p-Cymene and Rosmarinic Acid Ameliorate TNBS-Induced Intestinal Inflammation Upkeeping ZO-1 and MUC-2: Role of Antioxidant System and Immunomodulation

Abstract

p-Cymene (p-C) and rosmarinic acid (RA) are secondary metabolites that are present in medicinal herbs and Mediterranean spices that have promising anti-inflammatory properties. This study aimed to evaluate their intestinal anti-inflammatory activity in the trinitrobenzene sulphonic acid (TNBS)-induced colitis model in rats. p-C and RA (25-200 mg/kg) oral administration reduced the macroscopic lesion score, ulcerative area, intestinal weight/length ratio, and diarrheal index in TNBS-treated animals. Both compounds (200 mg/kg) decreased malondialdehyde (MDA) and myeloperoxidase (MPO), restored glutathione (GSH) levels, and enhanced fluorescence intensity of superoxide dismutase (SOD). They also decreased interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and maintained IL-10 basal levels. Furthermore, they modulated T cell populations (cluster of differentiation (CD)4⁺, CD8⁺, or CD3⁺CD4⁺CD25⁺) analyzed from the spleen, mesenteric lymph nodes, and colon samples, and also decreased cyclooxigenase 2 (COX-2), interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), and nuclear transcription factor kappa B subunit p65 (NFκB-p65) mRNA transcription, but only p-C interfered in the suppressor of cytokine signaling 3 (SOCS3) expression in inflamed colons. An increase in gene expression and positive cells immunostained for mucin type 2 (MUC-2) and zonula occludens 1 (ZO-1) was observed. Altogether, these results indicate intestinal anti-inflammatory activity of p-C and RA involving the cytoprotection of the intestinal barrier, maintaining the mucus layer, and preserving communicating junctions, as well as through modulation of the antioxidant and immunomodulatory systems.

Keywords: antioxidant; cytoprotection; immunomodulation; intestinal inflammation; p-cymene; rosmarinic acid.

Figure 1

Representative images of rat colons from non-colitic group (A); colitic groups (5% Tween 80 or 0.9% NaCl treated groups) (B); prednisolone 2 mg/kg (C); p-C or RA (RA) 25 mg/kg (D), 50 mg/kg (E), 100 mg/kg (F), and 200 mg/kg (G) subjected to TNBS-induced ulcerative colitis model. The figure also shows representative hematoxylin–eosin (HE)-stained photomicrographs (n = 3 per group) from (H) non-colitic; (I) colitic; (J) prednisolone 2 mg/kg; (K) p-C 200 mg/kg; and (L) RA 200 mg/kg. 40× magnification. Scale: 1500 µm.

Figure 2

Effect of oral administration of p-C and RA on glutathione (GSH) (non-protein sulfhydryl groups (NPSH)), malondialdehyde (MDA), and myeloperoxidase (MPO) levels in colonic tissue from TNBS-induced ulcerative colitis in rats. Data are expressed as mean ± standard error, having been analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s test. *** p < 0.001, compared to 5% Tween 80 or 0.9% NaCl control groups (n = 5–6).

Figure 3

Representative photomicrographs of superoxide dismutase (SOD) with immunoreactivity marked in green (white arrows) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstained in blue from rats submitted to TNBS-induced ulcerative colitis. (A) Non-colitic group; (B) colitic group; (C) prednisolone (2 mg/kg); (D) p-C (200 mg/kg); (E) RA (200 mg/kg). Scale: 100 μm. Magnification: 400×. Data are expressed as mean ± standard error, having been analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s test. *** p <0.001, compared to colitic control group (n = 5–9 fields/3 per group). MFI: median fluorescence intensity.

Figure 4

Effect of oral administration of p-C and RA on interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-10 levels in colonic tissue from TNBS-induced ulcerative colitis in rats. Data are expressed as mean ± standard error, having been analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s test. *** p < 0.001, compared to 5% Tween 80 or 0.9% NaCl control groups (n = 5–6).

Figure 5

Effect of oral administration of p-C and RA in mucin type 2 (MUC-2) and zonula occludens 1 (ZO-1) expression in a TNBS-induced ulcerative colitis rat model. Data are expressed as mean of cell percentages ± standard error, having been analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s test. *** p < 0.001, ** p < 0.01 compared to colitic control groups (n = 5–6).

Figure 6

Photomicrographs with immunohistochemical staining (→) for MUC-2 (I) and ZO-1 (II) in colon samples from rats submitted to TNBS-induced ulcerative colitis. (A) Non-colitic; (B) colitic (5% Tween 80); (C) colitic (0.9% NaCl); (D) prednisolone (2 mg/kg); (E) p-C (200 mg/kg); (F) RA (200 mg/kg). Scale: 50 μm. 400×. MUC: mucin; ZO: zonula occludens.

Figure 7

Effect of oral administration of p-C and RA in the modulation of T cell populations in a TNBS-induced ulcerative colitis rat model. (A) Representative dot plots from flow cytometry analysis (FACS) analysis of T lymphocytes gated for cluster of differentiation (CD)4⁺, CD8⁺, and CD3⁺CD4⁺CD25⁺ cells; (B) T cell percentages (%) in the assessed samples (colon, spleen, and mesenteric lymph nodes (MLN)). Data are expressed as mean ± standard error percentage of the total number of cells, having been analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s test. *** p < 0.001, ** p < 0.01, * p < 0.05 compared to colitic control groups (n = 5–6).

Figure 8

Effect of oral administration of p-C and RA in cyclooxigenase 2 (COX-2), interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), suppressor of cytokine signaling 3 (SOCS3), and nuclear transcription factor kappa B subunit p65 (NFκB-p65) expression on TNBS-induced ulcerative colitis rat model. Data are expressed as mean of cell percentages ± standard error, having been analyzed by one-way ANOVA followed by Dunnett’s or Tukey’s test. *** p < 0.001, ** p < 0.01, * p < 0.05 compared to colitic control groups (n = 5–6).