The Pharmacological Preconditioning Effect Of Ginkgolide B On Astrocytes Against Ischemic Injury And Its Mechanisms

Objective: To observe the effect of Ginkgolide B (Gin B) againstischemic injury on astrocytes in vitro and to approach the possiblemechanisms of its pharmacological preconditioning effect.Methods: We studied the effects of pretreatment of Gin B (120μM, 24h)or 0.5 h, 3 h ischemic preconditioning (Hank's medium without glucose,1ï¼…O₂) on astrocytes in vitro against ischemic injury for 24 h (Hank'smedium without glucose, 1ï¼…O₂) through the observation of cellularmorphological and immunofluorescent changes; detection of apoptoticcell nuclei analyzed by fluorescence microscopy with dye Hoechst 33342;analysis of cell viability by use of MTT and LDH assays; measurement ofthe expression of erythropoietin (EPO), bystin, glial fibrillary acidicprotein (GFAP), phosphorylated-Bad (p-Bad), phosphorylated-extracellular regulated protein(p-ERK), phosphorylated-glycogensynthase kinase-3 (p-Ser9-GSK-3β) by western-blot analysis.Results:(1) After exposed to ischemia for 0.5 h,3 h or treated with 120μM GinB for 24 h, astrocytes acquired a characteristic 'reactive' morphologysuch as enlarged cell bodies, prolonged and overlapped processes couldbe seen under light microscope and displayed a series of changesincluding: enhancement of cellular viability and up-regulation of EPO,bystin, p-Bad, p-Ser9-GSK-3β, p-ERK expression. (2) After ischemia for 24 h, cell bodies began to shrink, processesdecurtated or even disappeared, viabilities descended, fragmented andmicro-nuclei were stained by Hoechst 33342, and expression of EPO,bystin, p-Bad, p-Ser9-GSK-3β, p-ERK down-regulated.(3) Treated with 120μM Gin B or 0.5 h, 3 h IP with 24 h intervalbefore ischemic injury, the morphology of cell bodies were recovered andviabilities increased, fragmented and micro-nuclei stained by Hoechst33342 were seldomly found, and expression of EPO, bystin, p-Bad,p-Ser9-GSK-3β, p-ERK up-regulated.(4) Reactive astrocytes can be induced by ischemia for 0.5 h, 3 h,treatment with 120μM Gin B or chemical hypoxia. The expression ofbystin could discriminate the reactive stage of astrocytes conspicuously.(5) The up-regulated expression of apoptotic related protein ¹¹²p-Badis inhibited by PD98059, an inhibitor of MEK/ERK pathway and¹³⁶p-Bad by LY294002, an inhibitor of PI3K/AKT/GSK-3βpathway.Conclusions: Pretreatment with Gin B have protective effects againstischemic injury on astrocytes in vitro, which is similary to the effects ofIE The effects are related to the increased expression of EPO, p-Bad,activation of MEK/ERK and PI3K/AKT/GSK-3βpathways.