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Aim This study is aimed to determine whether SPK2 (Sphingosine kinase2) is involved in isoflurane preconditioning induced autophagy activation in primary cultured neurons.We also examined whether SPK2 protects neurons from ischemic injury by activating autophagy, and explored the molecular mechanism of SPK2 contributing to the autophagy activation in neurons.Methods Isoflurane preconditioning (ISO) and oxygen glucose deprivation (OGD) model was established in primary cultured murine cortical neurons.Neurons were transfected by siRNA to interfere SPK2 and Beclin 1, or lentivirus to overexpress SPK2.Protein expression of SPK2, LC3, and Beclin1 were detected with immunofluorescence and Western blot analysis.The neurons were treated with lysosomal inhibitor ammonium chloride (NH4Cl) to test the autophagy flux.The protection of SPK2 on OGD/R induced neuronal death was detected with CCK8 (cell counting kit8) and LDH cytotoxicity assay kit.Autophagy inhibitor 3MA (3Methyladenine) was used to detect the protection of autophagy on SPK2 induced ischemic tolerance.Coimmunoprecipitation was used to detect the interaction between Beclin 1 and Bcl2.Results In primary cultured neurons, ISO enhanced SPK2 and LC3 immunofluorescence.SPK2 siRNA inhibits LC3Ⅱ upregulation induced by ISO.Beclin 1 siRNA also inhibits LC3Ⅱ upregulation induced by ISO.Lentivirusinduced SPK2 overexpression increased LC3Ⅱ/LC3Ⅰ ratio and enhanced the autophagy flux in neurons.SPK2 overexpression also exerted neuroprotection against OGD model in cortical neurons, as evidenced by improvement of neuronal morphology, increased cellular viability and reduced LDH leakage, while 3MA partly abolished the SPK2induced neuroprotection.After SPK2 overexpression, Beclin 1 siRNA inhibited SPK2 induced LC3Ⅱ upregulation, and the coimmunoprecipitation of Beclin 1 and Bcl2 was reduced..Conclusion ISO increases SPK2 and activates autophagy in neurons.SPK2or Beclin 1 interference cancels ISO induced autophagy activation.SPK2 overexpression activates autophagy, and protects the neurons against ischemic injury.SPK2 may induce autophagy by disrupting Beclin 1/Bcl2 interaction.