Protective Effects And Mechanisms Of SPK1on Cell Model Of Alzheimer’s Disease

Objective: To explore whether sphingosine kinase-1(SPK1) has protective effects onAβ-induced Alzheimer’s disease(AD) model in vitro, and the possible underlyingmechanisms.Methods:(1) Using differentiated N2a cells treated with Aβ25-35for24h as the modelof Alzheimer’s disease, add different concentrations of Aβ25-35into the culture of N2acells. Cell viability was detected by MTT; Apoptosis ratio was detected with Hoechst33258; Nucleusul trastructural changes were observed by transmission electronmicroscopy. Compare the diference between each groups.(2) The expression of SPK1levels in cells treated with or without20μM Aβ25-35for24hours was detected bywestern blot. Then, a recombinant adenovirus vector for SPK1overexpression and arecombinant adenovirus vector for silencing of SPK1expression were constructed.We investigated the effects of SPK1overexpression or SPK depletion on cell viabilityand cell apoptosis after24hours of Aβ25-35treatment.(3) Using western-blot assay thelevels of apoptosis-related proteins; Using transmission electron microscopy observethe ultrastructure of mitochondria. Compare the diference between each groups.Results：(1) With the increase in the concentration of Aβ25-35, cell viability wasdecersed and cell apoptosis was incresed. We established differentiated N2a cellstreated with Aβ25-35for24h as the model of Alzheimer’sdisease.(2) The expression levels of SPK1was lower in Aβ25-35group than that in the control group.Up-regulation of SPK1attenuated Aβ25-35-induced neurotoxicity, as evidenced by thedecreased cell viability and the increased apoptotic rate. whereas silencing theexpression of SPK1aggravated it.(3) Incubated with20μM Aβ25-35for24h caused arobust increase in Bax/Bcl-2rotio and much structural damage to mitochondria.Further, the SPK1-siRNA group mimicked the changes induced by Aβ25-35treatment.However, pre-treating cells with Ad-SPK1reversed the alternations of bcl-2and baxexpressions induced by Aβ25-35.Conclusion: The expression of SPK1was markedly decreased in Aβ25-35-inducedneurotoxicity and that up-regulation of SPK1could attenuate Aβ25-35-inducedneurotoxicity by regulating Bcl-2family. These findings suggest that SPK1could be apotential therapeutic target for treatment of AD.