Effects of Bone Marrow-Derived Mesenchymal Stem Cells on the Axonal Outgrowth through Activation of PI3K/AKT Signaling in Primary Cortical Neurons Followed Oxygen-Glucose Deprivation Injury

BACKGROUND: Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs. METHODOLOGY/PRINCIPAL FINDINGS: (1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen–Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×10(3), 5×10(5)/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours. RESULTS: Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×10(5)/ml and of 5×10(3)/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor LY294002. CONCLUSIONS/SIGNIFICANCE: BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway

IL-10 Protects Neurites in Oxygen-Glucose-Deprived Cortical Neurons through the PI3K/Akt Pathway.

IL-10, as a cytokine, has an anti-inflammatory cascade following various injuries, but it remains blurred whether IL-10 protects neurites of cortical neurons after oxygen-glucose deprivation injury. Here, we reported that IL-10, in a concentration-dependent manner, reduced neuronal apoptosis and increased neuronal survival in oxygen-glucose-deprived primary cortical neurons, producing an optimal protective effect at 20ng/ml. After staining NF-H and GAP-43, we found that IL-10 significantly protected neurites in terms of axon length and dendrite number by confocal microscopy. Furthermore, it induced the phosphorylation of AKT, suppressed the activation of caspase-3, and up-regulated the protein expression of GAP-43. In contrast, LY294002, a specific inhibitor of PI3K/AKT, reduced the level of AKT phosphorylation and GAP-43 expression, increased active caspase-3 expression and thus significantly weakened IL-10-mediated protective effect in the OGD-induced injury model. IL-10NA, the IL-10 neutralizing antibody, reduced the level of p-PI3K phosphorylation and increased the expression of active caspase-3. These findings suggest that IL-10 provides neuroprotective effects by protecting neurites through PI3K/AKT signaling pathway in oxygen-glucose-deprived primary cortical neurons

Differentiated capacity of BMSCs.

<p>(A) 200× The third passage of BMSCs were fibroblast-like cells and uniformly distributied on the bottom of a plastic flask. (B) 400× Cells stained with Oil-red-O dyes show that BMSCs differentiated into lipid laden adipocyte (red). (C) 400× Cell stained with Von kossa dyes show that BMSCs differentiated into osteocyte of calcium deposits (black).</p

Detection of GAP-43 and p-AKT expression.

<p>(A) The most representative image of wesrernblot analysis for GAP-43 and p-AKT expression. (B) Statistical graph of GAP-43 expression in different group (n = 6). (C) Statistical graph of p-AKT expression in different group (n = 6). ^★P<0.05 vs Control group, ^#P<0.05 vs CM group.</p

CM treatment promote axonal outgrowth.

<p>(A)OGD group: After OGD culture, axons of neurons were injured and shortened. (B)CM group: OGD-Injured axons of neurons were protected and enhanced by CM. (C) LY294002+CM group: Effect of promoting axonal outgrowth of CM was blocked by inhibitor LY294002. (D) LY294002 group. ^★P<0.05 vs control group; ^♦P<0.05 vs CM group. Scale bar = 20 µm.</p

BMSCs treatment promoted axonal outgrowth.

<p>(A) Immunofluorescence showing body and neurites of cell (red). (B) Manually traceing morphology of neurites by the ZEN2009 software (yellow). (C) Quantification of the longest axonal length of neurons. Data are expressed as mean±SD. ^#P<0.01 vs Normal control; ^★P<0.05 vs OGD; ^▴P>0.05 vs BMSCs (5×10³/ml). (D) OGD (E) OGD+BMSCs (5×10³/ml), (F) OGD+BMSCs (5×10⁵/ml). Scale bar = 20 µm.</p

Detection of neuronal survival by flow cytometry.

<p>After 48-plot graph. (A) OGD, (B) OGD+CM, (C) OGD+CM+LY294002, (D) OGD+LY294002. (E) ^#P<0.01 vs OGD, ^★P<0.01 vs OGD+CM.</p

Detection of GAP-43 expression.

<p>(A) The most representative image of wesrernblot analysis for GAP-43 expression (B) Statistical graph of GAP-43 expression in different group (n = 6) ^#P<0.05 vs Normal control; ^★P<0.05 vs OGD; ^▴P>0.05 vs BMSCs (5×10³/ml).</p