The protective effect of sonic hedgehog is mediated by the phosphoinositide [corrected] 3-kinase/AKT/Bcl-2 pathway in cultured rat astrocytes under oxidative stress.

In our previous study, we found that the sonic hedgehog (Shh) signaling pathway is activated in neurons under oxidative stress and plays a neuro-protective role [Dai RL, et al. (2011) Neurochem Res 36:67-75]; we are led to postulate that the Shh might be released by astrocytes, thereby protecting neurons against oxidant injury. In primary cultured astrocytes of rats, we found that treatment with 100 μM H₂O₂ for 24 h induced a significant increase in the mRNA and protein levels of Shh, Patched1, and Gli-1, and the increase was substantially greater in astrocytes than in neurons. In the coculture systems of astrocytes and neurons under the H₂O₂ treatment, blocking the Shh signaling pathway with 5E1 (an antibody against the N-terminal domain of Shh) could block the neuroprotective activity of astrocytes on cocultured neurons. In this study, we found that treatment with H₂O₂ (100-800 μM) for 24 h caused cell death of astrocytes in a concentration-dependent manner. MTT reduction and Trypan Blue exclusion assay showed that exogenous Shh increased survival rate of the H₂O₂-treated astrocytes, whereas pretreatment with cyclopamine (a specific inhibitor of the Shh signaling pathway) or 5E1 decreased the survival rate of the H₂O₂-treated astrocytes. Shh also inhibited H₂O₂-induced apoptosis of astrocytes, and this effect could be partially reversed by cyclopamine. We also found that Shh promoted the phosphorylation of AKT, but had no significant effect on p38 or extracellular signal regulated kinases 1 and 2 (ERK 1/2) in H₂O₂-treated astrocytes. Blocking Shh or phosphoinositide 3-kinases (PI3-K)/AKT signaling pathway with cyclopamine or LY294002 decreased the survival rate of astrocytes, induced cell apoptosis, upregulated the expression of Bax, and downregulated the expression of Bcl-2. We are led to conclude that the oxidative stress induces astrocytes to secrete endogenous Shh and exogenous administration of Shh might protect the astrocytes from oxidative stress by activating PI3-K/AKT/Bcl-2 pathway.