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Effects of Inhibiting Shh Signaling Pathway with Cyclopamine Pretreatment on Proliferation of Cortical Neural Stem Cells after Oxygen-Glucose Deprivation/Reoxygenation Injury in Rats

??OBJECTIVE To investigate the effect of inhibitting sonic hedgehog(Shh) signaling pathway with cyclopamine pretreatment on proliferation of rat cortical neural stem cells (NSCs) after oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. METHODS The suspended culture was used for the isolation and purification of NSCs in neonatal Sprague-Dawley (SD) rats. The third passage NSCs for adherent culture were deprived oxygen and glucose for 150 min and recovered oxygen and glucose for 24 h. There were three groups, including normal, model and cyclopamine pretreatment groups. NSCs were identified with immunofluorescence. CCK-8 assay was used to examine cell viability. The proliferation of NSCs was measured with BrdU assay and flow cytometry cell cycle. Western blot was used to detect the protein expressions of Ptc-1,Smo and Gli-1. RESULTS There were high expression of nestin protein in suspended and adherent cultured cells. The cell vitalities in model and cyclopamine groups were decreased significantly compared with the normal group. Especially, there was less cell vitality in cyclopamine group(P<0.05). There was significantly increased for NSCs proliferation and upregulated for Ptc-1,Smo and Gli-1 proteins in the model group. On the contrary, compared with the model group, NSCs proliferation and the expressions of Ptc-1,Smo and Gli-1 proteins in cyclopamine group were significantly decreased(P<0.05). CONCLUSION Cyclopamine pretreatment can inhibit NSCs proliferation after OGD/R injury. The result suggests that Shh signaling may participate in the regulation of NSCs proliferation after injury.