硫化氢在肠缺血-再灌注损伤大鼠肠黏膜屏障功能障碍中的作用

Role of hydrogen sulfide in gut mucosal barrier dysfunction during gut ischemia-reperfusion injury

目的 观察硫化氢（H_2S）在肠缺血.再灌注损伤大鼠肠黏膜屏障功能障碍中的作用.方法 雄性Wistar大鼠24只,分为S（假手术）组、Ⅰ（缺血-再灌注）组,N（缺血-再灌注＋NaHS）组（n=8）,N组在再灌注前10 min静脉注射100 μmol/kg NaHs后按每小时1 mg/kg持续静脉注射直到再灌注2 h,S和Ⅰ组静脉注射相同体积的生理盐水.采用改良的酶学分光光度法测定血浆D-乳酸水平,采用分光光度法检测小肠黏膜超氧化物歧化酶（SOD）和髓过氧化物酶（MPO）、丙二醛（MDA）、黄嘌呤氧化酶（XO）水平,敏感硫电极法检测硫化氢（H_2S）浓度.电镜下观察肠黏膜形态学改变,TUNEL染色观察小肠上皮细胞凋亡指数（AI）,逆转录.聚合酶链反应（RT-PCR）法检测小肠黏膜组织胱硫醚-γ-裂解酶（CSE）、胱硫醚-β-合酶（CBS）、多聚ADP核糖合成酶（PARP）和细胞凋亡蛋白酶（Caspase）-3 mRNA的表达,蛋白质印迹法检测小肠黏膜PARP和Caspase-3蛋白水平.结果 N组D-乳酸含量、AI分别为（2.35±0.26）mg/L、（24.41±2.76）%,低于Ⅰ组、高于S组（P〈0.01）,两者正相关（r=0.892,P〈0.01）;N组MDA、XO分别为（9.17±0.35）nmol/mg、（9.94±0.41）U/g,低于Ⅰ组、高于S组（P〈0.01）,两者正相关（r=0.995,P〈0.01）;N组CSE mRNA、H_2S、SOD、MPO分别为（0.33±0.02）μmol/L、（35.27±3.14）μmol/L、（8.83±0.29）U/mg、（5.95±0.49）U/mg;N组CSE mRNA、H_2S、SOD水平均低于S组（P〈0.01）,N组H2S、SOD水平均高于Ⅰ组（P〈0.01）,N组MPO水平高于S组、低于Ⅰ组（P〈0.01）;N组活化的Caspase-3、PARP蛋白表达量分别为11.50±1.25、9.37±1.18,高于s组、低于Ⅰ组（P〈0.01）,两者正相关（r=0.785,P〈0.01）.结论 H_2S对肠缺血再灌注损伤大鼠肠黏膜屏障功能障碍有保护作用,其机制之一是减少中性粒细胞浸润和激活、肠上皮细胞氧化损伤水平,增加SOD清除氧自由基的活性,下调活化的Caspase-3和PARP蛋白表达.

Objective To investigate the role of hydrogen sulfide in gut mucosal barrier dysfunc tion during gut ischemia-reperfusion injury in rats. Methods Twenty-four Wistar rats were randomly divided into three groups （8 in each group） ：sham-operated （S） ,ischemia-reperfusion （I） .ischemia-reper fusion and sodium hydrosulfide （N）. Rats in group N received sodium hydrosulfide （100 μmol/kg bolus ＋ 1 mg·kg^（-1）·h^（-1） infusion） 10 min prior to the onset of reperfusion, whereas rats in groups S and I received normal sodium of equal volume. The concentration of D-lactate in serum was measured by modified enzymol ogy spectrometry. Intestinal mucosa morphology was observed under an electron microscope. The contents of malondialchehyche （MDA） ,superoxide dismustase （SOD） , xanthine oxidase （XO） ,and myeloperoxidase （MPO） on mucous membrane of small intestine were measured by spectrophotometry. The contents of hydro gen sulfide in serum were determined by sensitive sulfur-electrode assay. Apoptosis index （AI） was tested by TdT-mediated dUTP nick end labeling （TUNEL） staining. The expression of cystathionine-γ-lyase （CSE） ,poly （ADP-ribose） polymerase （PARP）, Caspase-3, and cystathionine-β-synthase （CBS） mRNA was detected by Reverse Transcription Polymerase Chain Reaction （RT-PCR） ,and that of Caspase-3 and PARP by Western blot. Results Plasma D-lactate concentration and AI in small intestine epithelium in group N were （2.35 ±0.26） mg/L,and （24.41 ±2.76）% respectively,which were dramatically lower than in group I,significantly higher than in group S （P 〈0.01） ,and there was a positive correlationg among groups （r = 0.892,P〈0.01）.With positive correlation （r =0.995,P〈0.01） .the contents of MDA and XO in group N were （9.17 ±0.35） nmol/mg,and （9.94 ±0.41） U/g respectively .which were predominandy lower than in group I,but significantly higher than in group S （P 〈0.01）. The levels of CSE mRNA,H_2S,SOD and MPO in group N were （0.33 ±0.02）, （35. 27 ±3. 14） μmol/L, （8. 83 ±0. 29） U/mg,and （5.95±0.49） U/mg respectively. Those of H_2S and SOD were notably lower than in group S, but significantly higher than in group I （P〈0.01） .while that of MPO was strikingly lower than in group I,but significantly higher than in group S （P 〈 0.01）. The expression of cleaved Caspase-3 and PARP protein in group N was 11.50 ± 1.25, and 9.37 ±1.18 respectively .which was dramatically lower than in group I, but significantly higher than in group S （P 〈 0. 01）, and there was a positive correlation between Caspase-3 and PARP proteins （r = 0.785 ,P 〈0.01）. Conclusion H_2S plays an important role in preventing gut mucosal barrier dysfunction during gut ischemia-reperfusion injury in rats by attenuating enterocyte oxidative damage degree, inhibiting neutrophil infiltration and activation,up-regulating SOD activity to eliminate oxygen free radical,and down regulating the expression of cleaved Caspase-3 and PARP protein.