| Objective: In recent years, many studies have confirmed that pyruvateprotects several organs against intestinal I/R injury which induced by differentclinical settings, such as trauma, shock and burn, and reduced the intestinal originof systemic inflammation and remote organ dysfunction. The protection ofpruvate may correlate with Hypoxia-Inducible Factor-1(HIF-1). Several studieshas revealed that HIF-1stabilized by pyruvate may attenuated intestinal ischemiareperfusion (I/R) injury. The intestinal ischemia-reperfusion (I/R) model was usedto compared the protective of pyruvate, glucose and citrate solutions in intestinalbarrier induced intestinal I/R; thus to investigate the mechanism of pyruvateinfluences the expression of HIF-1and the downstream gene, furthermorepromating the expression of intestinal tight junction protein ZO-1.Methods:Groups and treatments:100male SD rats, weight220~260g, were randomlydivided into5groups of SMAO model: sham group (Group S), pyruvate group(Group IP), glucose group (Group IG), citrate group (Group IC) andphysiological saline control group (Group IS). According to the differentobservation time, each group were randomly divided into three subgroups SMAO30min (I30), SMAO60min (I60) and SMAO60min and reperfusion30min(R30), each with8rats.SMAO model: rats were intramuscularly anesthesised with0.5ml/kg ofmixture of ketamine and Sumianxin II (2:1). An upper-midline laparotomy wasperformed and the jejunum was identified5-cm distal to the ligament of Treitz.One8-cm intestinal sac was created in ench rat. The proximal of the sac wasinserted laser-Doppler probe to detect the intestinal mucosal blood flow (IMBF).It was used the rat in an isolated superior mesenteric artery occlusion (SMAO) intestinal I/R injury model about SMAO30min (I30), SMAO60min (I60) and30min of reperfusion after SMAO60min (R30). Immediate blanching of thesmall intestine verified that the blood supply to the intestinal segment had beenshut off. Sacs were filled with sodium pyruvate (10mmol/L, Group IP), glucose(10mmol/L, Group IG) or sodium citrate (10mmol/L, Group IC) inphysiological saline, or physiological saline (Group IS). The jejunums of differenttime points were harvested for the following assays. Animals in Group shamunderwent superior mesenteric artery isolation without arterial clamping as acontrol. At the end of the experiment, abdominal aorta puncture andexsanguination were used to achieve euthanasia.Measurements and methods: Morphological analysis of intestinal injury atdifferent time was measured. HIF-1, erythropoietin (EPO), pyruvatedehydrogenase kinase1(PDK1) and intestinal tight junction protein ZO-1wasdetected by Western blot assay. The activities of eNOS and iNOS and the contentof NO were measured by detection kits. The intestinal mucosal blood flow wasmonitored with a laser-Doppler flowmerer. The content of MDA, H2O2and theactivity of XOD were measured by detection kits. ZO-1detected by laserconfocal microscopy and the activity of DAO were measured to evaluate theintestinal mucosa barrier function.Results:1Morphological analysis of intestinal injury: Histological evaluations ofjejunum mucosal damage revealed extensive blunting of the villus tips in ratssubjected to SMAO compared with Group S. The mucosal damage score inGroup IP was lower than other I/R groups. In contrast, the mucosal damage scorein other groups was significantly aggravated. Thus, pyruvate conferred asignificant protection against SMAO-induced villus injury.2ZO-1: ZO-1protein levels by western blot assays in the intestinal sacs ofthe rat subjected to I60were significantly reduced. The expression of ZO-1waslower in Group IP than in Group S, but significantly higher than in other I/Rgroups. Frozen sections for immunofluorescence studies of the intestinalepithelial ZO-1were also conducted. In Group S, ZO-1showed a strong and continuous fluorescent signal along the crypt-villus unit while the fluorescentsignal of other groups was not obvious, uncontinuous. The expression of ZO-1protein was lower in Group IP than in Group S, but significantly higher than inother I/R groups.3DAO: DAO activities were markedly decreased at R30in I/R groups,compared with Group S. The DAO activity of Group IP was significantlyelevated, compared with Group IS (P<0.01). The DAO activity of Group IP washigher than Group IG (P<0.01). But there was no difference between Group ISand Group IC.4HIF-1, PDK1and EPO: The pyruvate highly stabilized HIF-1in theintestinal tissue that was subjected to SMAO. Western blot assays demonstratedthat each group subjected to I60was associated with an increase of HIF-1protein,compared with Group S. The HIF-1level in Group IP was also higher than that inother groups (P<0.05). Changes in the EPO and PDK1expression wereassociated with HIF-1protein levels. EPO and PDK1protein levels werestrikingly increased at I60. In addition, the EPO and PDK1protein in Group IPwere obviously higher than in Group S and other groups (P<0.05).5eNOS, iNOS and NO: Group pyruvate markedly induced eNOS and NOin I/R intestine higher than other I/R groups (P<0.01). The I/R groups alsomarkedly increased the iNOS compared to Group S, while iNOS activity ofGroup IP was lower than other I/R groups (P<0.01). Although no significantdifference was found in the sum of the iNOS and eNOS among other I/R groups,compared with Group IS the content of iNOS was markedly reduced in Group IP.6IMBF: Compared to Group S, the IMBF was significantly reduced in therats subjected to SMAO. The IMBF was significant higher in Group IP than inother I/R groups. The IMBF in Group IG was higher than in Group IS. there wasno difference between Group IS and Group IC.7XOD, H2O2and MDA: Compared with Group S, XOD activities werecaused various increases in rats at R30. Group IP significantly reduced intestinallevels of XOD, compared to Group IS (P<0.01). The XOD activity of Group IPwas higher than Group IG and Group IC (P<0.01). There was no difference between Group IC and Group IS. Production of H2O2in I/R groups wassignificantly increased compared with Group S (P<0.01). Hypoxia caused anincrease in intracellular H2O2in Group IS in sharp contrast to the reduction inH2O2levels that was observed in Group IP. The H2O2level in Group IC washigher than in other I/R groups.The intestinal MDA levels were significantlyincreased in I/R groups subjected to R30, compared with Group S (P<0.05). Thelevel of MDA was significantly reduced in Group IP, compared with Group IS(P<0.01) and Group IG (P<0.05). The MDA level in Group IC was higher than inGroup IS (P<0.05).Conclusion:1Pyruvate might contribute to protect intestinal mucosa barrier functionagainst I/R injury by lessening the degradation of ZO-1, then reduce the damageof intestinal mucosa permeability. The protective of pyruvate was significantbetter than glucose and citrate.2The mechanism of pyruvate attenuated the intestine I/R-induced intestinalbarrier function loss might been associated with the expression of HIF-1and thedownstream gene EPO and PDK1, could activate eNOS producted NO whichcould increase intestinal mucosa blood flow, and inhibit the activity of PDH, toreduce ROS production. |
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