大麻素2型受体对癫痫持续状态大鼠海马CA1区自噬相关蛋白LC3、Beclin-1表达的影响

Effect of cannabinoid receptor type 2 on the expression of autophagy-related proteins LC3 and Beclin-1 in the hippocampal CA1 region of rats with status epilepticus

目的建立癫痫持续状态(status epilepticus,SE)大鼠模型,探讨大麻素2型受体(canna-binoid receptor type 2,CB2R)对自噬相关蛋白LC3、Beclin-1 表达的影响.方法 SD大鼠随机分为4组:对照组,癫痫组( Epilepsy 组),CB2R 激动剂 JWH-133 组(Epilepsy +JWH-133 组),CB2R 拮抗剂AM630组(Epilepsy+AM630组).采用腹腔注射氯化锂-匹鲁卡品的方法建立SE模型.匹鲁卡品腹腔注射前1 h各组大鼠经侧脑室注射等量不同药物,对照组及Epilepsy组注射二甲基亚砜,Epilepsy+JWH-133组注射JWH-133,Epilepsy+AM630组注射AM630,在SE后24 h,观察各组大鼠癫痫症状学表现,采用HE 染色观察各组大鼠海马CA1 区神经元数量变化,应用双重免疫荧光和Western blot技术检测各组大鼠海马CA1区自噬相关蛋白LC3和Beclin-1表达的变化.结果大鼠癫痫症状学观察显示:对照组无明显癫痫发作,Epilepsy组大鼠的抽搐潜伏期为(13.50±0.61) min,抽搐强度评分为(4.33 ± 0.47)分;Epilepsy+JWH-133 组的抽搐潜伏期为( 19.33±0.42 ) min,较 Epilepsy 组明显延长( P <0.05);Epilepsy+JWH-133抽搐强度评分为(3.33 ± 0.59)分,较Epilepsy组低(P<0.05),而Epilepsy+AM630组的作用正好相反.HE染色结果显示:Epilepsy组大鼠海马CA1 区神经元数量明显减少,与Epilepsy组相比,Epilepsy+JWH-133组神经元数量明显增多(P<0.05),而Epilepsy+AM630组神经元数量明显减少(P<0.05).免疫荧光及蛋白印迹结果显示:LC3和Beclin-1主要表达在神经元,Epilepsy组大鼠各组LC3和Beclin-1的表达均比对照组升高(P<0.05),与Epilepsy组相比,Epilepsy+JWH-133组LC3和Beclin-1的表达升高更明显( P<0.05),而Epilepsy+AM630组的LC3和Beclin-1的表达降低(P<0.05).结论在SE后神经元损伤修复的早期阶段CB2R对海马神经元的自噬活动可能产生影响,暗示CB2R可能成为癫痫治疗的一个潜在靶点.

Objective To explore effect of cannabinoid receptor type 2(CB2R)on the expression of autophagy-related proteins LC3 and Beclin-1 in the hippocampal CA1 region of rats with status epilepticus(SE).Methods SE model was established by treating lithium chloride-pilocarpine intraperitoneally injection in Sprague-Dawley(SD)rats.All the rats were randomly divided into four groups including Control group, Epilepsy group, Epilepsy+ JWH-133 group and Epilepsy+ AM630 group.JWH-133, AM630, and DMSO were injected 60 min before pilocarpine injection by intracerebroventricular injection(Control group and Epilepsy group with DMSO, Epilepsy+ JWH-133 group with JWH-133 and Epilepsy+ AM630 group with AM630).At 24 h after SE, the epileptic symptoms were observed.HE staining was performed to observe the number changes of neurons in the hippocampal CA1 region.The spatiotemporal expressions of LC3 and Beclin-1 in the hippocampal CA1 region were observed using Double-label immunofluorescence and Western blot.Results Epileptic symptoms showed that rats in the control group had no obvious epileptic seizures.Those in Epilepsy group presented seizures about (13.50±0.61)min after treated with pilocarpine and racine scale was (4.33±0.47) scores.The latency period in Epilepsy+ JWH-133 group was(19.33±0.42)min and longer than that in the Epilepsy group(P<0.05);Racine scale in Epilepsy+ JWH-133 group was 3.33±0.59 and less than the Epilepsy group(P<0.05).The opposite effect was observed in Epilepsy+ AM630 group.HE staining showed that compared with the Epilepsy group, the number of the hippocampal CA1 neurons increased in Epilepsy+ JWH-133 group and decreased in Epilepsy+ AM630 group.Immunofluorescence and Western blot showed that LC3 and Beclin-1 were mainly expressed in neurons, and the expression of LC3 and Beclin-1 in Epilepsy group were up-regulated dynamically than that in the control group at 24 h after SE(P<0.05).Furthermore compared with the Epilepsy group, the expression of LC3 and Beclin-1 in Epilepsy+ JWH-133 group were higher but lowered in Epilepsy+ AM630 group.Conclusion We demonstrate that CB2R may play a role in autophagy of the hippocampal neurons at the early stage of pilocarpine-induced SE and hinted CB2R may become a treatment target for epilepsy.