The Protective Effects Of Valproic Acid On Gut Barrier Function After Major Burn Injury And Its Underlying Mechanisms

ObjectiveGut barrier dysfunction is one of common complications following major burninjury, and burn-induced gut barrier dysfunction plays an important role in thedevelopment of endotoxemia leading to sepsis and multiple organ dysfunction.Mechanisms responsible for burn-induced gut barrier dysfunction remain unclearhowever, hypoxia-inducible factor-1-mediated vascular endothelial growth factor(VEGF) and myosin light chain kinase (MLCK) expression are critical inhypoxia-induced paracelluar barrier dysfunction. Recent studies have demonstratedthat valproic acid (VPA), a histone deacetylase inhibitor (HDACI), can protect cellsfrom insults including hypoxia and inflammation, and can improve survival inhemorrhagic and septic shock models. Furthermore, recent studies have also shownthat VPA can protect the paracelluar barrier by preventing the degradation of multipletight junction proteins, and thus attenuating the blood-brain and blood-spinal cordbarrier disruption in traumatic or ischemic-reperfusion injury models. This study aimsto investigate the potential protective effects of valproic acid on gut barrier functionafter major burn injury and its underlying mechanisms.Methods1. Rats were randomly assigned to four groups: Sham+NS, Sham+VPA, Scald+NS,Scald+VPA. Rats were subjected to third degree55%TBSA burns or sham-burns andthen treated with/without VPA (300mg/Kg) accordingly. Intestinal barrier dysfunctionwas evaluated by permeability of intestinal mucosa to fluorescein isothiocyanate(FITC)-dextran and by Chiu’s grading system. Levels of acetylated lysine at the ninthposition of histone3protein (Ac-H3K9), hypoxia-inducible factor-1α(HIF-1α), zona occludens-1(ZO-1) and MLCK were measured by immunofluorescence stainingand/or immunoblotting. Protein levels of VEGF were measured by enzyme-linkedimmunosorbent assay.2. Caco2cells were stimulated with CoCl2(1mM) for24hours with/without VPA(2mM) followed by analysis of HIF-1α, VEGF, MLCK and ZO-1. In addition, Caco2cells were transfected with siRNA directed against HIF-1α, and the protein levels ofVEGF，MLCK and ZO-1were measured in order to confirm the role of HIF-1αin gutbarrier function and the mechanisms for the protective effects of VPA on gut barrierfunction.Results1. Burn insults resulted in a significant increase in intestinal permeability (2.61-fold),elevation of Chiu’s score (7.76-fold), protein levels of HIF-1α(3.88-fold), VEGF(1.94-fold) and MLCK (1.71-fold), and a lowering in the levels of AC-H3K9(0.73-fold) and ZO-1(0.65-fold) at2hours after injury (all P<0.05). These changeswere much more prominent at6hours after injury: intestinal permeability (4.80-foldincrease), Chiu’s score (17.33-fold increase), HIF-1α(4.42-fold increase), VEGF(2.43-fold increase), MLCK (2.80-fold increase), AC-H3K9(0.48-fold decrease) andZO-1(0.43-fold decrease)(all P<0.05).2. VPA treatment significantly attenuated an increase in HIF-1α protein levels and thedecrease in ZO-1protein levels at2hours after injury, and these changes were moreprominent at6hours after injury (all P<0.05). VPA treatment also attenuated theincrease in the intestinal permeability, Chiu’s score, protein levels of VEGF andMLCK at6hours after injury (all P<0.05), but not at2hours after injury (all P>0.05).3. CoCl2stimulation resulted in a significant increase in protein levels ofHIF-1α(5.84-fold increase), VEGF (9.97-fold increase) and MLCK (3.49-foldincrease) in Caco2cells, accompanied by a decrease in ZO-1protein levels (0.42-folddecrease)(all P<0.05). VPA treatment significantly reduced HIF-1α, VEGF andMLCK production, and it prevented ZO-1loss in CoCl2-stimulated Caco2cells (all P <0.05).4. Transfection of siRNA directed against HIF-1α led to inhibition of VEGF andMLCK production in CoCl2-stimulated Caco2cells, accompanied by an upregulationof ZO-1(all P<0.05).ConclusionsVPA can protect against burn-induced histone deacetylation and gut barrierdysfunction. These protective effects may be due to its inhibitory action on HIF-1α,leading to a reduction in intestinal VEGF and MLCK expression and minimizingZO-1degradation.