Therapeutic and prophylactic thalidomide in TNBS-induced colitis: synergistic effects on TNF-alpha, IL-12 and VEGF production

Abstract

Aim: To evaluated the therapeutic and prophylactic effect of thalidomide on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Thalidomide has been reported to downregulate the expression of tumor necrosis factor alpha (TNF-alpha), IL-12, and vascular endothelial growth factor (VEGF), hallmarks of intestinal inflammation in Crohnos disease (CD).

Methods: Male Wistar rats were divided in five groups of ten animals each. Four groups received a rectal infusion of TNBS in ethanol. The first group was sacrificed 7 d after colitis induction. The second and third groups received either thalidomide or placebo by gavage and were sacrificed at 14 d. The fourth group received thalidomide 6 h before TNBS administration, and was sacrificed 7 d after induction. The fifth group acted as the control group and colitis was not induced. Histological inflammatory scores of the colon were performed and lamina propria CD4+ T cells, macrophages, and VEGF+ cells were detected by immunohistochemistry. TNF-alpha and IL-12 were quantified in the supernatant of organ cultures by ELISA.

Results: Significant reduction in the inflammatory score and in the percentage of VEGF+ cells was observed in the group treated with thalidomide compared with animals not treated with thalidomide. Both TNF-alpha and IL-12 levels were significantly reduced among TNBS induced colitis animals treated with thalidomide compared with animals that did not receive thalidomide. TNF-alpha levels were also significantly reduced among the animals receiving thalidomide prophylaxis compared with untreated animals with TNBS-induced colitis. Intestinal levels of TNF-alpha and IL-12 were significantly correlated with the inflammatory score and the number of VEGF+ cells.

Conclusion: Thalidomide significantly attenuates TNBS-induced colitis by inhibiting the intestinal production of TNF-alpha, IL-12, and VEGF. This effect may support the use of thalidomide as an alternate approach in selected patients with CD.

Figure 1

HE staining of tissue obtained from the left colon of animals: untreated colitis (A), therapeutic-thalidomide (B), respectively. Sample from untreated colitis group shows gross ulceration and intense cellular infiltration, congestion and edema, while thalidomide-treated animal presents less inflammation and no ulceration (Original magnification × 100).

Figure 2

Histological inflammatory scores of the colon. Total score of histological parameters analyzed in all groups: untreated colitis, therapeutic- and prophylactic-thalidomide, placebo-treated, and normal controls, respectively (n = 10 in each group). Horizontal bars represent medians, boxes represent the 25^th and 75^th percentiles, and vertical bars represent ranges. Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. Significant differences are presented.

Figure 3

Macrophages in the colonic lamina propria. Immunoperoxidase staining for macrophages is shown in placebo-treated colitis (A), therapeutic-thalidomide (B), and in a normal control (C), respectively. Arrowheads show immunoreactive cells (Original magnification × 400).

Figure 4

Quantitative analysis of CD4+ T-cells and macrophages in the colonic lamina propria of animals with untreated colitis, animals receiving therapeutic or prophylactic thalidomide, placebo-treated, and normal controls, respectively (n = 10 in each group). Horizontal bars represent medians, boxes represent the 25^th and 75^th percentiles, and vertical bars represent ranges. Significant values are presented. Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. Significant differences are presented.

Figure 5

Quantitative analysis of CD4+ T-cells and macrophages in the colonic lamina propria of animals with untreated colitis, animals receiving therapeutic or prophylactic thalidomide, placebo-treated, and normal controls, respectively (n = 10 in each group). Horizontal bars represent medians, boxes represent the 25^th and 75^th percentiles, and vertical bars represent ranges. Significant values are presented. Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. Significant differences are presented.

Figure 6

Quantitative analysis of VEGF+ cells in the colonic mucosa of the groups: untreated colitis, therapeutic- and prophylactic-thalidomide, placebo-treated, and normal controls, respectively (n = 10 in each group). Horizontal bars represent medians, boxes represent the 25^th and 75^th percentiles, and vertical bars represent ranges. Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. Significant differences are presented.

Figure 7

TNF-α production by the colonic mucosa. Levels of TNF-α are measured in supernatants of organ cultures and presented as pg/μg of tissue protein, in the groups: untreated colitis, therapeutic- and prophylactic-thalidomide, placebo-treated, and normal controls, respectively (n = 10 in each group). Horizontal bars represent medians, boxes represent the 25^th and 75^th percentiles, and vertical bars represent ranges. Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. Significant differences are presented.

Figure 8

IL-12 production by the colonic mucosa. Levels of IL-12 are measured in supernatants of organ cultures and presented as pg/μg of tissue protein, in the groups: untreated colitis, therapeutic- and prophylactic-thalidomide, placebo-treated, and normal controls, respectively (n = 10 in each group). Horizontal bars represent medians, boxes represent the 25^th and 75^th percentiles, and vertical bars represent ranges. Differences were analysed using one-way ANOVA with the Dunnett’s test for multiple comparisons. Significant differences are presented.