A2BAR Involved In Hypoxia Induced-intestinal Barrier Dysfunction

BackgroundIntestinal barrier (IEB), which consisit of mechanical barrier, biologic barrier,immunologic barrier and chemical barrier, plays a central role in maintaining internalhomostasis of the body. All sorts of acute or chronic pathological conditions, such assurgical stress, war injury, burn, inflammatory bowel diseases (IBD),ischemia/reperfusion (I/R) and total parenteral nutrition (TPN) can lead to intestinalmucosa barrier damage, increases the intestinal permeability, and often causes fatalconditions including sepsis and multiple organ failure, resulting in a high degree ofmorbidity and mortality. Although plenty of research which focus on regulationmechanism of intestinal mucosal barrier function under different pathological conditionshave been done, but intestinal mucosa injury and repair mechanism under acute andchronic conditions has not yet been elucidated.Adenosine (Ado), a precursor and a metabolites of adenine nucleotide that isinvolved in a variety physiological and pathological processes, is being increasinglyrecognized to modulate a wide variety of inflammation and I/R and hypoxic condition.After being released from cells or after being formed extracellularly duringinflammation, I/R and hypoxia, adenosine mediates its effect by activating andcombining with Adenosine Receptor: A1, A2A, A2B and A3, and launchs a series ofsignal transduction process. A1receptors coupling to Gi/G0protein, while A3receptorscoupling to Gi protein, which both of them inhibiting activity of adenylate cyclase (AC)and reducing levels of the adenosine monophosphate (cAMP); Both A2A and A2Bcoupling to Gs protein, activating the activity of AC and increasing levels of the cAMP.Low concentration of adenosine can activate A2A receptor, but only a high concentrationof adenosine can activate the A2B receptor, which means that A2B receptors is not easilybeen activated in normal condition. A2B receptor (A2BAR) involved in regulating variety of physiological andpathological processes, including activating mast cells, inhibiting the growth of vascularsmooth muscle cells and stimulating growth of endothelial cells and vasodilation ofblood vessels, regulating the release of inflammatory factors and intestinal secretion ofchloride ion, etc. Study found that acute ischemia and hypoxia conditions can induceintestinal A2BAR expression, and then participate in regulation of and repair processafter ischemia and hypoxia. In the model of IBD, intestinal mucosa injury in A2BAR/KOmice is more serious than those in WT mice. These data suguest A2BAR plays a pro-inflammatory role by strengthening macrophages secrete IL-6and promotes intestinalinflammation. However, the mechanism of A2BAR regulates intestinal barrier functionunder acute or chronic conditions is not cleare.MethodsFirstly, adult C57BL/6J mice were treated with A2BAR antagonist PSB1115beforesurgery, then undergoing intestinal I/R injury, and the small bowel was harvested. Theexpressions of A2BAR and Claudin-1,Occludin and ZO-1were detected byimmunohistochemistry， RT-PCR and Western Blot method. The transepithelialresistance (TER) was mesured by using Ussing Chamber system.Secondly, intestinal epithelial cells (Caco-2cells) were treated with A2BARantagonist PSB1115under hypoxia, the expressions of Claudin-1、Occludin and ZO-1were measured with RT-PCR、 western blot and confocal microscopy analysis. Thetransepithelial resistance (TER) was mesured by using a Millicell ERS.Thirdly, Caco-2cells were treated with FSK and SQ22536, the activator andinhibitor of adenylate cyclase, cAMP concentration was assessed using the cAMPenzyme immunoassay kit, the expressions of tight junction proteins were measured withwestern blot and RT-PCR analysis. The transepithelial resistance (TER) was mesured byusing a Millicell ERS.Results and Conclusion1. The expression of A2BAR significantly increased after the intestinal I/R injuryand hypoxia, and the level of tight junction proteins were decreased after intetinal I/Rand hypoxia. 2. The administration of A2BAR antagonist PSB1115to mice and Caco-2cellsunder intestinal I/R and hypoxia resulted in the increase of tight junction proteinsexpression and attenuate the loss of tight junction structural damage and TER caused byhypoxia and I/R.3. The administration of SQ22536to Caco-2cells under hypoxia increased theexpression of Claudin-1and Occludin, and attenuated the decreasing of TER caused byhypoxia.