Anti-Inflammatory Effects of Spexin on Acetic Acid‑Induced Colitis in Rats via Modulating the NF-κB/NLRP3 Inflammasome Pathway

Abstract

Ulcerative colitis is a chronic inflammatory bowel disease characterized by inflammation and ulcers in the lining of the colon and rectum. Spexin is a novel peptide with antioxidant and anti-inflammatory properties. This study aims to elucidate the therapeutic effects and underlying mechanisms of spexin in mitigating acetic acid-induced colitis in rats. Male Sprague Dawley rats were assigned to control (n = 14) and colitis (n = 21) groups. Colitis was induced via 5% acetic acid (AA) administration (1 mL, intrarect). Post-induction, rats received subcutaneous saline (1 mL/kg), spexin (50 µg/kg/day), or oral sulfasalazine (500 mg/kg) for 5 days. Control groups received saline or spexin. After 24 h of the final treatment, colons were evaluated macroscopically, and levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-18 were determined by ELISA, oxidative stress markers myeloperoxidase (MPO), malondialdehyde (MDA) and glutathione (GSH) levels were measured spectrophotometrically and NOD-like receptor pyrin domain-containing 3 (NLRP3), nuclear factor-κB (NF-κB), caspase-1 proteins were analyzed with Western Blot alongside histopathological assessments. Colitis induction significantly elevated macroscopic damage scores, stool consistency, inflammatory cytokines, MDA, MPO, and NLRP3, NF-κB, caspase-1, while reducing GSH levels (p < 0.001-0.01). Microscopic evaluations confirmed increased necrosis, submucosal edema, and inflammatory cell infiltration (p < 0.001). Spexin reversed these effects by enhancing GSH levels (p < 0.01), reducing macroscopic/microscopic scores, cytokines, MDA, and MPO levels (p < 0.05-0.001), and suppressing NLRP3, NF-κB, and caspase-1 activation (p < 0.01-0.001). For the first time that spexin ameluates acetic acid-induced colitis in rats by modulating the NF-κB/NLRP3 signaling pathway, reducing oxidative damage, enhancing antioxidant capacity, and suppressing inflammation.

Keywords: NLRP3; inflammation; oxidative damage; spexin; ulcerative colitis.

FIGURE 1

(A) Malondialdehyde (MDA), (B) Glutathione (GSH) and (C) Myeloperoxidase (MPO) activity levels in colon tissues of experimental groups. Results represent the mean ± S.E.M. Each group consists of seven rats. **p < 0.01, ***p < 0.001, * compared to saline‐treated control group; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺ compared to saline‐treated colitis group.

FIGURE 2

The colonic TNF‐α levels, and IL‐18 and IL‐1β levels in serum and colon samples of experimental groups. Results represent the mean ± S.E.M. Each group consists of seven rats. *p < 0.05, **p < 0.01, ***p < 0.001, * compared to saline‐treated control group; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺ compared to saline‐treated colitis group.

FIGURE 3

Effect of Spexin on NLRP3, caspase‐1 and NF‐κB p65 protein levels in acetic acid‐induced ulcerative colitis. (A) Representative Western blot bands of protein levels. (B–D) NLRP3, caspase‐1 and NF‐κB protein expression levels in the colon samples, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, * compared to saline‐treated control group; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺ compared to saline‐treated colitis group.

FIGURE 4

Representative colon microscopic images of experimental groups. Black arrow: Damage/necrosis; black arrowhead: Submucosal edema; black star: inflammatory cell infiltration; red arrowhead: vasculitis, Hematoxylin and eosin, Magnification: 20×; Scale bar: 200 μm.

FIGURE 5

Histopathological scoring results of experimental groups. (A) Damage/necrosis score, (B) Submucosal edema score, (C) Inflammatory cell infiltration, (D) Vasculitis score. Results represent the mean ± S.E.M. Each group consists of seven rats. *p < 0.05, ***p < 0.001, * compared to saline‐treated control group; ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺ compared to saline‐treated colitis group.