Prevention and Mitigation of Radiation‐Induced Colonic Epithelial Barrier Dysfunction by Lysophosphatidic Acid Receptor‐2 Agonists

Abstract

BackgroundLysophophatidic acid (LPA), a lipid growth factor, regulates secretion, absorption, cell survival and migration in the intestinal epithelium. Altered LPA activity is associated with many gastrointestinal (GI) diseases such as inflammatory bowel disease, colon cancer and GI acute radiation syndrome (GI‐ARS). Disruption of intestinal epithelial tight junctions (TJ) and gut barrier dysfunction is associated with the pathogenesis of many GI diseases. In this study, we evaluated the effect of LPA2 receptor on radiation‐induced disruption of intestinal epithelial barrier by in vivo and in vitro studies.MethodsWT and LPA2−/− mice were subjected to total body irradiation (TBI; 4Gy) or partial body irradiation (PBI‐BM5; 16 Gy). RP‐1, a stable, LPA2‐specific, LPA analog was administered (0.1 mg/kg, s.c.) 30 min prior to TBI or 24 h after PBI‐BM5. For in vitro studies, Caco‐2 and mICC12 cell monolayers were treated with LPA (10 mM) 30 min prior to irradiation (4 Gy). The barrier function was evaluated by measuring transepithelial electrical resistance (TER) and FITC‐inulin flux. TJ integrity was analyzed by immunofluorescence confocal microscopy for occludin, ZO‐1, F‐actin and claudins. Protein thiol oxidation was evaluated by measuring reduced and oxidized protein thiols by fluorescence staining using BODIPY FL‐N‐(2‐aminoethyl)maleimide as a probe. Colonic sections were also stained for Nrf2, the transcription factor responsible for regulation of antioxidant gene expression.ResultsTBI induced redistribution of TJ and AJ proteins from the epithelial junctions disruption in ileum and colon within 2 hours post irradiation (post‐IR), and the damage was sustained at least until 24 hours. TBI‐induced disruption of TJ and AJ was more severe in LPA2 −/− mice. On the other hand, RP‐1 administration in WT mice completely significantly attenuated TBI‐induced TJ and AJ disruption. PBI induced TJ disruption at 48 hours post‐IR, which was sustained at least until 100 hours. RP‐1 administered 24 h after IR mitigated PBI‐induced loss of TJ integrity. Both TBI and PBI depleted reduced protein thiols in colonic epithelium, which was blocked by RP‐1 administration. This effect of RP‐1 was associated with mitigation of PBI‐induced reduction of Nrf2 levels in colonic epithelium. In Caco‐2 and mICC12 cell monolayers, IR reduced TER, elevated inulin permeability and induced redistribution of TJ proteins in Caco‐2 and mICC12 cell monolayers. LPA (10 mM) administration, blocked IR‐induced decrease in TER, increase in inulin permeability and redistribution of TJ proteins.ConclusionThese results indicate that LPA2 receptor agonists prevent and mitigate radiation‐induced colonic epithelial barrier dysfunction. Studies in Caco‐2 and mICC12 cells indicate that LPA agonists may directly impact the intestinal epithelial cells. Supported by NIH grant DK55532