Protective effect of Schisandra chinensis lignans on hypoxia-induced PC12 cells and signal transduction.

It is well-known that hypoxia induces neuronal injury; however, the mechanisms underlying this observed effect remain to be determined. Schisandra chinensis lignans (SCL). The aim of this study was thus to examine the ability of Schisandra chinensis lignans (SCL) to prevent hypoxia-induced neuronal injury using a human adrenal pheochromocytoma cell line (PC12). Exposure to hypoxia significantly reduced cell survival rate in cultured PC12 cells. However, pretreatment with SCL at 10, 20 or 40 µmol/L followed by hypoxia prevented loss of cellular viability. Flow cytometry demonstrated that the apoptotic rate in PC12 cells following hypoxia was significantly increased. Pretreatment with SCL 20 or 40 µmol/L in hypoxia-exposed cells resulted in significantly reduced apoptotic rates compared to hypoxia. Immunocytochemical staining showed that protein expression of p-Akt was significantly diminished by hypoxia. Following pre-treatment with different concentrations of SCL, PC12 cells were markedly stimulated as evidenced by elevated protein expression of p-Akt in a concentration-dependent manner. The expression of p-Akt protein in the presence of PI3K/Akt signaling pathway inhibitor LY294002 and SCL was not markedly changed indicating that signal transduction was affected by this Chinese herb. There were no significant differences in total Akt protein expression following hypoxia or pretreatment with SCL. Western blot demonstrated that expression levels of caspase-3 protein were significantly increased while expression levels of Bcl-2 protein were decreased in hypoxic cells. Pretreatment with SCL followed by hypoxia significantly lowered expression levels of caspase-3 protein accompanied by elevated expression levels of Bcl-2 protein in a concentration-dependent manner. After co-incubation with LY29004 and SCL, down-regulation of expression of caspase-3 protein and up-regulation of the expression of Bcl-2 protein noted with SCL alone were suppressed. Data suggest that the protective effect exerted by SCL in hypoxia-induced PC12 cell injury involves enhanced cell proliferation and inhibition of apoptosis mediated by activation of PI3K/Akt signaling pathway. The increased protein Akt phosphorylation expression levels resulted in consequent reduced downstream caspase-3 expression and enhanced Bcl-2 expression.

Royal jelly-induced neurite outgrowth from rat pheochromocytoma PC12 cells requires integrin signal independent of activation of extracellular signal-regulated kinases.

We showed earlier that neurite outgrowth of rat pheochromocytoma PC12 cells was stimulated by royal jelly extract (PERJ) or its unique component, AMP N(1)-oxide, via adenosine A2a receptors. In this study, we found that stimulated neurite outgrowth occurred in medium supplemented with serum, but not in serum-free medium. The pentapeptide GRGDS, which includes the RGD sequence commonly shared by extracellular matrix (ECM) components, could attenuate the effect of serum, suggesting that integrin receptor signaling was essential for the neurite outgrowth induced by PERJ or AMP N(1)-oxide. PERJ or AMP N(1)-oxide also activated extracellular signal-regulated kinases 1 or 2 (ERK1/2); however, this activation was not associated with the neurite outgrowth. As it is known that Mn(2+) induces neurite outgrowth from PC12 cells and activates ERK1/2 through integrin signals and that activation of ERK1/2 is essential for Mn2+-induced neurite outgrowth, a difference in the mechanism between Mn(2+)-induced and PERJ- or AMP N(1)-oxide-induced neurite outgrowth is suggested. Furthermore, we demonstrated that PERJ contained no ECM component-like substances. These results demonstrate that AMP N(1)-oxide and its analogues were the only entities in PERJ with neurite outgrowth-inducing activity and that they required integrin signaling in addition to activation of A2a receptors to induce neurite outgrowth.