Functional Analysis of the Synthetic Compatible Solute Homoectoine in the Stabilization of Intestinal Epithelial Barrier During Inflammation

Abstract

Background and AimInflammatory bowel disease (IBD) is a multifactorial disorder that comprises Ulcerative Colitis (UC) and Crohn's disease (CD) affecting millions of people worldwide with alarmingly increasing incidences every year. Dysfunction of the intestinal epithelial barrier has been associated with the pathogenesis of IBD. IBD therapies include anti‐inflammatory drugs that enhance intestinal barrier function but they frequently have adverse side effects. Compatible solutes such as bacterial ectoines have been shown to stabilize cell membranes and proteins. Here, we test if homoectoine (4,5,6,7‐tetrahydro‐2‐methyl‐1H‐(1,3)‐diazepine‐4‐carboxylic acid), a synthetic derivative of ectoine, prevents excessive intestinal epithelial permeability during inflammation.MethodsConfluent Caco‐2 cells in transwell filters were incubated with the inflammatory cytokines TNF‐α and IFN‐γ with or without parallel administration of homoectoine. In order to observe changes in epithelial permeability, transepithelial electrical resistance (TEER) was measured every 24 hours. Paracellular flux of 4kDa FITC‐dextran was measured when differences in TEER were observed. For in vivo experiments, homoectoine was administered orally to C57BL/6 mice in parallel to the induction of colitis with dextran sulfate sodium (DSS) during 7 days. To determine the clinical severity of colitis, the disease activity index (DAI) was evaluated and hematoxylin/eosin stainings of colon tissues were analyzed. Intestinal epithelial permeability was determined by means of an Evans Blue assay.ResultsIn vitro assays showed that homoectoine protects against the increase of epithelial permeability induced by pro‐inflammatory cytokines such as TNF‐α and IFN‐γ as indicated by increased TEER. We also observed that cells receiving homoectoine were able to recover faster from inflammation. However, paracellular flux of macromolecules was not improved significantly. In vivo, homoectoine treatment alone did not affect the DAI suggesting that homoectoine has no negative effects on healthy mice. Importantly, homoectoine treatment in colitic mice significantly reduced the DAI score with greater effects on intestinal bleeding and stool consistency. Moreover, intestinal epithelial permeability was reversed to control levels in the group of colitic mice receiving homoectoine. Histologically, homoectoine reduced edema formation, leukocyte influx and tissue damage induced by DSS.ConclusionsHomoectoine has protective effects on the epithelial barrier during inflammation and may thus serve in the future as diet supplementation in IBD patients to reach or extend phases of remission.Support or Funding InformationCONACyT Grants; 179895, 207268 and 233395