon. C: Numbers of goblet cells based on AB-PAS staining. D-F: Western blotting for detecting expression levels of ZO-1 and claudin-1 in colonic mucosal tissue. *P<0.05 vs WT; #P<0.05 vs TNBS.

Fig.5 Effect of CIM on balance of Th1/Th2 cell response in mice with TNBS-induced colitis. A, B: Assessment of Th1 cell percentage in mouse mesenteric lymph nodes by flow cytometry. C, D: Assessment of Th2 cell percentage in mouse mesenteric lymph nodes by flow cytometry. *P<0.05 vs WT; #P<0.05 vs TNBS.

Fig.6 Effect of CIM on balance of Th17/Treg cell response in mice with TNBS-induced colitis. A, B: Assessment of Th17 cell percentage in mouse mesenteric lymph nodes by flow cytometry. C, D: Assessment of Treg cell percentage in mouse mesenteric lymph nodes by flow cytometry. *P<0.05 vs WT; #P<0.05 vs TNBS.

Fig.7 CIM network pharmacology and molecular docking. A:Venn diagram. B:PPI network diagram. C:KEGG analysis. D:GO analysis(CC, BP, MF).

Tab.2 Molecular docking results

Compound	Target	PDB	Energy (kcal/mol)
CIM	MAP3K14	8YHW	-8.4
CIM	MAP2K1	3EQI	-8.3
CIM	MAPK14	6SFO	-8.2
CIM	MAPK1	6SLG	-7.6
CIM	IKBKB	4KIK	-7.5
CIM	BRAF	5VYK	-6.7
CIM	CASP3	1RE1	-6.5
CIM	JUN	6Y3V	-5.7

Fig.8 Verification of the role of the MAPK signaling pathway in mediating the therapeutic effect of CIM on TNBS-induced colitis in mice. A, B: Expressions of key proteins of the MAPK pathway (p-p38, p-ERK and p-JNK) detected in the intestinal mucosal tissues of the mice by Western blotting. *P<0.05 vs WT; #P<0.05 vs TNBS.

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