一氧化氮通过环磷酸鸟苷激活细胞外信号调节激酶/P^(90RSK)信号通路在大鼠脑缺血再灌注损伤中的作用

Influence of nitric oxide on cerebral ischemical reperfusion injury in rats through cyclic guanosine monophosphate mediating extracellular signal-regulated kinase/P^(90RSK) kinase signal pathway

目的:观察大鼠脑缺血再灌注损伤时诱导型一氧化氮合酶源性一氧化氮、细胞外信号调节激酶激酶/细胞外信号调节激酶的信号转导通路,分析神经细胞损伤之间的联系。方法:实验于2005-03/2006-03在中国医科大学第一临床学院麻醉实验室完成。32只Wistar大鼠随机摸球法分为4组,即对照组、模型组、诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组,每组各8只。采用4血管阻断法制作大鼠全脑缺血再灌注模型,对照组行假手术。诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组夹闭两侧颈总动脉前30min分别腹腔注射N-3-苯甲基-乙脒(诱导型一氧化氮合酶特异性抑制剂)45μmol/kg或SL327(细胞外信号调节激酶激酶特异性抑制剂)100mg/kg,对照组和模型组腹腔注射等量生理盐水。缺血20min再灌注24h后处死大鼠取海马,检测大鼠海马组织中一氧化氮、环磷酸鸟苷量的变化及诱导型一氧化氮合酶mRNA和细胞外信号调节激酶1/2、P90RSK蛋白表达水平;电镜观察海马线粒体的变化。结果:Wistar大鼠32只全部纳入结果分析。①模型组大鼠海马中一氧化氮、环磷酸鸟苷的量、诱导型一氧化氮合酶mRNA水平比对照组增高[(0.42±0.03),(0.21±0.02)μmol/g;(44.7±4.1),(19.6±1.8)nmol/L;1.07±0.04,0.25±0.02;P<0.01];诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组大鼠海马中一氧化氮、环磷酸鸟苷、诱导型一氧化氮合酶mRNA水平比模型组降低[(0.31±0.02),(0.44±0.03)μmol/g;(29.5±2.4),(46.3±4.2)nmol/L;0.70±0.03,1.10±0.04;P<0.01]。②模型组大鼠海马中细胞外信号调节激酶1/2,P90RSK蛋白表达水平较对照组升高;诱导型一氧化氮合酶抑制剂组、细胞外信号调节激酶激酶抑制剂组较模型组降低(P<0.01)。③电镜观察模型组大鼠海马线粒体变性率比对照组升高(P<0.01),诱导型一氧化氮合酶抑制剂组和细胞外信号调节激酶激酶抑制剂组大鼠海马线粒体变性率比模型组低(P<0.01)。结论:脑缺血再灌注损伤时,诱导型一氧化氮合酶源性的一氧化氮可能通过环磷酸鸟苷激活了细胞外信号调节激酶激酶/细胞外信号调节激酶/P90RSK信号转导通路诱导了神经细胞的损伤。

AIM： To investigate the cell signal pathway of inducible nitric oxide synthase （iNOS）-derived nitric oxide, extracellular signal-regulated kinase （ERK） kinase/ERK in rats with cerebral ischemical reperfusion injury and analyze the relationship with injury among nerve cells. METHODS： The experiment was conducted at the Anesthesia Laboratory, First Clinical College, China Medical University from March 2005 to March 2006. Totally 32 Wistar rats were randomly divided into 4 groups： control group, model group, iNOS inhibitor group and ERK kinase inhibitor group with 8 rats in each group. Global ischemia-reperfusion rat models were＇established with four-vessel occlusion （4VO） method. The rats in the control group did not receive any operation. The rats in the iNOS inhibitor group and ERK kinase inhibitor group received 45μmol/kg N-triphenyl- methyl-acetamidine （iNOS specific inhibitor） and 100 mg/kg SL327 （ERK kinase specific inhibitor） by intraperitoneal injection, respectively 30 minutes before common carotid artery ligation at the two sides. Saline of the same volume was injected in the control group and model group by intraperitoneal injection. The rats were killed at ischemia for 20 minutes and reperfusion for 24 hours so as to gain hippocampus. Changes of nitric oxide （NO） and cyclic guanosine monophosphate （cGMP） as well as the expressions of iNOS mRNA, ERK 1/2, P90RSK protein in hippocampal tissues of rats were examined. Change of hippocampal mitochondrion was observed under electron microscope. RESULTS： Totally 32 Wistar rats were involved in the result analysis. ①The expressions of NO, cGMP and iNOS mRNA in hippocampus increased in the model group than those in the control group [（0.42±0.03）, （0.21±0.02） Ixmol/g; （44.7±4.1）, （19.6±1.8） nmol/L;1.07±0.04, 0.25±0.02;P 〈 0.01]. The expressions of NO, cGMP and iNOS mRNA in the iNOS inhibitor group and ERK kinase inhibitor group reduced as compared with the model group [（0.31±0.02）, （0.44±0.03）μmol/g; （29.5±2.4）, （46.3±4.2） nmol/L;0.70±0.03,1.10±0.04;P 〈 0.01]. ② The expressions of ERK 1/2, P90RSK protein in hippocampal tissues of rats in the model group ＇increased as compared with the control group. It was lower in the iNOS inhibitor group and ERK kinase inhibitor group than that in the model group （P 〈 0.01）. ③Degenerative rate of hipocampal mitochondrion of rats in the model group increased as compared with the control group observed under electron microscope （P 〈 0.01）. Degenerative rate of hippocampal mitochondrion of rats in the iNOS inhibitor group and ERK kinase inhibitor group was lower than that in the model group （P 〈 0.01）. CONCLUSION： The iNOS derived-NO through cGMP activating ERK kinase/ERK/P90RSK cell signal pathway induces injury of nerve cells after cerebral ischemical reperfusion injury.