Palmitoylethanolamide regulates development of intestinal radiation injury in a mast cell-dependent manner

Abstract

Background: Mast cells and neuroimmune interactions regulate the severity of intestinal radiation mucositis, a dose-limiting toxicity during radiation therapy of abdominal malignancies.

Aim: Because endocannabinoids (eCB) regulate intestinal inflammation, we investigated the effect of the cannabimimetic, palmitoylethanolamide (PEA), in a mast competent (+/+) and mast cell-deficient (Ws/Ws) rat model.

Methods: Rats underwent localized, fractionated intestinal irradiation, and received daily injections with vehicle or PEA from 1 day before until 2 weeks after radiation. Intestinal injury was assessed noninvasively by luminol bioluminescence, and, at 2 weeks, by histology, morphometry, and immunohistochemical analysis, gene expression analysis, and pathway analysis.

Results: Compared with +/+ rats, Ws/Ws rats sustained more intestinal structural injury (p = 0.01), mucosal damage (p = 0.02), neutrophil infiltration (p = 0.0003), and collagen deposition (p = 0.004). PEA reduced structural radiation injury (p = 0.02), intestinal wall thickness (p = 0.03), collagen deposition (p = 0.03), and intestinal inflammation (p = 0.02) in Ws/Ws rats, but not in +/+ rats. PEA inhibited mast cell-derived cellular immune response and anti-inflammatory IL-6 and IL-10 signaling and activated the prothrombin pathway in +/+ rats. In contrast, while PEA suppressed nonmast cell-derived immune responses, it increased anti-inflammatory IL-10 and IL-6 signaling and decreased activation of the prothrombin pathway in Ws/Ws rats.

Conclusions: These data demonstrate that the absence of mast cells exacerbate radiation enteropathy by mechanisms that likely involve the coagulation system, anti-inflammatory cytokine signaling, and the innate immune system; and that these mechanisms are regulated by PEA in a mast cell-dependent manner. The eCB system should be explored as target for mitigating intestinal radiation injury.

Figure 1

Protein levels of CB1 and CB2 receptors in vehicle-treated unirradiated and irradiated intestines from +/+ rats (N=9) and Ws/Ws (N=8) rats. Irradiated and unirradiated intestines from vehicle-treated +/+ and Ws/Ws rats were procured 2 weeks after the completion of irradiation. Protein levels were measured ELISA. A. Comparison of CB1 and CB2 levels in unirradiated intestines from +/+ and Ws/Ws rats; B. Levels of CB1 receptors in unirradiated and irradiated intestines from +/+ and Ws/Ws rats; C. Levels of CB2 receptors in unirradiated and irradiated intestines.

Figure 2

Radiation effects on intestines in +/+ rats and Ws/Ws rats (mean, standard error of mean). p values refer to difference between +/+ rats and Ws/Ws rats. Irradiated intestines from vehicle-treated +/+ and Ws/Ws rats were procured 2 weeks after the completion of irradiation. A. Radiation injury score (a composite histopathologic score); B. Mucosal surface area; C. Intestinal wall thickness; D. MPO activity by bioluminescence; F. Collagen deposition.

Figure 3

Effect of PEA (10 mg/kg/d) on radiation-induced intestinal injury in +/+ rats and Ws/Ws rats (mean, standard error of mean). Irradiated intestines from vehicle-treated and PEA-treated +/+ and Ws/Ws rats were procured 2 weeks after the completion of irradiation. A. Radiation injury score; B. Intestinal wall thickness; C. Mucosal surface area; D. Collagen deposition. Representative histological images of intestines from Ws/Ws rats treated with vehicle or PEA are shown.

Figure 4

Bioluminescence image of in vivo MPO activity on vehicle- and PEA-treated (10 mg/kg/d) +/+ rats and Ws/Ws rats (mean, standard error of mean). Quantitative total MPO flux 12 days after radiation exposure.