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

Radiation therapy (RT) plays an important role both for curative and palliative purposes in a wide variety of malignancies, including gastrointestinal, gynecological and urological tumors [1]. One of the complications of RT for gastrointestinal, gynecological, and urological tumors is radiation-induced colitis (RIC). In the acute form of RIC, symptoms such as hemorrhage, pain, incontinence and diarrhea are self-limiting, while in the chronic form, symptoms such as rectal bleeding, stricture, diarrhea and incontinence may persist [1]. Colitis cystica profunda (CCP) is the abnormal displacement of normal mucosal crypts into the submucosa and muscle layer, sometimes containing mucin pools [1, 2]. CCP is often mistaken for malignancy due to ectopic gland localization, but it lacks cellular dysplasia. It is a rare complication of chronic RIC, typically following pelvic RT [2].

Mast cells play a primary role in the pathogenesis of acute and chronic RIC [3‒5]. A previous study reported that in a genetically mast cell-deficient mouse model, chronic RIC including CCP and other histopathologic changes was markedly improved compared to that in normal littermates [3]. In radiation-induced proctitis after RT for prostate cancer, the activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in rectal mucosa increased [6]. Mast cells are activated in parallel with elevation of MMP-9 levels [7] and mast cell-fibroblast interactions in atopic conditions [8].

Tranilast, a cell membrane stabilizer, has been widely used in the treatment of inflammatory diseases and it can inhibit the release of histamine and other chemical mediators such as MMP-2 and MMP-9 [9, 10]. Previous studies have demonstrated that tranilast attenuates inflammation by modulating mast cell infiltration and fibrosis in inflammatory diseases, such as diabetic kidney disease [11] and renal fibrosis [12]. However, the role and mechanisms underlying the effects of tranilast on chronic RIC are unclear. To evaluate the therapeutic potential of tranilast in chronic RIC, we investigated the association between its histologic effect on mast cell infiltration and MMP-9 expression in rats.

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) guidelines and were performed in accordance with guidelines approved by the Institutional Ethics Committee of the Uijeongbu St. Mary’s Hospital, Korea (UJA2008-01A) and performed in strict compliance with the Committee’s guidelines on animal care.

In this study, we obtained 23 five-week-old female Sprague-Dawley rats from ORIENT BIO Co. (Seongnam, South Korea). The rats were maintained 7 days on a 12-hour light and dark cycle, with a temperature maintained within 23 ± 3°C and relative humidity within 50% ± 10%. All animals were fed a standard chow diet. They were randomly divided into the following three groups: control group (n = 5), radiation group (RG, n = 9), and tranilast-pretreated radiation group (TG, n = 9). Rats in the TG (n = 9) were given tranilast (100 mg/kg of body weight, in phosphate-buffered saline solution [PBS]) by intraperitoneal injection once per day for 10 days starting from the day of pelvis radiation. Tranilast was kindly donated by JW Pharmaceutical (Seoul, South Korea). Excluding the control group, the rats in the RG and TG were irradiated in the pelvic area (1.5 cm from the anus) with a single dose of 20 Gy after collimation. Gamma rays were irradiated with a 6 MV linear accelerator (Mevatron MX-2, Siemens, USA) at a dose rate of 300 cGy/min under general anesthesia with an intraperitoneal injection of 30 mg/kg Zoletil (Virbac Korea, Seoul, South Korea). Ten weeks after radiation, all rats were euthanized after being anesthetized with pentobarbital (40 mg/kg).

After euthanasia, their abdominal cavities were opened, and a 5-cm portion of the colon was removed from each animal, which was then divided and stored as samples (one from each animal; total n = 18; RG, n = 9; TG, n = 9). The samples were fixed using 10% formalin and stained with hematoxylin-eosin (one from each animal; total n = 18; RG, n = 9; TG, n = 9). The hematoxylin-eosin-stained samples were scanned using a 3DHistech Pannoramic p250 Flash III digital slide scanner (3DHISTECH, Ltd.) and viewed using SlideViewer software (3DHISTECH, Ltd.). From the scanned images, one high-power field (HPF, ×400) that showed the most severe inflammatory features histopathologically was selected and photographed, and the total inflammation score (TIS) of RIC was histopathologically assessed according to the method used in a previous study [13]. Briefly, the histopathological features of inflammation were evaluated by a pathologist who was blinded to the samples. The TIS of RIC, consisting of the following seven components was measured according to that reported in a previous study: thickening of subserosa (TS, scores 0, 1, 2, 3; 0 = no TS, 1 = slight TS, 2 = marked TS, 3 = extreme TS), mucosal ulceration (MU, scores 0, 1, 2; 0 = no MU, 1 = small superficial MUs, 2 = ulcerations involving more than half of the intestinal circumference), epithelial atypia (EA, scores 0, 1, 2, 3; 0 = no EA, 1 = abnormally oriented crypts, 2 = irregular crypt regeneration with atypical epithelial cells, 3 = adenocarcinoma), vascular sclerosis (VS, scores 0, 1, 2, 3; 0 = no VS, 1 = slight thickening and hyalinization of vessel wall, 2 = vessel wall double normal thickness or hyalinization and stenosis, 3 = extreme sclerosis with marked stenosis or complete occlusion; fibrinoid necrosis), intestinal wall fibrosis (IWF, scores 0, 1, 2, 3; 0 = no IWF, 1 = submucosa double normal thickness or broadened and hyalinized collagen fibers, 2 = submucosa three to four times normal thickness, 3 = massive fibrosis including muscularis), lymphocytic congestion (LC, scores 0, 1; 0 = no LC, 1 = dilated lymph vessels or cystic collections of lymphocytes), and CCP (scores 0, 1, 2, 3; 0 = no submucosal glandular inclusions, 1 = submucosal glandular inclusions, 2 = submucosal cysts with polypoid elevation of the mucosa, 3 = large cysts extending into the muscularis). TIS was calculated as the sum of the scores of the seven components.

We performed modified toluidine blue staining to detect mast cells (one from each animal; total slide number = 18; RG, n = 9; TG, n = 9) [4]. Briefly, the paraffin sections were deparaffinized and rehydrated. The sections were stained in toluidine blue for 5 min, washed in distilled water, and dehydrated. The sections were then cleared in xylene and mounted. A single image at medium magnification (×100) was randomly selected to count the number of metachromatic mast cells and photographed under a light microscope with a digital camera (Olympus BX53 microscope coupled to a DP22 digital camera).

One slide was stained for each marker (MMP-9, MUC2, MUC5AC) from each animal; the total number of slides was 54 (3 markers × 18 animals; RG, n = 9; TG, n = 9).

Tissue sections 3 μm thick were deparaffinized, rehydrated and washed twice in buffer. To reduce nonspecific background staining due to endogenous peroxidase, the slides were incubated in a Hydrogen Peroxide Block for 10 min and washed twice with PBS (pH 7.4). The primary antibody MMP-9 (1:10,000, Abcam, UK, ab76003) was applied and incubated according to the manufacturer's recommended protocol, and the slides were washed four times with PBS (pH 7.4). The slides were then treated with Primary Antibody Enhancer, incubated for 30 min at a temperature maintained at 23 ± 3°C, and then washed four times in buffer. The slides were then incubated with horseradish peroxidase for 40 min at room temperature and rinsed with PBS. 3,3′-Diaminobenzidine (DAB) substrate (DAKO, USA) was used to visualize the bound antibodies. Counterstaining was performed with Mayer’s hematoxylin, and the staining was blued with a 0.75% ammonium-water solution. These MMP-9 immunohistochemically stained slides were scanned using a 3DHistech Pannoramic p250 Flash III digital slide scanner (3DHISTECH, Ltd.) and viewed using SlideViewer software (3DHISTECH, Ltd.). From the scanned images, the HPF (×400) that showed the densest MMP-9-positive cells was selected and photographed.

Immunohistochemical staining was performed on an automated Ventana Benchmark XT platform (Roche Diagnostics, Basel, Switzerland) using monoclonal antibodies, anti-MUC2 antibody (1:100, Leica Biosystems Nussloch GmbH, Germany, Ccp58), anti-MUC5AC antibody (1:100, Leica Biosystems, CLH2) and the Ventana ultraVIEW DAB Detection Kit (Roche Diagnostics, Basel, Switzerland). A single image depicting CCP at medium magnification (×100) was selected and photographed under a light microscope with a digital camera (Olympus BX53 microscope coupled to a DP22 digital camera).

Data are presented as mean ± standard deviation or median and range. MedCalc Statistical Software v. 18.2.1 (MedCalc Software, Ostend, Belgium) was used for the statistical analysis. First, the Mann-Whitney U test with Bonferroni correction and Fisher’s exact test were performed to determine the differences in the seven components of TIS between the RG and TG. The Mann-Whitney Utest was performed to determine differences in the number of infiltrating mast cells between the RG and TG. Spearman’s correlation analysis was performed to calculate the correlation coefficients between TIS and MMP-9-positive mast cell count or TIS and infiltrating mast cell counts, which were classified as excellent (>0.80), good (0.60–0.79), fair (0.40–0.59), or poor (<0.40). Differences were considered statistically significant at p < 0.05.

Histologically, the median TIS score was significantly higher in the RG (12 [7−16]; n = 9) than in the TG (7 [4−7]; n = 9) (median [range]; p < 0.05; Fig. 1), and the mean TIS score was also higher in the RG (11.33 ± 3.00) than in the TG group (6.89 ± 2.89) (mean ± standard deviation; p< 0.05; Table 1). Among the seven components of the TIS (TS, MU, EA,VS, IWF, LC, and CCP), EA, VS, and CCP were significantly higher in RG than in TG (EA: 1.67 ± 0.50, 1.11 ± 0.33; VS: 2.00 ± 0.71, 1.00 ± 0.71; CCP: 1.89 ± 1.17, 0.33 ± 1.00; mean ± standard deviation; p < 0.05) (Table 1). LC showed no significant difference between RG and TG (8/9 vs. 7/9; p > 0.05; Table 2). Moreover, positive cases of CCP were found almost exclusively in the RG (8/9) group compared to those in the TG (1/9) and control (0/5) groups (Table 1; Fig. 1, 2).

The number of infiltrating mast cells was significantly higher in the RG than in the TG (median [range]: 20 [3.0−54.0] and 6.0 [3.0−25.0], respectively; p = 0.034) (Fig. 3). The number of infiltrating mast cells did not differ between the area near the CCP (23.67 ± 18.91) and the remaining areas in the RG (23.00 ± 20.05) (mean ± standard deviation; p > 0.05).

Immunohistochemical analysis showed that the total 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 = 0.024). The number of MMP-9-positive cells in one HPF of RG, case no. 7, was 210 (Fig. 4a), whereas in TG, case no. 9, it was 12 (Fig. 4b).

The TIS and the number of infiltrating MMP-9-positive cells exhibited a strong association (correlation coefficient r = 0.56, p = 0.025) (Fig. 5; Table 3). In contrast, the TIS and infiltrating mast cell counts were not significantly correlated (r = 0.3, p > 0.05) (Table 3).

Immunohistochemically, the CCP lesions showed strong positivity for MUC2 (Fig. 6a), but were negative for MUC5AC (Fig. 6b).

RIC can develop during radiotherapy for pelvic malignancies and presents as a persistent and difficult-to-treat condition [1]. Consequently, prevention through a thorough understanding of the mechanisms of RIC is clinically significant. Mast cells play a primary role in the pathogenesis of acute and chronic RIC [3‒5]. A previous study using a genetically mast cell-deficient mouse model showed that loss of mast cells alleviated radiation-related damage [3], which was consistent with our data, although the function of mast cells in the genetic model is different from that in a pharmacologic intervention, as in our study. Radiation exposure causes an increase in vascular permeability in a dose-dependent manner, with mast cells playing a role in this process [14, 15]. The pro-inflammatory environment attracts mast cells to the site of injury. Chemokines, such as CCL2 (MCP-1) and CXCL8 (IL-8), play a significant role in the recruitment of mast cells from the circulation to damaged tissue. Infiltrated mast cells promote inflammation by degranulation to release various chemical mediators such as histamine, cytokines, proteoglycans, and proteases like tryptases or MMP-9, as well as by attracting other immune cells [16, 17].

Tranilast has been used since 1982 for its anti-allergic, anti-inflammatory, anti-fibrotic, anti-cancer, anti-angiogenic, and antioxidant properties via suppression of tumor growth factor-beta and inflammasome pathways [9, 18]. It is licensed for the treatment of bronchial asthma in Japan and South Korea. However, its effect on RIC has not been explored. We hypothesized that tranilast, a mast cell stabilizer, prevents chronic RIC. Our study demonstrated that tranilast pre-treatment can prevent chronic RIC by decreasing mast cell infiltration and inhibiting MMP-9 expression. Conversely, in acute RIC, mast cells are not crucial and might even be protective [19‒21]. This debate arises because mast cells release both harmful and beneficial mediators of wound healing [22, 23].

The severity of RIC was histopathologically assessed based on seven factors comprising the TIS. The current study revealed that the TIS and its sub-scores, including EA, VA, and CCP, were significantly lower in the TG than in the RG (Fig. 1 and Table 1). Our findings suggest that tranilast plays a protective role and can reduce the inflammation associated with radiation injury. A previous study reported that tranilast administration effectively reduced the mRNA expression of pro-inflammatory cytokines and increased that of anti-inflammatory cytokines in mice with dextran sulfate sodium-induced colitis [24]. Our results are consistent with those of previous studies that reported the protective effect of tranilast in rat models of colitis [25, 26]. Tranilast has another pharmacological effect as an anti-allergic agent, reducing the production of inflammatory cytokines [27, 28], suppressing fibrosis, and inhibiting tumor cell growth [29, 30].

In this study, we found that the number of infiltrating mast cells was significantly higher in the RG than in the TG (Fig. 2, 3). This finding indicates that mast cells are key players in the pathogenesis of acute and chronic RIC, which is consistent with previous studies [3, 4]. Interestingly, the number of infiltrating mast cells did not differ between areas near the CCP and other regions of the RG. Although the exact reason for this is still unclear, we propose several explanations. It may be plausible that the mast cells are localized near CCP at some points during the course of colitis, although they were not found at the 10th week post-radiation. Additionally, because CCP is considered benign, mast cell infiltration near the CCP might not be as significant as in cases of cancer invasion. Further investigation is needed to clarify these findings.

In terms of MMP-9 expression in RIC, the total number of infiltrating MMP-9-positive cells was significantly higher in the RG than in the TG, as shown in Figure 4. In the present study, we demonstrated that TIS and MMP9 were closely correlated, indicating that elevated MMP-9 levels are associated with the severity of inflammation in RIC (Fig. 5). These findings are consistent with previous studies: the relationship between the activities of inflammatory mediators in animal models of colitis and MMPs [31‒35], the activation of mast cells associated with the elevation of MMP-9 levels [7], and even mast cell-fibroblast interactions [8]. In accordance with the present results, tranilast reduces the production of MMPs in various cell types, including cancer cells and fibroblasts [9, 36, 37].

In the present study, we showed that CCP occurred in the rectum at a radiation dose of 20 Gy (Fig. 6). However, a higher radiation dose (>20 Gy) is required for the occurrence of CCP in other reports [3, 5]. Interestingly, the number of rats with CCP was significantly higher in the RG (8/9) than in the TG (1/9), and the mean CCP score per rat was significantly higher in the RG (1.89 ± 1.17) than in the TG (0.33 ± 1.00) (mean ± standard deviation, p < 0.05), as shown in Table 1. Thus, it is presumed that tranilast pretreatment could prevent CCP formation during pelvic RT. CCP is a rare intestinal disease associated with several conditions, including rectal prolapse syndrome, inflammatory bowel disease, trauma, chronic RIC, and malignant disease [1, 2, 38]. Pathologists occasionally misinterpret it as a malignant disease. In the mucosa of the normal colon, only MUC2 is expressed, without the expression of MUC5AC. However, both MUC2 and MUC5AC may be expressed in colon cancer [39]. In ulcerative colitis in humans and experimental colitis in rats induced by dextran sulfate sodium, both MUC2 and MUC5AC may be expressed as protective colonic barriers [40, 41]. Therefore, we conducted mucin staining of this chronic RIC to evaluate the malignant potential of CCP [42]. Since only MUC2 and not MUC5AC, were positively stained in CCP, we found that CCP has characteristics similar to those of normal colon mucosa (Fig. 6), unlike ulcerative colitis.

In the present study, we demonstrated that tranilast reduced the infiltration of mast cells into the rectal wall and exerted an anti-inflammatory effect on RIC. Therefore, tranilast pre-treatment may prevent RIC in patients receiving pelvic RT. Further clinical studies are required.

We would like to thank Editage (www.editage.co.kr) for English language editing.

All experimental procedures were carried out according to the Declaration of Helsinki and the Guide for the Care and Use of Laboratory Animals. The animal study was reviewed and approved by the Animal Ethics and Experimental Committee of the Uijeongbu St. Mary’s Hospital, Korea (UJA2008-01A).

The authors declared that they have applied for a patent for the use of tranilast for the treatment of RIC. No other potential conflict of interest relevant to this study exists.

This research was supported by the National Research Foundation of Korea (Grant No. NRF 2021R1A2C1093096).

Kyung Jin Seo, Hae Kyung Lee, and Hiun Suk Chae were involved in material preparation, data curation, and formal analysis. Kyung Jin Seo carried out investigation and methodology and wrote the original draft. Kyung Jin Seo and Hiun Suk Chae conducted project administration and resources. Mohammad Rizwan Alam, Jamshid Abdul-Ghafar, Sang Woo Kim, Hyung Keun Kim, Hyun Ho Choi, Seung Ho Sin, and Hae Kyung Lee reviewed and edited the manuscript.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.