Probing The Mechanism Of Sphingosine Kinase 1 And Sphingosine-1-phosphate Receptor 2 In Temporal Lobe Epilepsy

ObjectiveEpilepsy is one of the common neurological illnesses characterized by unpredictable,repetitive spontaneous seizures and affects approximately 50 million of people worldwide.Seizures are the results of hypersynchronous neuronal activity originating at restricted sites of the brain.About one-third of the patients affected suffer from intractable epilepsy(IE).Temporal lobe epilepsy(TLE)is one of the most prevalent types of IE in adults and its most important pathological feature is hippocampal sclerosis(HS).Hippocampal sclerosis may be the cause and the result of seizures.Recurrent seizures lead to progressive brain injury and cognitive dysfunction.Inflammation,oxidative stress,neurotoxicity,neural cell death,gliosis,and blood brain barrier(BBB)dysfunction are the most common causes of epilepsy.Sphingosine kinase 1 is a rate-limiting enzyme that converts sphingosine into sphingosine-1-phosphate(S1P)by phosphorylation.Most of the known actions of SIP are mediated by a family of five specific G protein-coupled receptors(S1PR1-S1PR5).SphK1 and sphingosine-1-phosphate receptor 2(S1PR2)are important signal molecules in SphKl/S1P signal pathway.SphKl/S1P signal pathway has been widely studied in tumors.The expression level of SphKl in tumor cells is closely related to the prognosis of patients,and has been used as a potential drug target for tumor therapy.SphK1/SIP signal pathway regulates lymphocyte trafficking,glial cell activation,vasoconstriction,endothelial barrier function,and neuronal death pathways which plays a critical role in numerous neurological conditions.However,the molecular mechanism by which SphKl and S1PR2 exert the activity in TLE remains poorly understood.The present study was designed to observe the expression of SphKl and S1PR2 in hippocampus of pilocarpine(PILO)induced TLE rats and in brain tissue of temporal lobe epilepsy(TLE)patients,and to investigate the pathogenesis of SphKl and S1PR2 in temporal lobe epilepsy.Methods1.Lithium-pilocarpine animal model:A robust convulsive SE was induced in rats by the application of pilocarpine hydrochloride.Control rats were injected with respective of saline.For one part of the rats,after anesthetized with 10%chloral hydrate(300 mg/kg),the brain was removed after sacrificed.The hippocampus were dissected from brain,rapidly frozen in liquid nitrogen and extracted protein(store at-80 ?).Another part of the rats,after anesthetized with 10%chloral hydrate,the rat was perfused transcardially with heparinized saline followed by 4%paraformaldehyde in 0.1M PBS.Brains were extracted and post fixed in 4%PFA overnight and then transferred to 30%sucrose in PBS at 4? until they sank.The coronal brain sections were prepared at the level of the hippocampus.Western blot technique was used to measure the expression of SphKl and S1PR2 in hippocampus at different point of time after pilocarpine treatment.Immunofluorescence was applied to detect the proliferation of hippocampal astrocytes and the expression of SphKl or S1PR2 in rat hippocampal astrocyte and neuron.2.Temporal lobe cortex samples from 16 patients with TLE were collected as epilepsy group and temporal lobe cortex samples from 10 patients with brain trauma were used as control group.The tissue was post fixed in 4%PFA overnight and then transferred to 30%sucrose at 4? until they sank.The brain sections were prepared for immunohistochemistry and immunofluorescence.Immunohistochemical technique was used to measure the expression of SphKl and S1PR2 in brain tissue.Immunofluorescence was applied to detect the proliferation of astrocytes and the expression of SphKl or S1PR2 in astrocyte and neuron.Results1.A robust convulsive SE was induced in 48 rats after the application of pilocarpine hydrochloride.Epileptic rats in acute phase(E6h,Eld,E3d)showed seizures,mental depression,reduced eating,irritability,latent phase rats(E7d)showed no seizures and chronic phase rats(E30d,E56d)showed recurrent spontaneous seizures.2.The level of SphKl in acute phase(E3d),latent phase(E7d)and chronic phase(E30d,E56d)rats were significantly higher than control group(P<0.05)and the expression of S1PR2 decreased in the acute phase(E3d),latent phase(E7d)and chronic phase(E30d,E56d)(P<0.05).3.Immunofluorescence confirmed the astrocyte activation and proliferation in hippocampus of epileptic rats.The number of astrocytes in hippocampus of epileptic rats was significantly higher than that of control rats(P<0.05).Confocal microscopy confirmed the preferential expression of SphKl and S1PR2 in hippocampal astrocytes.SphKl was mainly expressed in astrocyte nucleus in control and epileptic rats.S1PR2 was expressed in astrocytes in both control and epileptic rats.The expression of SphKl and S1PR2 in hippocampal neurons was not detected by immunofluorescence.4.The number of SphKl positive cells in TLE patients was higher than control patients(P<0.05)and the number of S1PR2 positive cells was decreased in TLE patients(P<0.05).5.Confocal microscopy confirmed the astrocyte activation and proliferation in TLE patients.The result shows that SphKl and S1PR2 expressed in astrocyte and neuron in TLE patients.ConclusionSphKl overexpressed in TLE rat hippocampus and cortex of temporal lobe in TLE patients.The expression of S1PR2 was declined in TLE rat hippocampus and cortex of temporal lobe in TLE patients.SphKl and S1PR2 expressed in astrocytes in TLE rat hippocampus and cortex of temporal lobe in TLE patients.SphK1 and S1PR2 may play an important role in the pathogenesis of temporal lobe epilepsy.