脑缺血/再灌注期PI3K/AKT和MAPK/ERK信号传导通路的转换及意义

It is well known that a large amount of reactive oxygen species (ROS) is produced during reperfusion after brain ischemia. Intervention of cell damage during this period of time may be critical for the treatment of brain ischemia in clinic. Recently, we have found that EGF receptor is transactivated in both ischemia and reperfusion periods. However, the two intracellular signal pathways of EGF receptor, PI3K/AKT and MAPK/ERK1/2 are stimulated differently in the two time periods. During ischemia, AKT, but not ERK1/2 is phosphorylated, but it is reversed by reperfusion. ERK1/2 phosphorylation is actually dependent on reperfusion. Furthermore,during the period of brain ischemia it phosphorylated Raf-1 on Ser-259 which can decrease Raf-1 activity. We hypothesized that during the period of brain ischemia AKT may regulated the MAPK/ERK1/2 pathway through phosphorylation of Raf-1 at Ser-259 regulatory site. During the period of reperfusion, the production of ROS causing endoplasmic reticulum stress (ERS) is the main reason for supression of PI3K/AKT signaling pathways through the interaction between glucose-regulated protein 78 (GRP78) and AKT or hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1). Then it weaken the inhibition of AKT to MAPK/ERK1/2 signaling pathways.The different activities of and interaction between the two signal pathways may be the underlying mechanisms of cell damage during brain ischemia/reperfusion. In this study, we will focus on the regulation of these signal pathways in brain ischemia/reperfusion and their role in cell injury. This information will eventually contribute the new therapeutic strategies for brain ischemia in clinic.

脑缺血后再灌注期氧供给重新建立使缺血组织产生大量过氧化物。如何防治此时脑损伤不但是临床关注焦点，也依赖对脑缺血再灌注病理生理机制和信号传导途经了解。我们发现缺血与再灌注均间接激活EGF受体，但其两个主要信号传导通路PI3K/AKT和MAPK/ERK活性截然不同。缺血期AKT激活，ERK无变化；而再灌注期则反之。由此猜测：缺血期AKT高度激活抑制MAPK/ERK上游Raf；而再灌注期产生过氧化物引起内质网应激，后者通过GRP78和/或mTORC1负性调节AKT磷酸化，从而消除AKT对Raf抑制。由于AKT和ERK在细胞损伤发挥作用不同，缺血期与再灌注期两个信号传导通路转换和相互作用可能与脑缺血/再灌注引起脑损伤密切相关。本项目将对脑缺血/再灌注对两条信号传导通路影响和两条通路相互作用机制做系统研究。并针对信号通路的变化采用不同干扰手段，观察其对脑损伤影响，为脑缺血/再灌注临床治疗提供新思路。

PI3K/AKT和Raf/MAPK/ERK1/2分别是EGFR下游两条重要的信号传导通路。我们应用脑缺血模型研究脑缺血期和再灌注期EGFR、PI3K/AKT和Raf/MAPK/ERK1/2的激活。我们发现脑缺血期和再灌注期都引起EGFR间接激活。脑缺血期，AKT磷酸化水平升高，提示PI3K/AKT信号通路活化水平增加，并可以被EGFR抑制剂及金属蛋白酶抑制剂所抑制。然而脑缺血期并不引起ERK1/2磷酸化，ERK1/2磷酸化依赖于再灌注期。SOS1与EGFR免疫共沉淀实验结果说明脑缺血期EGFR与SOS1结合增加，表明其信号阻断位点在SOS1下游。而脑缺血期Raf-1抑制性位点Ser259磷酸化升高，而活性位点Ser338磷酸化无改变。并且引起Raf-1与AKT结合增加，而且PI3K和AKT抑制剂不仅能抑制AKT磷酸化，而且能恢复脑缺血期ERK1/2磷酸化。以上结果表明脑缺血期PI3K/AKT信号通路通过提高Raf-1抑制性位点Ser259磷酸化水平，抑制Raf-1活性，进而抑制Raf/MAPK/ERK信号传导通路。再灌注期，ERK磷酸化显著升高，但AKT磷酸化无改变。应用ROS和PTEN的抑制剂可以恢复再灌注期AKT磷酸化，并抑制ERK1/2磷酸化，表明再灌注期通过ROS引起PTEN活性增加抑制AKT磷酸化，进而解除AKT对ERK1/2磷酸化的抑制。