Protective Effect of Cashew Gum (Anacardium occidentale L.) on 5-Fluorouracil-Induced Intestinal Mucositis

Abstract

Intestinal mucositis is a common complication associated with 5-fluorouracil (5-FU), a chemotherapeutic agent used for cancer treatment. Cashew gum (CG) has been reported as a potent anti-inflammatory agent. In the present study, we aimed to evaluate the effect of CG extracted from the exudate of Anacardium occidentale L. on experimental intestinal mucositis induced by 5-FU. Swiss mice were randomly divided into seven groups: Saline, 5-FU, CG 30, CG 60, CG 90, Celecoxib (CLX), and CLX + CG 90 groups. The weight of mice was measured daily. After treatment, the animals were euthanized and segments of the small intestine were collected to evaluate histopathological alterations (morphometric analysis), levels of malondialdehyde (MDA), myeloperoxidase (MPO), and glutathione (GSH), and immunohistochemical analysis of interleukin 1 beta (IL-1β) and cyclooxygenase-2 (COX-2). 5-FU induced intense weight loss and reduction in villus height compared to the saline group. CG 90 prevented 5-FU-induced histopathological changes and decreased oxidative stress through decrease of MDA levels and increase of GSH concentration. CG attenuated inflammatory process by decreasing MPO activity, intestinal mastocytosis, and COX-2 expression. Our findings suggest that CG at a concentration of 90 mg/kg reverses the effects of 5-FU-induced intestinal mucositis.

Keywords: 5-fluorouracil; heteropolysaccharide; inflammation; intestinal mucositis.

Figure 1

Body weight variation in mice subjected to intestinal mucositis (5-FU, 450 mg/kg, ip, single dose) and treated with CG (30, 60 and 90 mg/kg for 4 days). The results are expressed as the mean ± SEM of the weight evaluation percentage of the initial weight, of a minimum of 6 animals per group. Two-way ANOVA followed by the Tukey’s test were used for the statistical analysis, where # p < 0.05 vs. saline and * p < 0.05 vs. 5-FU.

Figure 2

Histopathological analysis. (A) Saline; (B) 5-FU; (C) CG 30; (D) CG 60; (E) CG 90. 5 FU induced inflammatory cell infiltrate (red arrow), decreased intestinal villi (black arrow), loss of intestinal crypt architecture (blue arrow). Pretreatment with CG (30, 60 and 90 mg/kg) decreased the inflammatory infiltrate and prevented the shortening of the villi (F), increased crypt depth (G) and decreased villus/crypt ratio (H), with greater reversion of the 5-FU effect in the CG 90 + 5-FU group. All panels were obtained on the 100 μm scale (×200). Values were expressed as mean ± SEM. One-way ANOVA followed by the Tukey’s test were used for the statistical analysis was used, where # p < 0.05 vs. saline group and * p < 0.05 vs. group 5-FU.

Figure 3

(A) Total leukocyte count; (B) Activity of myeloperoxidase (MPO); (C) Level of malondialdehyde (MDA); (D) concentration of glutathione (GSH). Values were presented as mean ± SEM. For the statistical analysis, one-way ANOVA followed by Tukey’s test was used, where # p < 0.05 vs. saline group and * p < 0.05 vs. group 5-FU.

Figure 4

Mast cell counts in the duodenum samples. In (B) demonstrates that 5-FU promoted increased mast cell counts (red arrows) when compared to saline group (A). CG 90 (C) reversed the 5-FU-induced mastocytosis. All the panels were obtained at ×400 magnification. (D) Values were presented as mean ± SEM of the number of mast cells per field. For the statistical analysis, tone-way ANOVA followed by Tukey’s test was used, where # p <0.05 vs. saline group and * p < 0.05 vs. group 5-FU.

Figure 5

Effect of GC on the cyclooxygenase-2 pathway (COX-2) in histopathological and morphometric. The histopathological analysis represented by the groups (A) Saline; (B) 5-FU; (C) CG 90; (D) Celecoxib (CLX); (E) CLX and CG 90, as well as morphometric analysis of villus height (F) showed that 5-FU caused a decrease in villi and loss of cellular architecture when compared to the saline group. C, D, and E reversed the effect of 5-FU. All panels were obtained at ×200 magnification. Values were expressed as mean ± SEM for villi height in μm. For statistical analysis, one-way ANOVA followed by Tukey’s test was used, where # p < 0.05 vs. saline group, * p < 0.05 vs. group 5-FU, ** p < 0.05 vs. group CLX.

Figure 5

Effect of GC on the cyclooxygenase-2 pathway (COX-2) in histopathological and morphometric. The histopathological analysis represented by the groups (A) Saline; (B) 5-FU; (C) CG 90; (D) Celecoxib (CLX); (E) CLX and CG 90, as well as morphometric analysis of villus height (F) showed that 5-FU caused a decrease in villi and loss of cellular architecture when compared to the saline group. C, D, and E reversed the effect of 5-FU. All panels were obtained at ×200 magnification. Values were expressed as mean ± SEM for villi height in μm. For statistical analysis, one-way ANOVA followed by Tukey’s test was used, where # p < 0.05 vs. saline group, * p < 0.05 vs. group 5-FU, ** p < 0.05 vs. group CLX.

Figure 6

Immunohistochemistry analysis for COX-2 and IL-1β. (A,B) Saline; (C,D) 5-FU; (E,F) CLX; (G,H) CG 90; (I,J) CLX + CG 90. (K) a number of cells immunolabelled for cox-2. (L) % immunolabelled for IL-1β. Values were expressed as mean ± SEM. For statistical analysis, one-way ANOVA followed by Tukey´s test was used, where # p < 0.05 vs. saline group, * p < 0.05 vs. group 5-FU, $ p < 0.05 vs. group CLX.

Figure 7

Hypothetical model of action of CG in intestinal mucositis induced by 5-FU. CG prevented the 5-FU-induced injury intestinal through inhibition of MDA formation, neutrophil recruitment (decreasing MPO levels), mast cells activation and IL-1β and COX-2 immunostaining marker and inhibited leucopenia. CG also stimulate villus enlargement and increase levels of GSH, an antioxidant. ROS: Reactive Oxygen Species; TNF-α: Tumor Necrosis Factor-alpha; NF-κB: transcription factor nuclear kappa b; IL-1β: Interleukin 1 beta; COX-2: Cyclooxygenase 2; MDA: Malondialdehyde; MPO: Myeloperoxidase; GSH: Reduced glutathione. Arrows green (stimulate / increase), red arrows (inhibit).