Sirt3Plays A Protective Role In Intestinal Epithelial Barrier Dyfunction

BackgroundIntestinal barrier function regulates transport and host defense mechanisms at themucosal interface with the outside world. Transcellular and paracellular fluxes are tightlycontrolled by membrane pumps, ion channels and tight junctions, adapting permeability tophysiological needs. Tight junctions are formed by many specific proteins includingclaudins, occludin and zonu-laoccludens and are connected with the cytoskeleton. In manyintestinal and systemic diseases, changes in intestinal permeability are related to alterationof tight junctions as an expression of intestinal barrier damage. Large number of clinicaland experimental studies have found that many factors can weaken or destroy the intestinalmucosal barrier function, thereby inducing bacterial translocation and endotoxemia. Thesefactors include: severe trauma, burns, hemorrhagic shock, stress, long-term use ofbroad-spectrum antibiotics or immunosuppressive agents, etc.Intestinal permeability has been investigated in several gastrointestinal disorders.These include inflammatory bowel disease IBD, food allergy and celiac sprue. Currentresearch supports the view of gut injury and loss of normal intestinal barrier function arekey elements in the paradigm of gut-origin systemic inflammatory response syndrome.A number of recent studies have revealed tissue hypoxia in both acute and chronicinflammatory diseases, particularly as they relate to mucosal surfaces involving epithelialcells, Hypoxia also plays a fundamental role in the pathophysiology of common causes ofmortality, including stroke, cancer, chronic lung disease and septic shock. In tissues andcells，adaptation to hypoxia leads to the transcriptional induction of a series of genes thatparticipate in angiogenesis, glucose metabolism,cell proliferation or survivaliron, andmetabolism. HIF-1is a transcriptional activator that is expressed in response to cellularhypoxia and mediates multiple cellular and systemic homeostatic responses to hypoxiamediating this response. HIF-1consists of an oxygen-regulated subunit HIF-1α(or its paralogs HIF-2αandHIF-3α)and a constitutively expressed HIF-1β subunit. in the hypoxia condition,HIF-1α subunit becomes stable and interacts with coactivators to modulate itstranscriptional activity. Its post-translational modifications the stability and activity of HIFαsuch as ubiquitination, acetylation, phosphorylation, and hydroxylation. in the past10years,the molecular mechanisms of HIF stabilization have been clarified. Four HiFhydroxylases—prolyl hydroxylase (PHD)1,2and3(also known as egl nine homolog1,2and3) and factor inhibiting HiF1(FIH1)—have been shown to be important in the hypoxicregulation of the HiF pathway. Three PHDs such as (PHD2/PHD3/PHD1)have thepotential to hydroxylate HIF-1α in vitro.HIF prolyl-hydroxylase2is the key oxygen sensorsetting low steady-state levels of HIF-1alpha in normoxia and The inverse relationshipbetween prolyl-hydroxylated HIFα had been reported by independent groups.Previous studies have indicated that HIF-1α activation is deleterious to intestinalbarrier function associated with hypoxia, ischemia/reperfusion, and inflam-mation.Sirtuins are a family of conserved proteins with deacetylase andADP-ribosyltransferase activity. In humans they are coded by seven genes (SIRT1-7). Themitochondrial deacetylase sirtuin-3(SIRT3) is one of the seven mammalian sirtuins, whichare homologs of the yeast Sir2gene.Recently, SIRT3as a novel HIF1α antagonist in cancercell metabolism has been described in numbers of studies and Previous studies have shownthat SIRT3is currently the only sirtuin family member that has been demonstrated todestabilize HIF1α through a PHD.Since HIF-1α and TJs play important roles in intestinal barrier function, and HIF-1αcould be destabilized by SIRT3,we explored the relationship of these3factors in thisprocess. The study was designed to further investigate the influence of SIRT3on theexpression of HIF-1α and epithelial barrier function in hypoxic or hypoxic-like caco-2cellmonolayers by silencing deacetylase sirt3and PHDs. Our data demonstrated that sirt3attenuated the barrier dysfunction by repressing HIF-1α and HIF-1α regulated intestinalbarrier protective factors in hypoxic caco-2cells. Partial PHDs deficiency increased theexpression of HIF-1α.the expression of HIF-1α Taken together, our finding, for the firsttime, reveals that Sirt3plays a protective role in intestinal epithelial barrier injure byPHD/HIF-1α pathway. Methods1. Cell culture and treatments.Caco-2cells was maintained in MEM medium(HyClone; Thermo Fisher Scientific, Inc.) supplemented with20%fetal bovine serum(Gibco, Life Technologies) and antibiotics. The cells were kept at37in a5%CO2environment. every2days,we change the culture medium. with0.53mM EDTA and0.25%trypsin subculture Caco-2cells after partial digestion in Hank’s balanced saline solution(HBSS) without Mg2+and Ca2+.Unless stated otherwise, in all experiments, Sirt3-siRNAand PHD-siRNA added to the basolateral compartment only and control monolayers wereincubated with the cell culture medium.2.Transient transfection assay.The inhibition of SIRT3，PHD1, PHD2, or PHD3function, cells were transiently transfected with short hairpin RNA (shRNA) plasmids,which aimed to interfere with SIRT3， PHD1,PHD2or PHD3expression, purchased fromGenecopoeia,Inc., according to the manufacturer’s instructions.3. real-time quantitative RT-PCR.Total RNA was isolated using a Trizol reagentaccording to the manufacturer’s protocol and quantified by NanoDrop2000. Real-timequantitative RT-PCR (qRT-PCR) was performed on50ng cDNA template, usingRotor-Gene Q (Qiagen) and the SYBR Premix Ex TaqTM II (TaKaRa), in a total volume of20mL. All data were normalized using b-actin as reference values and expressed as relativefold increases over control.The amplification efficiency ofHIF-1a andb-actin was*1.72andthe difference between them was lower than0.02.4.Measurement of transepithelial electrical resistance.Transepithelial electricalresistance (TER) values of monolayers grown on0.33cmTranswell supports weredetermined with a Millicell-ERS voltohmmeter (Millipore), andTER measurements werecalculated in Ohms cm2(O(cm2)to determine the TER of the epithelium alone (Wells andothers1998). To facilitate comparisons between conditions, TER was normalized to initialvalue, and expressed as percentage of initial resistance values.5.Western blot analysis. The cells were washed twice with phosphate-bufferedsaline(PBS) before lysis in cold RIPA buffer (PBS,1%NP-40,0.5%sodium deoxycholate,0.1%SDS,1mg/mL APMSF,1.0mM sodium orthovandate,1·mammalian protease inhibitorcocktail; Sigma-Aldrich). Where indicated, nuclear epithelial cell fractions were preparedusing the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer’s instructions. The protein concentration was determined according to theBradford method using BCA assay reagent (Beyotime). Samples (25mg protein) wereloaded onto8–12%SDS-PAGE, and the proteins were then electrophoretical transferred toa polyvinylidene fluoride membrane blocked by5%bovine serum albumin (BSA) for1h atroom temperature and then incubated with antibodies overnight at4℃:SIRT3(1:1,000)HIF-1a (1:1,000), Occludin (1:500),ZO-1(1:500), Claudin-1(1:1,000), anti-PHD1(1:500),PHD2(1:500), PHD3(1:500), and rabbit polyclonal anti-tubulin (1:1000). The secondaryantibodies at room temperature for2h and membranes were then washed thrice andincubated with horseradish peroxidase-conjugated. Membrane imaging was performedusing the enhanced chemiluminescence detection system (ECL; Boster) according to themanufacturer’s instructions.6.Statistical analyses.All experiments were repeated at least3individual experimentsto ensure reproducibility. Data were analyzed using SPSS18.0software. All experimentaldata are shown as mean–SD. Comparisons among3or more groups were made by analysisof variance, and the2groups were compared by Student’s-test. All reported significancelevels represent2-tailed Pvalues.ResultsHIF1α is detectable from SIRT3-deficient cells in normoxia demonstrated elevatedlevels of HIF1α relative to wide-type cells.Likewise,,when caco-2cell cultured in2%O2,The western blot studies of HFIα protein confirmed that HFIα protein expression inhypoxia reached the peak value at6h.An increase in the levels of HIF-1α protein by Western blotafter transfection ofSirt3-siRNA in normoxia and hypoxia cionditions. Sirt3expression was significantlyreduced after transfection of Sirt3-siRNA.The expression levels of HIF-1α protein(0.93±0.01)was increased in the hypox-ia plus Sirt3-siRNA group than those under hypoxia for6h and normoxia.The expression of TJs mRNA(ZO-1:0.88±0.03,Occludin:0.80±0.01,Claudin-1:0.84±0.01)(p<0.001),TJS protein(ZO-1:0.72±0.02,Occludin:0.71±0.03,Claudin-1:0.69±0.01)(p<0.001)was decreased after Sirt3-siRNA transfection as compared with that beforetransfection.4.Silencing SIRT3increased disruption of tight junction proteins.The TER value in the hypoxia plus Sirt3-siRNA group was decreased by30.8%compared with hypoxiafor6h.ConclusionsSirt3could regulate the expression of HIF-1αand TJs Under hypoxia,and theninfluenced the epithelial barrier function.