| Ischemic cerebro-vascular disease is one of the main diseases influencinghuman health, which is secondary to lack of cerebral blood supply due to severevascular stenosis or vascular blockage. Currently, many methods, such as carotidendarterectomy, stent implantation and intracranial and extracranial vascular bypass,could be selected to improve the blood flow to ischemic cerebral region. However,brain damaged induced by reperfusion has been thought to play a pivotal role inaffecting the prognosis of each treatment. Thus, it is needed to investigate themechanism underlying the brain injury caused by ischemia/reperfusion and todevelop preventive methods or medicines, which would be helpful to improve theoutcome of any treatment targeting to recover cerebral blood supply. In this study,from the point of neuronal damage and synaptic disconnections, we investigated thepotential mechanisms responsible for brain damage, and tried to found out amedicine that could be used for future treatment.Lethal autophagy is a pathway leading to neuronal death caused by transientglobal ischemia. In this study, we examined the effect of Ginsenoside Rb1(GRb1)on ischemia/reperfusion-induced autophagic neuronal death and investigated the roleof PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucosedeprivation (OGD) in SH-SY5Y cells, and transient global ischemia was producedby using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells wasassessed by MTT assay, and CA1neuronal death was evaluated byHematoxylin-eosin staining. Autophagic vacuoles were detected by using bothfluorescent microscopy in combination with acridine orange (AO) andMonodansylcadaverine (MDC) staining and transmission electronic microscopy.Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473wereexamined by western blotting analysis. GRb1inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuolesin SH-SY5Y cells. By contrast, PI3K inhibitor LY294002counteracted theprotection of GRb1against neuronal death caused by either OGD or transientischemia. LY294002not only mitigated the up-regulated protein level of phosphorAkt at Ser473caused by GRb1, but also reversed the inhibitory effect of GRb1onOGD and transient ischemia-induced elevation in protein levels of LC3II andBeclin1.The microtubule-dependent GEF-H1pathway controls synaptic re-networkingand overall gene expression via regulating cytoskeleton dynamics. Understandingthis pathway after ischemia is essential to developing new therapies for neuronalfunction recovery. However, how the GEF-H1pathway is regulated followingtransient cerebral ischemia remains unknown. This study employed a rat model oftransient forebrain ischemia to investigate alterations of the GEF-H1pathway usingWestern blotting, confocal and electron microscopy, dephosphorylation analysis, andpull-down assay. The GEF-H1activity was significantly upregulated by:dephosphorylation and translocation to synaptic membrane and nuclear structuresduring the early phase of reperfusion. GEF-H1protein was then downregulated inthe brain regions where neurons were destined to undergo delayed neuronal death,but markedly upregulated in neurons that were resistant to the same episode ofcerebral ischemia. Consistently, GTP-RhoA, a GEF-H1substrate, was significantlyupregulated after brain ischemia. Electron microscopy further showed that neuronalmicrotubules were persistently depolymerized in the brain region where GEFH1protein was downregulated after brain ischemia. The results demonstrate that theGEF-H1activity is significantly upregulated in both vulnerable and resistant brainregions in the early phase of reperfusion. However, GEF-H1protein isdownregulated in the vulnerable neurons but upregulated in the ischemic resistantneurons during the recovery phase after ischemia. The initial upregulation ofGEF-H1activity may contribute to excitotoxicity, whereas the late upregulation ofGEF-H1protein may promote neuroplasticity after brain ischemia. |
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