Extracellular signal-regulated kinase 1/2 (ERK1/2) pathway has been shown to be important for regulating cell proliferation and survival. The role of ERK1/2 signaling in the survival and growth of neural stem cells (NSCs) has not been addressed adequately. In this work, we aimed to provide evidence that proliferation of NSCs in vitro is controlled via ERK1/2-dependent pathway. NSCs were isolated from embryonic day 14.5 (E14.5) cortex of mouse forebrain. Cells were harvested at the desired times (1 d, 3 d and 5 d) and the total protein was extracted and analyzed by Western blot. It was observed that ERK1/2 was activated during the proliferation of NSCs. In addition, mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, which directly prohibited ERK1/2 phosphorylation, inhibited the formation of neurospheres, and this inhibitory effect was dose-dependent. After treatment with 20 mumol/L PD98059, the growth of NSCs was also inhibited with time-dependence. These data indicate that ERK1/2 is essential for the proliferation of NSCs derived from mouse embryonic cortex.
Animals ; Cell Proliferation ; Cerebral Cortex ; cytology ; embryology ; Flavonoids ; pharmacology ; Mice ; Mitogen-Activated Protein Kinase 3 ; physiology ; Neural Stem Cells ; cytology ; Phosphorylation ; Protein Kinase Inhibitors ; pharmacology

OBJECTIVETo investigate the protective effects of icaritin (ICT) on apoptosis of primarily cultured rat neurons induced by Abeta(25-35) peptide and its mechanism.

METHODSCortical neurons from rat embryonic cortical on d17 pregnancy were cultured in neural basal medium for 7 days. Icaritin (ICT) was pre-incubated for 24 h before adding Abeta(25-35) peptide and then the cells were incubated for 72 h. Neuroprotective effects of ICT were evaluated by MTT assay, LDH level in medium and cell morphological observation. Meanwhile, apoptosis was determined by JC-1 staining for mitochondria membrane potential (DeltaPsim) and AO/EB double staining for genetic damage of nucleoli in monolayer cells.

RESULTS0.1 micromol.L(-1) ICT pre-incubation for 24 h prevented rat neurons from Abeta(25-35) peptide induced apoptosis significantly as demonstrated by MTT, LDH assay and morphological observation. AO/EB double staining also indicated that ICT prevented neurons from apoptosis. JC-1 staining further showed that ICT prevented decreasing of mitochondrial DeltaPsim induced by Abeta(25-35) peptide.

CONCLUSIONICT could protect primarily cultured rat neurons from Abeta(25-35) peptide induced apoptosis.

Amyloid beta-Peptides ; toxicity ; Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; embryology ; Drugs, Chinese Herbal ; pharmacology ; Flavonoids ; pharmacology ; Neurons ; cytology ; drug effects ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

Bone morphogenic protein 4 (BMP4), a member of the TGF-beta superfamily, induced neural differentiation of neural stem cells (NSCs) grown in a medium containing basic fibroblast growth factor (bFGF). The Ras protein level and the activities of the downstream ERKs were increased by transfection of BMP4 or treatment with recombinant BMP4. The effects of BMP4, including activation of the Ras-ERK pathway and induction of the neuron marker beta-tubulin type III (Tuj1), were blocked by co-treatment of the BMP4 antagonist, noggin. The roles of the Ras-ERK pathway in neuronal differentiation by BMP4 were revealed by measuring the effect of the ERK pathway inhibition by dominant negative Ras or PD98059, the MEK specific inhibitor. BMP4 is a transcriptional target of Wnt/beta-catenin signaling, and both the mRNA and protein levels of BMP4 were increased by treatment of valproic acid (VPA), a chemical inhibitor of glycogen synthase kinase 3beta (GSK3beta) activating the Wnt/beta-catenin pathway. The BMP4- mimicking effects of VPA, activation of the Ras-ERK pathway and induction of Tuj1, also were blocked by noggin. These results indicate the potential therapeutic usage of VPA as a replacement for BMP4.
Animals ; Bone Morphogenetic Protein 4/genetics/*metabolism ; Cell Differentiation/drug effects ; Cells, Cultured ; Cerebral Cortex/cytology/embryology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Neurons/*cytology ; Rats ; Rats, Sprague-Dawley ; Stem Cells/*cytology ; Up-Regulation/drug effects ; Valproic Acid/pharmacology ; beta Catenin/metabolism ; ras Proteins/genetics/metabolism

In the present study, we examined the effect of oxygen glucose deprivation (OGD) post-conditioning (PostC) on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke's medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase (LDH) release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h (O4/R20). In addition, Western blotting was used to examine the expression of heat-shock protein 70 (HSP70), Bcl-2 and Bax. The ratio of Bcl-2 expression was (0.44±0.08)% and (0.76±0.10)%, and that of Bax expression was (0.51±0.05)% and (0.39±0.04)%, and that of HSP70 was (0.42±0.031)% and (0.72±0.045)% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was (28.96±3.03)% and (37.02±4.47)%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group (P<0.05). These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.
Animals ; Apoptosis ; drug effects ; Blotting, Western ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; embryology ; Flow Cytometry ; Glucose ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Ischemic Postconditioning ; methods ; Neurons ; cytology ; drug effects ; metabolism ; Oxygen ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control ; bcl-2-Associated X Protein ; metabolism