目的 应用体外培养的大鼠脑皮层星形胶质细胞,观察银杏叶提取物(EGb761)对氧糖剥夺再复氧导致细胞损伤的影响,并探讨其作用机制.方法 以连二亚硫酸钠和无糖DMEM液造成化学性氧糖剥夺(OGD)模型模拟体内脑缺血再灌注损伤.利用WST-1法检测细胞活性、细胞上清液中乳酸脱氢酶(LDH)检测细胞损伤程度、GFAP染色及Hoechst 33258染色观察细胞形态变化和凋亡,免疫蛋白印迹法测定bcl-2、bax、caspase-3蛋白的表达.结果 OGD处理90 min再复氧24 h能使细胞存活率明显下降,LDH漏出量增高,细胞形态损伤性改变、凋亡细胞增多,并能下调bcl-2/bax比值和上调caspase-3表达.而给予EGb761可逆转上述变化,减轻细胞损伤.结论 EGb761对OGD/R诱导的星形胶质细胞损伤具有保护作用,其作用机制可能与调整bcl-2/bax比值、抑制caspase-3激活对抗细胞凋亡有关.%Objective: To investigate the effect of EGb761 on the injury of astrocytes in rats after oxygen and glucose deprivation (OGD) and reoxygenation. Methods: Na2S2O4 and sugar free DMEM solution were used to establish the chemical oxygen glucose deprivation (OGD) model which was reoxygenation 24 h to mimic ischemia/ reperfusion injury in vivo. The cell proliferating ability was determined by the WST-1 cell proliferation and cyto-toxicity assay kit and the degree of cell damage was detected by the LDH release. The apoptotic cell number and the cell morphology were measured by Hoechst 33258 and GFAP staining. Meanwhile,protein expressions including the bcl—2,the bax and the caspase—3 were investigated by the Western blotting. Results: The OGD method decreased the cell viability, increased the LDH release rate and increased the number of apoptotic astrocytes. Down—regulation with the ratio of bcl—2/bax and up— regulation with the expression of caspase—3 were also treated through this method. However,the pre-treatment with EGb761 could reverse these injuries. Conclusion: EGb761 exhibits protective effect on OGD/R—induced astrocyte injury. One of the mechanisms might be related with its regulation of the balance ratio between bcl-2 and bax,reduction of the caspase-3 expression and counteraction of the astrocyte apoptosis.
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