中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
1期
71-74
,共4页
肠黏膜%缺血%再灌注损伤%硫化氢%脱噬作用
腸黏膜%缺血%再灌註損傷%硫化氫%脫噬作用
장점막%결혈%재관주손상%류화경%탈서작용
Intestinal mucosa%Ischemia%Reperfusion injury%Hydrogen sulfide%Apoptosis
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.
目的 觀察硫化氫(H_2S)在腸缺血.再灌註損傷大鼠腸黏膜屏障功能障礙中的作用.方法 雄性Wistar大鼠24隻,分為S(假手術)組、Ⅰ(缺血-再灌註)組,N(缺血-再灌註+NaHS)組(n=8),N組在再灌註前10 min靜脈註射100 μmol/kg NaHs後按每小時1 mg/kg持續靜脈註射直到再灌註2 h,S和Ⅰ組靜脈註射相同體積的生理鹽水.採用改良的酶學分光光度法測定血漿D-乳痠水平,採用分光光度法檢測小腸黏膜超氧化物歧化酶(SOD)和髓過氧化物酶(MPO)、丙二醛(MDA)、黃嘌呤氧化酶(XO)水平,敏感硫電極法檢測硫化氫(H_2S)濃度.電鏡下觀察腸黏膜形態學改變,TUNEL染色觀察小腸上皮細胞凋亡指數(AI),逆轉錄.聚閤酶鏈反應(RT-PCR)法檢測小腸黏膜組織胱硫醚-γ-裂解酶(CSE)、胱硫醚-β-閤酶(CBS)、多聚ADP覈糖閤成酶(PARP)和細胞凋亡蛋白酶(Caspase)-3 mRNA的錶達,蛋白質印跡法檢測小腸黏膜PARP和Caspase-3蛋白水平.結果 N組D-乳痠含量、AI分彆為(2.35±0.26)mg/L、(24.41±2.76)%,低于Ⅰ組、高于S組(P<0.01),兩者正相關(r=0.892,P<0.01);N組MDA、XO分彆為(9.17±0.35)nmol/mg、(9.94±0.41)U/g,低于Ⅰ組、高于S組(P<0.01),兩者正相關(r=0.995,P<0.01);N組CSE mRNA、H_2S、SOD、MPO分彆為(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組CSE mRNA、H_2S、SOD水平均低于S組(P<0.01),N組H2S、SOD水平均高于Ⅰ組(P<0.01),N組MPO水平高于S組、低于Ⅰ組(P<0.01);N組活化的Caspase-3、PARP蛋白錶達量分彆為11.50±1.25、9.37±1.18,高于s組、低于Ⅰ組(P<0.01),兩者正相關(r=0.785,P<0.01).結論 H_2S對腸缺血再灌註損傷大鼠腸黏膜屏障功能障礙有保護作用,其機製之一是減少中性粒細胞浸潤和激活、腸上皮細胞氧化損傷水平,增加SOD清除氧自由基的活性,下調活化的Caspase-3和PARP蛋白錶達.
목적 관찰류화경(H_2S)재장결혈.재관주손상대서장점막병장공능장애중적작용.방법 웅성Wistar대서24지,분위S(가수술)조、Ⅰ(결혈-재관주)조,N(결혈-재관주+NaHS)조(n=8),N조재재관주전10 min정맥주사100 μmol/kg NaHs후안매소시1 mg/kg지속정맥주사직도재관주2 h,S화Ⅰ조정맥주사상동체적적생리염수.채용개량적매학분광광도법측정혈장D-유산수평,채용분광광도법검측소장점막초양화물기화매(SOD)화수과양화물매(MPO)、병이철(MDA)、황표령양화매(XO)수평,민감류전겁법검측류화경(H_2S)농도.전경하관찰장점막형태학개변,TUNEL염색관찰소장상피세포조망지수(AI),역전록.취합매련반응(RT-PCR)법검측소장점막조직광류미-γ-렬해매(CSE)、광류미-β-합매(CBS)、다취ADP핵당합성매(PARP)화세포조망단백매(Caspase)-3 mRNA적표체,단백질인적법검측소장점막PARP화Caspase-3단백수평.결과 N조D-유산함량、AI분별위(2.35±0.26)mg/L、(24.41±2.76)%,저우Ⅰ조、고우S조(P<0.01),량자정상관(r=0.892,P<0.01);N조MDA、XO분별위(9.17±0.35)nmol/mg、(9.94±0.41)U/g,저우Ⅰ조、고우S조(P<0.01),량자정상관(r=0.995,P<0.01);N조CSE mRNA、H_2S、SOD、MPO분별위(0.33±0.02)μmol/L、(35.27±3.14)μmol/L、(8.83±0.29)U/mg、(5.95±0.49)U/mg;N조CSE mRNA、H_2S、SOD수평균저우S조(P<0.01),N조H2S、SOD수평균고우Ⅰ조(P<0.01),N조MPO수평고우S조、저우Ⅰ조(P<0.01);N조활화적Caspase-3、PARP단백표체량분별위11.50±1.25、9.37±1.18,고우s조、저우Ⅰ조(P<0.01),량자정상관(r=0.785,P<0.01).결론 H_2S대장결혈재관주손상대서장점막병장공능장애유보호작용,기궤제지일시감소중성립세포침윤화격활、장상피세포양화손상수평,증가SOD청제양자유기적활성,하조활화적Caspase-3화PARP단백표체.
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.
