Protective Role Of Sonic Hedgehog Signaling In Primary Cultured Cortical Astrocytes Against Oxidative Stress

Objectives: This study aimed to explore the protective role of Sonic Hedgehog signaling in primary cultured cortical astrocytes against oxidative stress.Methods: This experiment was performed in the central laboratory of union hospital and the laboratory of department of neurology, Tongji medical college, Huazhong University of Science and Technology. 1. The matter of cerebral cortex dissected from postnatal 1 to 3 days SD rats was dissociated for astrocytes primary culture. These cultured cells were treated with H2O2 to mimic oxidative stress. The endogenous expression of Shh signaling pathway components such as Shh, Patched 1 and Gli-1 were determined by Real-time PCR and western-blot. 2. The cells divided into the control, H2O2, cyclopamine or 5E1 (a selective inhibitor of the Shh signaling pathway) and/or exogenous Shh, and the changes of cell vitality and cell apoptosis were observed by MTT and flow cytometry assays. 3. The expression of Bcl-2 and Bax were determined by Western-blot and quantitative RT-PCR.Results: 1. Oxidative stress could up-regulate endogenous expression of Shh, Patched1 and Gli-1. 2. Treatment with exogenous Shh improved cell vitality and inhibited cell apoptosis, and the effect could be partly inhibited by cyclopamine or 5E1. 3. Exogenous Shh could up-regulated the expression of Bcl-2, but down-regulated Bax expression. These regulatory effects of Shh were partially rescued by cyclopamine.Conclusion: Oxidative stress could improve the expression of Shh and reactive Shh signal. Exogenous Shh improved cell vitality and inhibited cell apoptosis, indicating playing a role of neuroprotective effect.