鞘氨醇激酶1和1-磷酸鞘氨醇受体2在癫痫大鼠海马组织中表达的变化

Altered expressions of SphK1 and S1PR2 in hippocampus of epileptic rats

目的:检测鞘氨醇激酶1(SphK1)和1-磷酸鞘氨醇受体2(S1PR2)在癫痫大鼠海马中的表达,探讨SphK1和S1PR2在癫痫中的作用机制。方法:成年雄性SD大鼠108只,随机分为对照(Control)组(n=48)和癫痫(PILO)组(n=60)。癫痫组腹腔注射氯化锂(127mg/kg),18~20h后注射匹罗卡品,首剂量为30mg/kg,发作<IV级的大鼠重复注射匹罗卡品(10mg/kg);对照组给予等剂量的生理盐水代替匹罗卡品。根据造模后观察时间和行为学改变,随机分为3个大组,6个亚组:急性期组(E6h、E1d、E3d)、潜伏期组(E7d)和慢性期组(E30d、E56d),每个亚组中对照大鼠和癫痫大鼠各8只。每组取4只大鼠麻醉取海马,另4只取大脑组织。运用Western blot检测SphK1、S1PR2在大鼠海马组织中的表达变化,免疫荧光检测星形胶质细胞活化增生情况及SphK1、S1PR2在星形胶质细胞中的定位表达。结果:与Control组比较,SphK1在造模后急性期(E3d)、潜伏期(E7d)和慢性期(E30d、E56d)海马中的表达均明显升高(P<0.05或P<0.01);S1PR2在急性期(E3d)、潜伏期(E7d)和慢性期(E30d、E56d)海马组织中的表达均明显下降(P<0.05或P<0.01);癫痫大鼠(E7d)海马星形胶质细胞活化、增生明显(P<0.05),SphK1和S1PR2在E7d的表达到位为海马星形胶质细胞中。结论:SphK1和S1PR2可能通过调控海马星形胶质细胞活化增生和影响神经元兴奋性参与了癫痫的发病。

Objective: To observe the expressions of sphingosine kinase 1( SphK1) and sphingosine-1-phosphate receptor 2( S1PR2) in hippocampus of epileptic rats and to investigate the pathogenesis of Sph K1 and S1 PR2 in epilepsy. Methods: One hundred and eight male Sprague-Dawley( SD) rats were randomly divided into control group( n = 48) and pilocarpine( PILO) group( n =60). A robust convulsive status epilepticus( SE) was induced in PILO group rats by the application of pilocarpine. Control group rats were injected with respective of physiological saline. Pilocarpine group was randomly divided into 6 subgroups( n = 8): acute group( E6 h,E1 d,E3 d),latent group( E7 d) and chronic group( E30 d,E56 d). Each subgroup has 8 control rats and 8 epileptic rats.Hippocampal tissue and brain slices were obtained from control rats and rats subjected to the Li-PILO model of epilepsy at 6 h,1 d,3 d,7 d,30 d and 56 d after status epilepticus( SE). Western blot technique was used to determine the expressions of Sph K1 and S1 PR2 in hippocampus at different point of time after pilocarpine treatment. Immunofluorescence was applied to detect the activation and proliferation of hippocampal astrocytes and the localization of Sph K1 and S1 PR2 in rat hippocampal astrocytes. Results: Compared with control group,the levels of Sph K1 in acute phase( E3 d),latent phase( E7 d) and chronic phase( E30 d,E56 d) were significantly increased while the expressions of S1 PR2 were decreased in acute phase( E3 d),latent phase( E7 d) and chronic phase( E30 d,E56 d)( P<0. 05 or P<0. 01). Immunofluorescence results showed astrocyte activation and proliferation in hippocampus of epileptic( E7 d)rats( P<0. 05). Confocal microscopy confirmed the preferential expressions of Sph K1 and S1 PR2 in epileptic rat( E7 d) hippocampal astrocytes. Conclusion: The results indicate that Sph K1 and S1 PR2 may play an important role in the pathogenesis of epilepsy by regulating the activation and proliferation of hippocampal astrocytes and altering neuronal excitability.