Isovanillic acid alleviates dextran sulfate sodium-induced ulcerative colitis in mice by improving mitochondrial function via activating the PPARγ pathway

doi:10.12122/j.issn.1673-4254.2026.07.03

Abstract

Abstract:

Objective To investigate the protective effect of isovanillic acid (IVA) against dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice and its underlying mechanism. Methods Forty-eight male C57BL/6 mice were randomly divided into 6 groups (n=8), including a control group and 5 DSS model groups with daily gavage of saline containing 0.1% DMSO, low-, medium- or high-dose IVA (50, 100, and 150 mg/kg, respectively), or 5-ASA (100 mg/kg) for 10 days. Body weight and disease activity index (DAI) of the mice were monitored, and colon length and pathologies were assessed after the treatments. Immunofluorescence staining, Western blotting, TUNEL staining, and JC-1 staining were used to evaluate the effects of IVA on barrier function, apoptosis, and mitochondrial function in the mouse models and DSS-induced NCM460 cells. Network pharmacology was employed to predict potential signaling pathways. Results The DSS-treated mice showed significantly decreased body weight, increased DAI score, shortened colon length, elevated colonic IL-6 and IL-1β expressions, and severe mucosal damage. IVA, especially at the medium and high doses, obviously improved these changes. Treatment with medium-dose IVA-M significantly increased colonic expressions of ZO-1 and claudin-1, decreased intestinal epithelial cell apoptosis rate and expressions of Bax and cleaved caspase-3, and increased Bcl-2 expression, TOMM20-positive cell counts, and activities of mitochondrial respiratory chain complexes I and IV. In NCM460 cells, IVA treatment obviously reversed DSS-induced mitochondrial impairment, reduced epithelial cell apoptosis, and enhanced expressions of ZO-1 and claudin-1. Network pharmacology analysis suggested that IVA potentially targeted the PPARγ pathway, which was confirmed by increased PPARγ protein expression in IVA-treated mice and NCM460 cells. Treatment with the PPARγ antagonist GW9662 significantly attenuated the protective effect of IVA in DSS-induced NCM460 cells. Conclusion IVA alleviates DSS-induced colitis in mice by protecting mitochondrial function via activating the PPARγ pathway and suppressing inflammation and apoptosis.

Key words: isovanillic acid, ulcerative colitis, mitochondrial functions, PPARγ pathway, intestinal barrier, cell apoptosis

Tong QIAO, Longtao ZHANG, Yu ZHANG, Ju HUANG, Qingqing LI, Zhijun GENG, Jianguo HU, Jing Li. Isovanillic acid alleviates dextran sulfate sodium-induced ulcerative colitis in mice by improving mitochondrial function via activating the PPARγ pathway[J]. Journal of Southern Medical University, 2026, 46(7): 1487-1497.

Figures/Tables 9

Fig.1 Dose-dependent effects of IVA on DSS-induced colitis in mice. A: Body weight changes during the experiment. B: Disease activity index (DAI) scores. C: Representative images of mouse colons. D: Quantitative analysis of colon length. E, F: Concentrations of IL-6 (E) and IL-1β (F) in colonic mucosa measured by ELISA. G: Histopathological scores. H: HE staining of mouse colon sections (Original magnification: ×200). I: AB-PAS staining of mouse colon sections (×200). DSS: DSS model group; IVA-L: DSS+50 mg/kg IVA; IVA-M: DSS+100 mg/kg IVA; IVA-H: DSS+150 mg/kg IVA; 5-ASA: DSS+100 mg/kg 5-aminosalicylic acid. Data are presented as Mean±SD (n=8). *P<0.05 vs Con group; #P<0.05 vs DSS group.

Fig.2 IVA alleviates intestinal barrier injury in mice. A: Immunofluorescence staining of ZO-1 and claudin-1 in mouse colon tissues (×200). B, C: Quantitative analysis of ZO-1 and claudin-1 expression levels in the intestinal mucosa by Western blotting. Data are presented as Mean±SD (n=8). *P<0.05 vs Con group. #P<0.05 vs DSS group.

Fig.3 IVA ameliorates DSS-induced apoptosis of intestinal epithelial cells in mice. A: TUNEL staining of mouse colon tissues (×200). B: Quantitative analysis of intestinal epithelial cell apoptosis rate. C, D: Western blotting of Bcl-2, Bax, and cleaved caspase-3 (C-caspase3) expression levels in mouse colonic mucosa. Data are presented as Mean±SD (n=8). *P<0.05 vs Con group. #P<0.05 vs DSS group.

Fig.4 IVA alleviates DSS-induced intestinal mitochondrial dysfunction in mice. A: Immunofluorescence staining for detecting TOMM20-positive cells (×200). B: Complex I activity. C: Complex IV activity. *P<0.05 vs Con group. Data are presented as Mean±SD (n=8). #P<0.05 vs DSS group.

Fig.5 IVA protects mitochondrial function in NCM460 cells with DSS-induced stress. A: Immunofluorescence staining for JC-1 (×400). B: Complex I activity. C: Complex IV activity. Data are presented as Mean±SD (n=3). *P<0.05 vs C-Con group. #P<0.05 vs C-DSS group.

Fig. 6 Effect of IVA on DSS-induced apoptosis of NCM460 cells. A: TUNEL staining of NCM460 cells (×200). B: Quantitative analysis of apoptosis rate. C: Western blotting of Bcl-2, Bax, and C-caspase3 expression. D: Relative protein expression levels of Bcl-2, Bax, and C-caspase3. Data are presented as Mean±SD (n=3). *P<0.05 vs C-Con group. #P<0.05 vs C-DSS group.

Fig.7 IVA alleviates DSS-induced barrier damage in NCM460 cells. A: Immunofluorescence staining of ZO-1 and claudin-1 in NCM460 cells (×200). B, C: Western blotting for detecting relative protein expression levels. Data are presented as Mean±SD (n=3). *P<0.05 vs C-Con group. #P<0.05 vs C-DSS group.

Fig.8 IVA enhances mitochondrial respiratory function by regulating the PPARγ pathway. A: Venn diagram. B: PPI network diagram. C: GO-Biological process analysis. D: KEGG analysis. E: Western blotting of PPARγ expression in mouse intestinal epithelial cells. F: Relative protein expression levels of PPARγ in mouse colon tissues (n=8). G: Western blotting of PPARγ expression in NCM460 cells. H: Relative protein expression levels of PPARγ in NCM460 cells. Data are presented as Mean±SD (n=3). *P<0.05 vs Con/C-Con group. #P<0.05 vs DSS/C-DSS group.

Fig.9 IVA improves mitochondrial function, apoptosis, and intestinal barrier injury via PPARγ. A: JC-1 staining showing mitochondrial membrane potential in NCM460 cells (×400). B, C: Activities of mitochondrial respiratory chain complexes I (B) and IV (C). D: TUNEL staining and quantification of apoptotic cells (×200). E, F: Western blotting of Bcl-2, Bax, and C-caspase3 expressions (E) and quantitative analysis of relative protein expression levels (F). G: Immunofluorescence staining of ZO-1 and claudin-1 in NCM460 cells (×200). H, I: Western blotting of ZO-1 and claudin-1 expressions (H) and quantitative analysis of relative protein expression levels (I). J: Schematic illustration of the proposed mechanism by which IVA ameliorates DSS-induced colitis. The cells were treated with 2% DSS in the presence or absence of IVA (20 μmol/L). C-IVA: DSS+IVA; C-IVA+GW9662: DSS+IVA+GW9662. Data are presented as Mean±SD (n=3). #P<0.05 vs C-IVA group.

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