内源性H_2S和NO在大鼠脑缺血-再灌注损伤中的相互作用

Interaction Between Endogenous Hydrogen Sulfide and Nitric Oxide during Rat Global Cerebral Ischemic-Reperfusion

目的研究胱硫醚β-合酶(cystathionine beta synthase,CBS)/硫化氢(H2S)体系和诱导型一氧化氮合酶(inducible nitric oxide synthasea,iNOS)/一氧化氮(NO)体系在大鼠脑缺血/再灌注损伤中的相互作用,探讨脑保护策略.方法 24只Wister大鼠随机分为4组(n=6):对照组(C)、脑缺血/再灌注组(I/R)、脑缺血-再灌注+氨基胍(iNOS抑制剂)组(I/R+A)、脑缺血-再灌注+羟氨(CBS抑制剂)组(I/R+H).采用4血管阻断方法制作大鼠全脑缺血-再灌注模型.对照组行假手术,脑缺血/再灌注+氨基胍组夹闭两侧颈总动脉前30 min腹腔注射氨基胍500 mg/kg,脑缺血-再灌注+羟氨组夹闭两侧颈总动脉前30 min腹腔注射羟氨5 mmol/L,对照组和脑缺血/再灌注组腹腔注射等量生理盐水.缺血20 min再灌注6 h后处死大鼠取海马,检测大鼠海马组织中H2S、NO、GSH、SOD、MDA量的变化及CBS-mRNA和iNOS-mRNA表达水平;电镜观察海马线粒体的变化.结果脑缺血/再灌注组与对照组相比大鼠海马中H2S、NO的量增高,GSH、SOD的量降低,MDA的量增高,CBS-mRNA和iNOS-mRNA表达增高(P<0.01),电镜观察海马线粒体受损;脑缺血-再灌注+氨基胍组与脑缺血/再灌注组相比大鼠海马中NO、MDA的量降低,H2S、GSH和SOD的量增高,CBS-mRNA的表达增高,iNOS-mRNA表达降低(P<0.05或P<0.01),电镜观察线粒体损伤减轻;脑缺血-再灌注+羟氨组与脑缺血-再灌注组相比大鼠海马中H2S、GSH和SOD的量降低,NO、MDA的量增高,CBS-mRNA的表达降低,iNOS-mRNA表达增高(P<0.05或P<0.01),电镜观察线粒体损伤加重.结论脑缺血/再灌注损伤过程中,iNOS/NO体系参与了神经细胞的损伤,而CBS/H2S体系具有抗损伤的作用,两体系间存在相互的调节;iNOS抑制剂在脑缺血-再灌注损伤过程中对神经细胞有保护作用.

Objective To investigate the interaction between CBS/H2S and iNOS/NO systems during global cerebral ischemia-reperfusion and explore brain protection methods.Methods Twenty-four Wistar male rats were randomly divided into 4 groups（n=6 in each gorup）：control（C）group,ischemia-reperfusion（I/R） group,ischemia-reperfusion＋aminoguanidine（I/R＋A）group,and ischemia-reperfusion＋hydroxylamine（I/R＋H） group.Global cerebral ischemia-reperfusion model was established by 4-vessel occlusion.The rats in group C were carried out sham operation.The rats in group I/R＋A and group I/R＋H were respectively intraperitoneal injected 500 mg/kg Aminoguanidine or 5 mmol/L Hydroxylamine before 30 min of clasping bilateral carotid artery.The rats in group C and group I/R were injected equation saline.The carotid clasps were removed after 20 min of 4-vessle occlusion and rats were killed at 6 h after reperfusion.The concentration of H2S,NO,GSH,MDA,the activity of SOD,and expression level of CBS-mRNA and iNOS-mRNA in rats＇ hippocampus were measured.The mitochondrial structure was observed by electron microscope.Results The concentration of H2S,NO and MDA was increased,GSH and SOD was decreased,iNOS-mRNA and CBS-mRNA expression was increased in group I/R as compared with group C（P〈0.01）.Meanwhile the structure of mitochondrial was destroyed.The levels of NO and MDA were decreased,but H2S,GSH and SOD were increased,iNOS-mRNA expression was decreased and CBS-mRNA was increased in group I/R＋A as compared with group I/R（P〈0.05 or P〈0.01）,meanwhile the injury of mitochondria was alleviated.The levels of GSH and SOD were significantly decreased,but levels of NO and MDA were increased,iNOS-mRNA expression was increased and CBS-mRNA was decreased in group I/R＋H as compared with group I/R（P〈0.05 or P〈0.01）,meanwhile the injury of mitochondria was aggravated.Conclusions iNOS/NO system can induce nerve cells damage,but CBS/H2S has an ahti-damage role in cerebral ischemia-reperfusion,they may interact with each other.iNOS inhibitor has neuroprotective effects.