EGb761 Ameliorates Neuronal Apoptosis and Promotes Angiogenesis in Experimental Intracerebral Hemorrhage via RSK1/GSK3 beta Pathway

摘要

Neuronal apoptosis after intracerebral hemorrhage (ICH) plays an essential role in neurological deterioration. Preclinical studies have shown that EGb761, an extract of Ginkgo biloba, is neuroprotective in some other neurological diseases with apoptosis. This study was conducted to investigate the potential neuroprotective effect of EGb761 on neuronal apoptosis in experimental ICH. A model of ICH was induced in C57BL/6 mice by injecting collagenase. EGb761 was administered for 21 days and neurologic behaviors were assessed at 1, 3, 7, 14, and 21 days after ICH. RNAi-mediated knockdown of p90 ribosomal S6 kinase 1 (RSK1) was used to further investigate the role of RSK1 in EGb761-induced neuroprotective effects. Neuronal death was determined by TUNEL staining. The image datasets of neurovascular networks were acquired via micro-optical sectioning tomography (MOST). The glycogen synthase kinase-3 beta (GSK3 beta) activity was assayed using commercial kit. Primary cultured cortical neurons were exposed to ferrous iron and treated with EGb761. Apoptotic neurons were counted by flow cytometry. RSK1, GSK3 beta, phosphorylated-GSK3 beta (pGSK3 beta), Bcl2, Bax, cleaved-caspase3 (CC3), and VEGF were measured by Western blot. The pGSK3 beta was also detected by immunofluorescence staining. We found that mice in EGb761 group performed better on rotarod test. Reduced TUNEL-positive neurons and richer microvascular networks were observed in mice treated with EGb761. EGb761 attenuates neuronal apoptosis induced by ferrous iron counted by flow cytometry in vitro. Decreased GSK3 beta activity was observed in EGb761-treated mice compared with mice with ICH. EGb761 increased the expression of pGSK3 beta (Ser9), RSK1 and the Bcl2/Bax ratio, and VEGF and decreased CC3 expression. In conclusion, EGb761 reduces neuronal apoptosis and promotes angiogenesis in experimental intracerebral hemorrhage via RSK1/GSK3 beta pathway.