缺血后处理对肠缺血/再灌注损伤大鼠回肠胱硫醚-γ-裂解酶/硫化氢通路的影响

Effects of post ischemic conditioning on cystathionine-y-lyase/hydrogen sulfide pathway in rats with intestinal ischemic/reperfusion injury

目的探讨缺血后处理(PC)对肠缺血/再灌注(I/R)损伤大鼠回肠组织胱硫醚-γ-裂解酶/硫化氢(CSE/H2S)通路的影响.方法将30只健康雄性Wistar大鼠按随机数字表法分为假手术组(Sham组)、I/R组和I/R+PC组,每组10只.采用无损伤血管夹阻断肠系膜上动脉(SMA)后恢复血流的方法建立肠I/R损伤模型;Sham组仅分离SMA后关腹.I/R+PC组于制模1h后采用反复阻断SMA并恢复血流的方法进行PC;I/R组持续阻断SMA血流2h.实验结束后处死大鼠,取下腔静脉血和回肠组织,苏木素-伊红(HE)染色后光镜下观察回肠组织病理学改变,并进行病理学评分;采用原位末端缺刻标记法(TUNEL)观察回肠上皮细胞凋亡情况;采用敏感硫电极法测定血浆和回肠组织H2S水平;采用反转录-聚合酶链反应(RT-PCR)及蛋白质免疫印迹试验(Western Blot)检测回肠组织CSE、胱硫醚-β-合成酶(CBS)的mRNA和蛋白表达.各指标间相关性采用直线相关分析.结果与Sham组比较,I/R组HE染色显示有大量腺上皮细胞脱落,绒毛水肿、出血,TUNEL染色显示有大量棕黄色的凋亡回肠上皮细胞,病理学评分和凋亡指数(AI)均显著升高,血浆和回肠组织H2S水平明显降低,回肠组织CSE的mRNA和蛋白表达显著下降.给予PC干预后,I/R+PC组回肠组织病理学改变明显减轻,凋亡细胞明显减少,定量分析显示病理学评分和AI均较I/R组明显降低〔病理学评分(分):2.34±0.23比4.25±0.37,AI:0.32±0.07比0.55±0.04,均P<0.01〕;同时I/R+PC组血浆和回肠组织H2S水平均较I/R组明显升高〔μmol/L:血浆为31.25±3.62比21.43±3.17,回肠组织为38.24±3.48比25.39±3.41,均P<0.01〕;RT-PCR和WesternBlot结果显示,I/R+PC组回肠组织CSE的mRNA和蛋白表达均较I/R组明显上调〔CSEmRNA(CSE/GAPDH):0.45±0.03比0.27±0.04,CSE蛋白(CSE/GAPDH):0.62±0.02比0.42±0.04,均P<0.01〕.各组回肠组织CBS的mRNA和蛋白表达差异均无统计学意义(均P>0.05).相关性分析显示,血浆和回肠组织H2S水平与回肠病理学评分、AI均呈显著负相关(血浆:r值分别为-0.59、-0.67;回肠组织:r值分别为-0.65、-0.61,均P<0.01);CSE的mRNA和蛋白表达与回肠病理学评分、AI均呈显著负相关(CSEmRNA:r值分别为-0.59、-0.62,CSE蛋白:r值分别为-0.63、-0.56,均P<0.01).结论PC可以通过上调肠I/R损伤大鼠CSE/H2S通路表达对肠组织发挥保护作用.

Objective To investigate the effects of post ischemic*conditioning(PC)on the expression nf ryslathionine-γ-lyase/hydrogen sulfide(CSE/H2S)pathway in rats with intestinal ischemic/reperfusion(I/R)injury.Methods Healthy female Wistar rats(n=30)were randomly divided into sham operation(Sham)group,I/R group,and I/R+PC group with 10 rats in each group.Phe animal model of intestinal I/H injury was reproduced by blocking superior mesenteric artery(SMA)with non-invasive vascular clamp followed by restoration of blood flow.SMA in I/R+PC group was continually restored blood flow after l-hour isrhemia while SMA in I/R group was blocked for 2 hours.Rats were sarrificed 2 hours after experiment.Blood in inferior vena cava and ileum were harvested.The pathologic change in ileum was observed with hematoxylin-eosin(HE)staining,and the pathology score was evaluated.Apoptosis index(AI)of ileum epithelial cells was measured by TdT—mediated dUTP Nick—end labeling(TUNEL)assay.The levels of H2S in plasma and ileum were measured hy sensitive sulfur electrode.The expressions of CSE and cystathionine—β—synthase(CHS)at mRNA and protein levels were determined by reverse transcription-polymerase rhain reaction(RT-PCR)and Western Blot.Linear correlation analysis was used to analyze the correlation among the parameters.Results Compared with Sham group,there were apoptotic,exfoliated glandular epithelial cells and edematous,hemorrhagir villi shnwed by HE and TUNEL staining.Ileum pathologic score and AI sharply inc reased,while H2S in plasma and ileum and CSE mRNA in ileum strikingly rlerreaserl.As PC was adopted,pathologic change in I/R+PC group ameliorated and apoptotic epithelial cells diminished.Quantitative analysis showed that ileum pathologic score and AI in I/R+PC group were strikingly lower than those in I/R group(pathologic score:2.43±0.23 vs.4.25±0.37,AI:0.32±0.07 vs.0.55±0.04.both P<0.01).The H2S levels in plasma and ileum in I/R+PC group were significantly higher than those in I/R group(μmol/L:31.25±3.62 vs.21.43±3.17 in plasma,38.24±3.48 vs.25.39±4.41 in ileum,both P<0.01).The results of RT-PCR and Western Blot showed that the expressions of CSE mRNA and protein in I/R+PC group were sharply higher than those in I/R group[CSE mRNA(CSE/GAPDH):0.45±0.03 vs.0.27±0.04,CSE protein(CSE/GAPDH):0.62±0.02 vs.0.42±0.04,both P<0.01].There was no statistical significance in CBS mRNA or protein among three groups(all P>0.05).Correlation analysis showed that H2S levels in plasma and ileum were negatively correlated with ileum pathologic score and AI(plasma:r value was-0.59 and—0.67,ileum:r value was—0.65 and-0.61,respectively,all P<0.01),and the expressions of CSE mRNA and protein were negatively conelated with ileum pathologic score and AI(CSE mRNA:r value was-0.59 and-0.62,CSE protein:r value was-0.63 and-0.56,respectively,all P<0.01).Conclusion PC plays a protective role in attenuating intestinal I/R injury by up-regulating CSE/H:S pathway.