Effect And Mechanism Of Resveratrol On Neurite Outgrowth And Synaptogenesis Of Rat Primary Cortical Neurons After Oxygen-glucose Deprivation/Reoxygenation (OGD/R) In Vitro

Background: Neurite outgrowth and synaptogenesis is a critical step for function outcome after stroke.Resveratrol can promote neurite outgrowth and synaptogenesis.However,its mechanism is less well understood.The Sonic hedgehog(Shh)signaling pathway is crucial for neuritogenesis and synaptogenesis in the embryonic,adult and ischemic brain,but relatively little is known about the role of Shh signaling in resveratrol-enhanced neuritogenesis and synaptogenesis after stroke.Moreover,resveratrol is a specific activator of Sirtuins1(Sirt1).whether does the Shh signaling pathway interact with Sirt1? It remains unclear.Objective: To investigate whether Shh signaling pathway mediates resveratrol to enhance neurite outgrowth and synaptogenesis of neurons after oxygen-glucose deprivation / reoxygenation(OGD/R)in vitro,and whether Shh signaling pathway interacts with Sirt1.Methods: Primary cortical neurons were cultured under oxygen and glucose deprivation for 150 minutes and reoxygenation for 24 hours.(1)To examine whether resveratrol enhanced neuronal viability after OGD/R injury in vitro.five groups were studied: normal group(Norm),control group(Ctrl),vehicle group(Veh),resveratrol group(1 ?mol/L,5 ?mol/L,and 20 ?mol/L;Res 1,Res 5,Res 20),blank group.(2)To test whether resveratrol reduced injury and promoted the neurites growth and synaptogenesis of neurons after OGD/R injury in vitro through the Shh signaling pathway.there were six groups: normal group(Norm),control group(Ctrl),resveratrol group(Res),resveratrol + cyclopamine group(R+C),cyclopamine group(Cyc),purmorphamine group(Pur).(3)To further investigate whether the Shh signaling pathway affect Sirt1 activation.five groups were studied: normal group(Norm),control group(Ctrl),resveratrol group(Res),cyclopamine group(Cyc),purmorphamine group(Pur).(4)To examine whether Sirt1 affect the Shh signaling pathway.four groups were studied: normal group(Norm),control group(Ctrl),resveratrol group(Res),Sirtinol group(Res).Cell Viability and apoptosis was assessed by the CCK8 assay and TUNEL Staining.Neurite outgrowth and synaptogenesis were detected by immunocytochemistry and Western blotting.The expressions of growth-associated protein-43(GAP43),synaptophysin(SYP),Shh,Ptc-1,Smo,Gli-1,and Sirt1 proteins were detected by Western blotting.Results: We observed that resveratrol significantly increased viability(P=0.008),reduced apoptosis(P=0.010),promoted neurite outgrowth(P=0.005;P=0.008)and synaptogenesis(P=0.001;P=0.011)of neurons after OGD/R injury in vitro.On the contrary,cyclopamine,a Smo inhibitor which inhibits the Shh signaling pathway,completely reversed the above effects of resveratrol.purmophamine,a Smo agonist which activates the Shh signaling pathway,had similar effects with resveratrol.Moreover,resveratrol increased the expression of GAP43,(SYP),Shh,Ptc-1,Smo,Gli-1,and Sirt1 proteins.In addition,resveratrol and purmophamine up-regulated the expression of Sirt1 protein,and cyclopamine inhibited the expression of Sirt1 protein.Sirt1 inhibitor sirtinol significantly down-regulated the expressions of Shh,Ptc-1,Smo,and Gli-1proteins.Conclusions: our results suggest that resveratrol reduce injury and enhance neurite outgrowth and synaptogenesis of neurons after OGD/R injury in vitro through activating the Shh signaling pathway.Moreover,the Shh signaling pathway may interact with Sirt1.