Dual Role Of Sphk1in Cerebral Ischemia

Objective: To investigate the role of Sphingosine kinase1(Sphk1) in cerebralischemia.Methods: Transient middle cerebral artery occlusion (MCAO) was induced inmice with a filament technique. Quantitative real-time PCR (RT-PCR) was used toexamine the mRNA expression levels of Sphk1, Sphk2, and inflammatory cytokines inthe cortical penumbra after MCAO. The expression of Sphk1protein in the corticalpenumbral was assessed with western blotting. Colocalization between Sphk1and theneuronal or microglial markers in the cortex were examined by doubleimmunofluoresence staining. To investigate the role of Sphk1in cerebral ischemicpathology, mice were randomly divided into the control group and the experimentalgroup after MCAO to receive vehicle or Sphk1inhibitors5C (2mg/kg) at6h、12h、24h、48h and72h after reperfusion. Brain sections were stained with1%2,3,5-tripheyltetrazolium chloride (TTC) for the analysis of the infarct sizes at24h and96h after MCAO. Z-Longa scores were determined to assess the effect of Sphk1inhibitoron neurological outcomes at96h after MCAO. Intracerebroventricular injection ofsmall-interfering RNA (siRNA) was employed to knock down Sphk1, Sphk2andTRAF-2in the cortical penumbra. The integrity of the BBB was investigated bymeasuring the extravasation of Evans blue dye (2%in saline,4mL/kg) at96h afterMCAO. Hemorrhagic transformation was assesessed by spectrophotometricallyquantifying hemoglobin content in the ischemic brain.at96hours after MCAO. The interaction between Sphk1and TRAF-2wasinvestigated with Co-Immunoprecipitation(Co-IP).Results: We founded that Sphk1played a dual role in focal cerebral ischemicpathology. Sphk1mRNA was induced in the cortical penumbra vs. the contralateral cortex over96h after MCAO, but the expression of Sphk2mRNA was not alteredfollowing MCAO. Western blotting analysis showed that Sphk1protein was induced inthe cortical penumbra after reperfusion. Double immunofluorescence staining showedthat Sphk1co-localized with neuronal and microglial markers， but mainly with theneuronal marker, in the cortical penumbra. As indicated by the results of TTC staining,the Sphk1inhibitor, Sphk1siRNA and TRAF-2siRNAsignificantly reduced theinfarct volumes. Moreover, the Sphk1inhibitor improved neurological outcome at96h after reperfusion. The Sphk1inhibitor and Sphk1siRNA also blunted MCAO-inducedmRNA expression of inflammatory factors in the ischemic brain. The results of EvansBlue extravasation showed that the permeability of the blood-brain barrier wasenhanced in the mice pretreated with Sphk1siRNA vs. the mice injected with nonsensesiRNA at96h reperfusion following MCAO. Moreover, cerebral knockdown of Sphk1with siRNA increased hemoglobin content in the ischemic brain. Co-IP results showedthat Sphk1binded with TRAF-2in the cortical penumbra.Conclusion: We showed that cerebral Sphk1expression was up-regulated byischemic insults for the first time, with nerons and microglia as the major cellular sourcefor Sphk1induction. Sphk1inhibition or blocking Sphk1induction not only alleviatedthe ischemic damage, improved neurological deficits, but also increased the blood-brainbarrier disruption. Our study showed that Sphk1was induced and may play a dual rolefollowing cerebral ischemia.