The Preconditioning Effects Of Ginkgolide B On Ischemic Injury Induced Apoptosis In Cultured Cortical Neurons And Its Mechanism

Objective: To observe the effects of Ginkgolide B (GB) against apoptosis induced by ischemic injury in cortical neurons and to approach the possible mechanisms.Methods: We studied the effects of pretreatment with (1)GB(120μM, 24h); (2) Ischemia(DMEM medium without glucose, 1%O₂) for 0.5h or 1h; (3) astrocyte-conditioned medium (ACM) collected from astrocytic cultures being treated with GB(120μM,24h)or ischemia for 0.5h,3h in primary cortical neurons against ischemic injury for 5h through detection of the viability of neurons by MTT and LDH methods; observation of apoptotic cell nuclei analyzed through fluorescence microscopy with dye Hoechst33342; assessment of fluorochromes for apoptotic stages of neurons by flow cytometry; surveying the contents of EPO in ACM by enzyme-linked immunosorbent assay (ELISA) and analysis of RTP801 mRNA expression by RT-PCR assays, measuring the expression of hypoxia inducible factor-1α(HIF-1α), EPO (erythropoietin), p-Bad, and caspase-3 proteins by Western-blot analysis.Results:(1) Pretreatment with GB has the preconditioning effects against ischemic injury in cortical neurons similar to ischemic pretreatment by elevating neuronal viability.(2) Apoptosis of cortical neurons can be induced by ischemic injury. Both pretreatment with GB and ischemic preconditioning have anti-apoptosis effects.(3) Increased expression of HIF-1αand EPO proteins can be induced by both pretreatment with GB and IP.(4) Elevated expression of apoptosis related gene RTP801 and protein caspase-3 are induced by ischemic injury, which can be inhibited by pretreatment with GB and IP. Augmented expression of protein p-Bad is also be induced by these pretreatments.(5) Phosphatidylinositol 3-kinase (PI3K)/ AKT (PKB) signaling inhibitor (LY294002) can inhibit the expression of EPO and p-Bad induced by GB or IP in neurons.(6) Treatment with GB can stimulate highly expression of EPO secreted by astrocytes into ACM, which has anti-apoptosis effect on ischemic injury through increasing the expression of p-Bad in neurons.Conclusions: Pretreatment with GB, similar to IP, has protective effect against ischemic injury through either direct anti-apoptosis on cortical neurons or indirect effect by stimulating astrocytes. The mechanisms of the anti-apoptosis of GB and IP may be related to elevated expression of HIF-1α, EPO, p-Bad proteins, activation of PI3K signaling pathway and down-expression of RTP801 mRNA and caspase-3 protein.