Resveratrol Mitigates Inflammation by Modulating Tumor Necrosis Factor-Alpha Receptors (TNFRs) in a 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-Induced Rat Model of Colitis

Abstract

Several substances with antioxidant and anti-inflammatory properties are currently being investigated as potential adjunctive or standalone treatments for inflammatory bowel disease (IBD). One such substance is resveratrol (RES), also known as 3,5,4'-trihydroxy-trans-stilbene, a natural dietary polyphenol with diverse health-promoting effects. In this study, male Wistar-Hannover rats received oral RES supplementation at doses of 5, 10, or 20 mg/kg/day for 28 days. On day 25 colitis was induced using intracolonic administration of 2,4,6-trinitrobenzene sulphonic acid (TNBS). Based on histological and planimetric analysis, the 10 mg/kg dose significantly reduced colonic ulceration and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) expression compared to the TNBS group. Immunohistochemistry also revealed that RES at this dose attenuated the intensity of TNF-α receptors, namely TNFR1 and TNFR2. Furthermore, the concentration of lipocalin-2 (Lcn-2) was significantly elevated in TNBS-induced colitis. In conclusion, our findings suggest that RES may exert its protective effects partly through the modulation of TNF receptor signaling in TNBS-induced colitis.

Keywords: IBD; TNBS-induced colitis; TNF-alpha; TNFR-α receptor; colon inflammation; oxidative stress; pro-inflammatory cytokine; resveratrol.

Figure 1

(a) Effect of resveratrol (RES) on the severity of inflammation and (b) colon weight in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Representative images of the colonic damage: (c) CTRL (no treatment); (d) 50% ethanol (EtOH enema); (e) TNBS enema; (f) TNBS + RES 5 mg/kg; (g) TNBS + RES 10 mg/kg; (h) TNBS + RES 20 mg/kg; (i) TNBS + sulfasalazine (SASP). Data represented by mean ± S.E.M.; n = 4–12/group; statistical significance: * p < 0.05 TNBS vs. TNBS + treatment; # p < 0.05 and #### p < 0.001 CTRL vs. TNBS; and & p < 0.05 CTRL vs. EtOH.

Figure 2

Histopathological changes in rat colon in 2,4,6-trinitrobenzene sulfonic acid (TNBS) model of colitis. Representative images of rat colon after haematoxylin and eosin staining: (a,b) CTRL (no treatment); (c,d) TNBS enema; (e,f) TNBS enema + resveratrol (RES) 5 mg/kg; (g,h) TNBS + RES 10 mg/kg; (i,j) TNBS + RES 20 mg/kg; (k,l) TNBS + sulfasalazine. Arrow: hemorrhage sm: submucosa, mm: muscularis mucosae, lp: lamina propria, el: epithelial layer. Scales: 50 μm (a,c,e,g,i,k), 20 μm (b,d,f,h,j,l).

Figure 3

The effects of resveratrol (RES) supplementation on colonic tumor necrosis factor-alpha concentration (TNF-α, expressed as pg/mg protein) in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. CTRL (no treatment); EtOH (50% ethanol, the solvent of TNBS); TNBS enema; TNBS + RES 5 mg/kg; TNBS + RES 10 mg/kg; TNBS + RES 20 mg/kg; TNBS + sulfasalazine (SASP). Results are presented as mean ± S.E.M.; n = 4-6/group; statistical significance: * p < 0.05 TNBS vs. TNBS + treatment; # p < 0.05 CTRL vs. TNBS.

Figure 4

Representative fluorescent micrographs of paraffin sections from (a) control, (b) 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated, and (c) TNBS + resveratrol (RES) 10 mg/kg rats showing different intestinal layers after TNF-α immunohistochemistry. Nuclei were counterstained with DAPI (blue). muc—mucosa, smuc—submucosa, scale bars: 50 µm.

Figure 5

Representative fluorescent micrographs of paraffin sections from control (a), 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated (b), and TNBS + resveratrol (RES) 10 mg/kg (c) rats showing different intestinal layers after TNFR1 immunohistochemistry. Nuclei were counterstained with DAPI (blue). muc—mucosa, smuc—submucosa, cm—circular smooth muscle, lm—longitudinal smooth muscle, scale bars: 100 µm.

Figure 6

Representative fluorescent micrographs of paraffin sections from control (a), 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated (b), and TNBS + resveratrol (RES) 10 mg/kg (c) rats showing different intestinal layers after TNFR1 immunohistochemistry. Nuclei were counterstained with DAPI (blue). cm—circular smooth muscle, lm—longitudinal smooth muscle, arrows—myenteric ganglia, scale bars: 50 µm.

Figure 7

Representative fluorescent micrographs of paraffin sections from control (a), 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated (b), and TNBS + resveratrol (RES) 10 mg/kg (c) rats showing different intestinal layers after TNFR2 immunohistochemistry. Nuclei were counterstained with DAPI (blue). muc—mucosa, smuc—submucosa, cm—circular smooth muscle, lm—longitudinal smooth muscle, arrows—myenteric ganglia, scale bars: 100 µm.

Figure 8

Representative fluorescent micrographs of paraffin sections from control (a), 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated (b), and TNBS + resveratrol (RES) 10 mg/kg (c) rats showing different intestinal layers after TNFR2 immunohistochemistry. Nuclei were counterstained with DAPI (blue). cm—circular smooth muscle, lm—longitudinal smooth muscle, arrows—myenteric ganglia, scale bars: 50 µm.

Figure 9

The effects of resveratrol (RES) on colonic lipocalin-2 concentration (Lcn-2, expressed as ng/mg protein) in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. CTRL (no treatment); EtOH (50% ethanol, the solvent of TNBS); TNBS enema; TNBS + RES 5 mg/kg; TNBS + RES 10 mg/kg; TNBS + RES 20 mg/kg; TNBS + sulfasalazine (SASP). Results are presented as mean ± S.E.M.; n = 4–8/group; statistical significance: # p < 0.05 CTRL vs. TNBS.

Figure 10

The experimental protocol of the study.