脑缺血再灌注对海马神经MMP-9表达及ERK信号通路的影响

Cerebral ischemia induce upregulation of MMP9 function in rat hippocampus and its underlying mechanisms

目的研究脑缺血诱导的海马神经基质金属蛋白酶(MMP)-9表达变化及其相关分子调节机制。方法①取雄性SD大鼠20只随机分为模型组15只和假手术组5只,模型组采用四动脉阻断法建立前脑缺血模型(再灌注时间分别为6、12、24 h),假手术组手术步骤同上、但不阻断动脉;两组均断头取脑,采用电泳和免疫印迹法检测胞外信号调节激酶(ERK)、磷酸化(p)-ERK、前酶原(pro)-MMP-9蛋白表达,选择表达最显著的再灌注时间点用于下述实验的模型制备。②取30只SD大鼠随机分为氯胺酮组、U0126组、腹腔溶剂组、脑室溶剂组各5只和假手术组10只,前四组均采用上述方法建立前脑缺血模型,在动脉阻断前30 min氯胺酮组腹腔注射氯胺酮50 mg/kg,U0126组用微量注射器通过单侧脑室注射U0126(0.5μg/2μL),腹腔溶剂组和脑室溶剂组分别经腹腔和脑室注射等量生理盐水,假手术组处理同上,各组处理完毕后均同上检测三种蛋白表达。结果前脑缺血再灌注后p-ERK和pro-MMP-9蛋白水平持续上调(P<0.05),且24 h最高;氯胺酮组p-ERK和pro-MMP-9蛋白水平显著高于腹腔溶剂组(P<0.05),U0126组pro-MMP-9蛋白表达显著高于脑室溶剂组(P<0.05)。结论脑缺血可诱导海马脑区pro-MMP-9基因表达和ERK活性上调,胞内N-甲基-D-门冬氨酸(NMDA)受体介导的钙信号增强及其激活的ERK级联是其重要机制;此为缺血后脑损伤的临床治疗提供了新的思路。

Objective To investigate cerebral ischemia-induced upregulation of matrix metalloproteinase （MMP）-9 function in rat hippocampus and its underlying mechanisms. Methods ① Total of 20 SD rats were divided randomly into model group （n = 15 ） and sham group（ n = 5 ）, the model group was established forebrain ischemia model by using four ar-tery occluding method（the reperfusion was 6,12 and 24hs）, the sham group was treated as above, only the arteries were not occluded;the two groups were both killed and the brain tissues were obtained, immunoblotting assay was employed to observe the activation of extracellular signal-regulated kinase （ERK）, phosphorylated ERK （p-ERK） and prozymogen （pro）-MMP-9, the most significant reperfusion time pot of expression was chosen and used in the follow experiment. ②Total of 30 SD rats were divided randomly into ketamine group（ n = 5 ）, U0126 group（ n = 5 ）, celiac solvent group（ n = 5 ）, ventricle solvent group（ n = 5 ） and sham group （n = 10）, the former four groups were all made forebrain ischemia model as above, and 50 mg/kg ketamine and 0.5 μg/2 μL U0126, equal normal saline were given respectively 30 mins before the obstruction, the sham group was treated as above, then the 3 indices were detected as above. Results The level of p-ERK and pro-MMP-9 protein exhibited a significant elevation in response to reperfusion in hippocampus after ischemia （P 〈 0.05 ）, and reached the peak at 24 h ; the level of p- ERK and pro-MMP-9 protein in ketamine group were both significant-ly higher than those in celiac solvent group（ P 〈 0.05 ）, the level of pro-MMP-9 protein in U0126 group was significantly higher than that in ventricle solvent group（P 〈 0.05）. Conclusion Cerebral ischemia-reperfusion can induce MMP-9 gene expression and protein increase dependent on ERK pathway, the enhancement of calcium signal and its ERK cascade play important role in the mechanism; this can provide new ways for the treatment of the cerabral ischemia injury.