大鼠局灶性脑缺血再灌注损伤后自噬的初步研究

Preliminary Study on the Autophagy after Focal Cerebral Ischemia Reperfusion in Rat

目的:研究大鼠局灶性脑缺血再灌注损伤后自噬溶酶体相关蛋白随损伤时间表达的规律,检测自噬体、溶酶体的变化。方法:采用线栓法建立大鼠大脑中动脉缺血再灌注损伤模型,免疫组织化学法检测损伤周边区(皮质)自噬溶酶体相关蛋白Beclin 1、cathepsin B的表达,透射电镜观察脑缺血再灌注损伤后神经细胞内自噬体的形成和溶酶体的激活。结果:免疫组织化学检测显示,脑缺血再灌注损伤后4 h Beclin 1阳性细胞增加(P<0.01),24 h达高峰,5 d仍有较多阳性细胞(P<0.01)。脑缺血再灌注损伤后2h cathepsin B阳性细胞增加(P<0.01),12h达高峰,5 d仍有较多阳性细胞(P<0.01)。透射电镜显示,脑缺血再灌注损伤后2 h即可见到损伤周边区神经细胞内自噬体形成、溶酶体增多,12～24 h最为明显,持续到5 d。脑缺血再灌注损伤后表现为自噬溶酶体相关蛋白Beclin 1、cathepsin B阳性细胞数随损伤时间而变化,以及缺血半暗带内自噬体的形成和溶酶体的激活。结果:大鼠局灶性脑缺血再灌注损伤能激活自噬溶酶体途径。

Aim： To investigate the expression of autophagy/lysosomal associated protein and detect the changes of autophagy and lysosome after focal cerebral ischemia reperfusion injury in rats. Methods： The middle cerebral artery ischemia and reperfusion model in rats was achieved by an intraluminal filament embolism. Immunohistochemistry was employed to determine the expression of Beclin I and cathepsin B in ischemic penumbra （cortex）, the formation of autophagosome and the activation of lysosome were observed in neuron after cerebral ischemia reperfusion by using transmission electron microscope （TEM）. Results： The expression of Beclin I detected by immunohistochemistry was significantly increased at 4 h （P〈0.01） and peaked at 24 h after cerebral ischemia reperfusion, its expression still could be found at 5 d （P〈0.01）. Cathepsin B was activated at 2h （P〈0.01 ）, getting its peak at 12 h, and keeping activation at 5 d after the cerebral ischemia reperfusion （P〈0.01）. The formation of autophagosomes and enhanced presence of lysosomes were detected by TEM at 2 h in ischemic cortex after cerebral ischemia reperfusion, its number was significantly increased at 12~24 h, and still could be found at 5 d. The robust elevation of Beclin 1, cathepsin B, the formation of autophagosomes and the activation of lysosomes were manifested in ischemic penumbra with the time challenged by cerebral ischemia reperfusion injury. Conclusion： Autophagy/lysosomal pathway can be activated after focal cerebral ischemia reperfusion in rats.