缺血性脑中风钙依赖性激活Raf-1/ERK的级联机制研究

Mechanisms underlying calcium dependent activation of the Raf-1/ERK cascade following cerebral ischemia

目的:研究Raf-1磷酸化与脑缺血诱导的ERK钙依赖性激活的联系。方法:采用4动脉阻断法诱导大鼠前脑缺血,用免疫印迹技术观察缺血/再灌后海马组织Raf-1和ERK磷酸化的变化,以及钙通道抑制剂氯胺酮对它们活性的影响。结果:缺血/再灌15 min,海马内ERK活性显著升高,而Raf-1的Ser259而不是Tyr340和Ser338位点磷酸化显著降低;氯胺酮能阻断缺血后Raf-1(Ser259)的去磷酸化,继而抑制Raf-1/ERK活性。结论:脑缺血通过钙依赖性地诱导Raf-1Ser259位点的去磷酸化,暴露其催化结构域,从而导致ERK级联的活性上调。

AIM： To investigate the relationship between Raf - 1 phosphorylation and calcium - dependent ERK activation following cerebral ischemia. METHODS ： Four - vessel occlusion method was used to induce forebrain ischemia in rats. Immunoblotting assay was employed to observe the phosphorylation level of Raf- 1 and ERK in hippoeampus after ischemia - reperfusion, and their activity after the treatment of ketamine. RESULTS ： The activation of ERK exhibited a significant elevation in response to 15 min reperfusion in hippocampus after ischemia, while the phosphorylation of Raf - 1 at Ser259 rather than Ser338 or Tyr340 decreased significantly. Ketamine blocked the dephosphorylation of Ser259 on Raf- 1 after ischemia, and then suppressed the activation of Raf- 1/ERK. CONCLUSION： Cerebral ischemia induces the dephosphorylation of Raf - 1 at the site of Ser259 in calcium - dependent manner, induces the formation of its catalytic domain, and finally results in up - regulation of ERK cascade.