Resveratrol (trans-3,5,4'-trihydroxystilbene) induces silent mating type information regulation-1 and down-regulates nuclear transcription factor-kappaB activation to abrogate dextran sulfate sodium-induced colitis

Abstract

Inflammatory bowel disease is a chronic, relapsing, and tissue-destructive disease. Resveratrol (3,4,5-trihydroxy-trans-stilbene), a naturally occurring polyphenol that exhibits beneficial pleiotropic health effects, is recognized as one of the most promising natural molecules in the prevention and treatment of chronic inflammatory disease and autoimmune disorders. In the present study, we investigated the effect of resveratrol on dextran sodium sulfate (DSS)-induced colitis in mice and found that it effectively attenuated overall clinical scores as well as various pathological markers of colitis. Resveratrol reversed the colitis-associated decrease in body weight and increased levels of serum amyloid A, tumor necrosis factor-alpha, interleukin (IL-6), and IL-1beta. After resveratrol treatment, the percentage of CD4(+) T cells in mesenteric lymph nodes (MLN) of colitis mice was restored to normal levels, and there was a decrease in these cells in the colon lamina propria (LP). Likewise, the percentages of macrophages in MLN and the LP of mice with colitis were decreased after resveratrol treatment. Resveratrol also suppressed cyclooxygenase-2 (COX-2) expression induced in DSS-exposed mice. Colitis was associated with a decrease in silent mating type information regulation-1 (SIRT1) gene expression and an increase in p-inhibitory kappaB expression and nuclear transcription factor-kappaB (NF-kappaB) activation. Resveratrol treatment of mice with colitis significantly reversed these changes. This study demonstrates for the first time that SIRT1 is involved in colitis, functioning as an inverse regulator of NF-kappaB activation and inflammation. Furthermore, our results indicate that resveratrol may protect against colitis through up-regulation of SIRT1 in immune cells in the colon.

Fig. 1.

Change in body weight of mice after DSS induction and resveratrol treatment. C57BL/6 mice were given no treatment (Δ, control), resveratrol (100 mg/kg b.wt.) alone suspended in 100 μl of distilled water by oral gavage (□, RES), DSS alone (3%) in drinking water (●, DSS+vehicle), or a combination of DSS and resveratrol (DSS+RES) at 10 (⧫), 50 (☒), and 100 (○) mg/kg b.wt. of resveratrol for 14 days. After 7 days, DSS was replaced with a water cycle (ad libitum) for another 7 days. The body weight of the mice was recorded daily. Changes in body weight were expressed as a percentage of the weight minus the change in weight divided by the weight before the onset of colitis at day 1 (current weight-weight of previous day/day 1 weight). The statistical significance between values of each group was assessed by Student's t test. Data represent the mean of three experiment involving six mice per group. Asterisks indicate statistically significance difference (P < 0.01) between DSS+vehicle and DSS+100 mg/kg RES-treated mice.

Fig. 2.

Effect of resveratrol on changes in SAA levels, colitis scores, and colon lengths in mice with DSS-induced colitis. In mice treated with DSS and resveratrol, as described in the legend for Fig. 1, the persistence or improvement of colitis was monitored by evaluating SAA levels (A), inflammation scores (B), and colon lengths (C). Asterisks indicate statistically significant differences (P < 0.01) between groups of six mice treated with DSS+vehicle and DSS+resveratrol.

Fig. 3.

Resveratrol-mediated reduction of serum levels of IL-1β, IL-6, TNF-α, and IFN-γ in DSS-induced colitis. Colitis was induced in mice that were exposed to vehicle or 100 mg/kg b.wt. of resveratrol as described in the legend to Fig. 1. Serum cytokines were measured 14 days after the DSS induction of colitis by ELISA assay. The data presented are the mean concentrations from six mice ± S.E.M. in serum. Asterisks indicate statistically significance difference (P < 0.01) between DSS+vehicle and DSS+RES-treated mice.

Fig. 4.

Changes in the number of CD4⁺ T cells expressing IFN-γ and TNF-α in mice with colitis mice given resveratrol treatment. Splenic, MLN, and LP lymphocytes were isolated from the four groups of C57BL/6 mice as described in the legend to Fig. 1. Changes in the numbers of CD4⁺ T cells expressing IFN-γ and TNF-α were determined by flow cytometry and expressed as the total number of cells/mice ± S.E.M. Data shown are from a representative experiment; three independent experiments involving six mice/group yielded similar results. Asterisks indicate statistically significant differences (P < 0.01) between groups treated with DSS+vehicle versus DSS+resveratrol (100 mg/kg).

Fig. 5.

Histological characterization of DSS-induced colitis after resveratrol treatment. Histological sections of colons from the four groups of mice were presented (as described in Fig. 1). DSS+vehicle-treated mice showed significant lymphocyte infiltration and distortion of glands, whereas DSS+resveratrol-treated mice showed colon lumen having markedly decreased lymphocyte infiltration. Other pathologic changes during DSS-induced colitis included diffuse leukocyte infiltrates, distorted crypts, and thickening of the LP in the area of distorted crypts in the colon. These changes were significantly reversed in DSS+resveratrol (100 mg/kg) groups.

Fig. 6.

Effect of resveratrol on effector T cells in MLNs and LPs after DSS-induced colitis. MLN and LP lymphocytes were isolated from the four groups of mice as described in Fig. 1 and stained for CD4⁺CD69⁺ T cells. Changes in the mean fluorescent intensity of CD69⁺ expressed by CD4⁺ T cells from MLNs and LPs were compared from various groups (A and C). The number (mean of six samples ± S.E.M.) of CD69-expressing CD4⁺ T cells from each group was counted, as shown in B and D. Asterisks indicate statistically significant differences; i.e., P < 0.01 between DSS+vehicle and the DSS+resveratrol (100 mg/kg)-treated group.

Fig. 7.

Resveratrol reduced DSS-induced macrophages in MLN and LP. Splenic, MLN, and LP lymphocytes were isolated from the four groups of mice (as described in Fig. 1 legend) and stained for CD11b⁺ and CD11c⁺ markers using flow cytometry. The numbers in the bottom right quadrant indicate the total percentage of CD11b⁺ cells; numbers in the upper left quadrant indicate the total percentage of CD11c⁺ cells (A). The numbers (mean of six samples ± S.E.M.) of total CD11b⁺ T cells from MLN and LP in each group were counted (B). Data are shown from a representative of three independent experiments. Resveratrol was used at 100 mg/kg. Asterisks indicate statistically significance difference (P < 0.01) between DSS+vehicle and DSS+RES-treated mice.

Fig. 8.

Resveratrol modulates DSS-induced CD4⁺ and CD8⁺ T cells during colitis. Splenic, MLN, and LP lymphocytes were isolated from the four groups of C57BL/6 mice (as shown in Fig. 1 legend) and stained for CD4⁺ and CD8⁺ T cells using flow cytometry. The numbers in the bottom right quadrant indicate the total percentage of CD4⁺ T cells (A); the numbers in the upper left quadrant indicate the total percentage of CD8⁺ T (A) cells. The number (mean of six samples ± S.E.M.) of total CD4⁺ T cells from MLN and LP in each group was counted (B). Data are shown from a representative of three independent experiments. Resveratrol was used at 100 mg/kg. Asterisks indicate statistically significance difference (P < 0.01) between DSS+vehicle and DSS+RES-treated mice.

Fig. 9.

Effect of resveratrol on COX1 and COX-2 expression in DSS-induced colitis. A, splenic, MLN, and LP lymphocytes were isolated from the four groups of C57BL/6 mice (as described in Fig. 1 legend) and analyzed for COX-1 and COX-2 expression by RT-PCR. B, expression. The 18S housekeeping gene was used as a positive control. Expression levels were compared with 18S and normalized. Data from multiple experiments on COX-2 expression are shown as mean ± S.E.M. Asterisks indicate statistically significant differences (P < 0.01) between groups treated with DSS+vehicle versus DSS+resveratrol (100 mg/kg).

Fig. 10.

Resveratrol up-regulates SIRT1 and down-regulates p-IκBα activity at the effector site of colitis. Colon LP lymphocytes were isolated from various groups of mice (as described in Fig. 1 legend) and analyzed for p-IκBα and SIRT1 expression (A and B) by Western blot and electrophoretic mobility shift assay analysis of NF-κB motif (C). Double-stranded wild-type and mutant NF-κB oligonucleotide probes were generated. Nuclear proteins (3–5 μg) generated from colon LP lymphocytes of various groups of mice (as described previously) were used in each reaction. Radiolabeled (³²P) wild-type NF-κB or mutant NF-κB probes were either directly used or used after incubation with nuclear protein. Arrow 1 shows wild-type and mutant NF-κB probe DNA bands. Arrows 2 and 3 indicate NF-κB-nuclear protein complexes. Data from multiple experiments have been depicted as mean ± S.E.M. (B). The 18S housekeeping gene was used as the positive control. The expression levels were compared with 18S and normalized. Asterisks indicate statistically significant differences (P < 0.01) between groups treated with DSS+vehicle versus DSS+resveratrol (100 mg/kg).