The Role Of SIRT1in The Maintance Of Intestinal Epithelial Barrier Function

BackgroundThe Intestinal epithelial barrier (IEB), which composed by intestinal epithelial cellsand tight junctions, plays an important role in maintaining the homeostasis of the body andresisting bacterial pathogens. All sorts of chronic or acute pathological conditions, such asburn, war injury, surgical stress, ischemia/reperfusion (I/R), inflammatory bowel diseases(IBD) and total parenteral nutrition (TPN) can lead to intestinal mucosa barrier damage,increases the intestinal permeability, and often causes fatal conditions including systemicinflammatory response syndrome and multiple organ dysfunction syndrome, resulting in ahigh degree of morbidity and mortality. IEB function depends on the oxygen concentrationof local mucosal tissue. Local mucosal hypoxia and inflammation may induce the loss ofintestinal barrier function.Sirtuins, the class III histone deacetylases (HDACs), are widely distributed and havebeen shown to regulate a variety of physiopathological processes, such as inflammation,cellular senescence/aging, cellular apoptosis/proliferation, differentiation, metabolism.There are seven mammalian enzymes belonging to class III HDACs: SIRT1to SIRT7. Thebest characterized and well-studied among the human sirtuins is sirtuin1(SIRT1), a nuclearprotein reported to regulate critical metabolic and physiological processes. SIRT1, amammalian ortholog of yeast silent information regulator2, plays an important role inregulation of pathogenesis of chronic inflammatory pulmonary diseases, neurodegenerative,cardiovascular and renal diseases. SIRT1, as a protein deacetylase, transfers the acetylgroup of lysines in a protein substrate to the ADP-ribose moiety of nicotinamide adeninedinucleotide to produce a deacetylated protein, nicotinamide, and2’-O-acetyl-ADP ribose.The mechanism in regulation of SIRT1on these processes is due to its ability todeacetylate histones and non-histone proteins, such as nuclear factor NF-κB, forkhead boxclass O3, p53, peroxisome proliferator-activated receptor-c and endothelial nitric oxide synthase. Increased activity of SIRT1results in the cellular resistance to oxidative stress viamodulation of forkhead box class transcription factors. In renal tubular cells, SIRT1protects cells against oxidative stress induced apoptosis via improving the activity ofcatalase. Recent evidence suggests that stimulation of Sirt1protects the heart from I/Rinjury, suggesting an possibility to treat cardiac disease by using SIRT1agonist.Resveratrol(agonist of SIRT1) could reverse the colitis-associated increased levels of serumamyloid A, interleukin-6, and interleukin-1.Therefore, in this study we developed an intestinal epithelial barrier model in vitro toinvestigate the protective effect of SIRT1on the intestinal barrier function under hypoxiaand TNF-α. This study would be helpful to provide a new theoretical basis for theprevention of the loss of intestinal epithelial barrier function.MethodsFirstly, intestinal epithelial cells (Caco-2cells) were treated with SIRT1agonistResveratrol under hypoxia. Caco-2cells were randomly divided into three groups:normoxia group(Nx), hypoxia group (Hx,1%O2for6h) and hypoxia plus40μMResveratrol (agonist of SIRT1) group (Hx+Res), the expressions of Claudin-1、Occludinand ZO-1were measured with RT-PCR、western blot and confocal microscopy analysis.The transepithelial resistance (TER) was mesured by using a Millicell ERS.Secondly, Caco-2cells were randomly divided into three groups: normoxia group(Nx),hypoxia group (Hx,1%O2for6h) and hypoxia plus40μM Resveratrol (agonist of SIRT1)group (Hx+Res), the expressions of NF-κB were measured with western blot.Thirdly, intestinal epithelial cells (Caco-2cells) were treated with TNF-α and SIRT1agonist Resveratrol. Caco-2cells were randomly divided into three groups: Control group,TNF-α group (TNF-α,50ng/ml for24h) and40μM Resveratrol (agonist of SIRT1) plusTNF-α group (Res+TNF-α), the expressions of Occludin and ZO-1were measured withconfocal microscopy analysis and western blot. The transepithelial resistance (TER) wasmesured by using a Millicell ERS.Results1. Both mRNA and protein expressions of SIRT1were significantly reduced afterhypoxia. The level of tight junction proteins were decreased after hypoxia. Theadministration of SIRT1agonist Resveratrol to Caco-2cells under hypoxia resulted in the increase of tight junction proteins expression and attenuate the decrease of TER caused byhypoxia. SIRT1could maintain the epithelial barrier function through regulating theexpression of TJs.2. Hypoxia induced the activation of NF-κB, whereas pretreatment withResveratrol(SIRT1agonist) inhibited hypoxia-induced activation of NF-κB.3. The protein expression of SIRT1were significantly reduced under the treatment ofTNF-α. The level of tight junction proteins were significantly decreased. Theadministration of SIRT1agonist Resveratrol to Caco-2cells under the treatment of TNF-αresulted in the increase of tight junction proteins expression and attenuate the decrease ofTER caused by TNF-α.Conclusion1.This study suggested that hypoxia induced the loss of epithelial barrier function anddisruption of tight junction proteins, by downregulation of SIRT1expression throughNF-κB pathway.2.We find that TNF-α induced the loss of epithelial barrier function and disruption oftight junction proteins by downregulation of SIRT1.