Tranilast Treatment Prevents Chronic Radiation-Induced Colitis in Rats by Inhibiting Mast Cell Infiltration

Abstract

Introduction: Mast cells are the principal cells involved in acute and chronic colitis due to radiation, known as radiation-induced colitis (RIC). In this study, we investigated whether pretreatment with tranilast, a mast cell inhibitor, could alleviate chronic RIC.

Methods: A total of 23 Sprague-Dawley rats were randomly divided into three groups: control group (n = 5), radiation group (RG, n = 9), and tranilast-pretreated radiation group (TG, n = 9). The rats in the RG and the TG were irradiated in the pelvic area (1.5 cm from the anus) with a single dose of 20 Gy under general anesthesia. Tranilast (100 mg/kg) was administered intraperitoneally to the rats of the TG for 10 days, starting from the day of pelvic radiation. Ten weeks after radiation, the rats were euthanized. Rectal tissue samples were histologically evaluated for the total inflammation score (TIS) and mast cell count. The expression of MUC2, MUC5AC, and matrix metalloproteinase-9 (MMP-9) was also assessed immunohistochemically.

Results: Both the TIS and specific components of TIS such as epithelial atypia, vascular sclerosis, and colitis cystica profunda (CCP) were significantly higher in the RG than in the TG (p = 0.02, 0.038, 0.025, and 0.01, respectively). Thein number of infiltrating mast cells was significantly higher in the RG than in the TG (median [range]: 20 [3-54] versus 6 [3-25], respectively; p = 0.034). Quantitatively, the number of MMP-9-positive cells was significantly higher in the RG (23.67 ± 19.00) than in the TG (10.25 ± 8.45) (mean ± standard deviation; p < 0.05). TIS and MMP-9 exhibited a strong association (correlation coefficient r = 0.56, p < 0.05). Immunohistochemically, the mucin-lake of CCP showed no staining for MUC5AC but was stained positive for MUC2.

Conclusion: Tranilast pretreatment of chronic RIC showed an anti-inflammatory effect associated with the reduction of mast cell infiltration and MMP-9 expression.

Keywords: Colitis cystica profunda; Mast cell; Matrix metalloproteinase; Radiation; Radiation-induced colitis; Tranilast.

Fig. 1.

Comparison of semi-quantitative TIS between two groups of rats with colitis: RG and TG. The TIS of the RG (12 [7−16]; n = 9) was significantly higher than that of the TG (7 [4−7]; n = 9) (median [range]; p = 0.02). TIS, total inflammation score; RG, radiation group; TG, tranilast-pretreated radiation group; SD, standard deviation.

Fig. 2.

Representative micrographs of prominent colitis cystica profunda (CCP) formation (a) and the absence of CCP development (b); representative micrographs of high mast cell infiltration (MCI) (c) and low MCI (d). Prominent CCP formation (arrow in (a)) compared to absence of CCP formation (b) (from case no. 7 in RG (a) and case no. 3 in TG (b)). High MCI (arrows in (c)) compared with low MCI (arrow in (d)) (from case no. 9 in the RG (c) and case no. 3 in the TG (d); H&E staining (a, b), toluidine blue staining (c, d); magnification: 400× (a, b), 100× (c, d); scale bar = 100 μm; inset: 400×; scale bar = 25 μm). RG, radiation group; TG, tranilast-pretreated radiation group.

Fig. 3.

Comparison of the number of infiltrating mast cells between the RG and TG. The number of infiltrating mast cells was significantly higher in the RG (20 [3−54]; n = 9) than in the TG (6 [3−25]; n = 9) (median [range]; p = 0.034). RG, radiation group; TG, tranilast-pretreated radiation group.

Fig. 4.

Representative micrographs of densely infiltrated MMP-9-positive cells in colon tissue of RG (a) and sparsely infiltrated MMP-9-positive cells in colon tissue of TG (b). A total of 210 MMP-9-positive cells were observed in one HPF from slide RG no. 7 (arrows in (a)), whereas only 12 MMP-9-positive cells were observed in one HPF from slide TG no. 9 (arrow in (b)) (a, b; magnification: 400×; scale bar = 50 μm). RG, radiation group; TG, tranilast-pretreated radiation group; MMP-9, matrix metalloproteinase-9; HPF, high-power field.

Fig. 5.

Correlation between TIS and the number of MMP-9-positive cells. Semi-quantitatively, the TIS and the number of MMP-9-positive cells showed a good correlation (correlation coefficient r = 0.56, p = 0.025): excellent (>0.80), good (0.60–0.79), fair (0.40–0.59), or poor (<0.40). TIS, total inflammation score; MMP-9, matrix metalloproteinase-9.

Fig. 6.

Mucin expression profiles in CCP lesions. Immunohistochemically, CCP lesions showed strong positivity for MUC2 (a) but were negative for MUC5AC (b) (100×; a, b; scale bar = 50 μm). CCP, colitis cystica profunda.