OX1/CB1-GPCR逆行信使抑制离体/在体海马神经元持续放电作用机制研究

Status epilepticus (SE) is a life-threatening neurological disorder associated with a significant morbidity and mortality. Benzodiazepines are the initial drugs of choice for the treatment of SE. Development of resistance to treatment with benzodiazepines with increasing SE duration has also been observed in experimental models and patients of SE..Cannabinoids have recently been demonstrated to regulate seizure mediated by retrograde endocannabinoid bind to presynaptic CB1 receptors located on certain types of axon terminals and suppress neurotransmitter release. The use of cannabinoids in the treatment of SE may offer a unique approach to controlling SE without the development of pharmacoresistance observed with conventional treatments. There are still not clear whether CB1 receptors expressed on excitatory glutamatergic or GABA axon terminals are responsible for the anti-convulsant effect.However, concern over the dangers of abuse led to the banning of the medicinal use of cannabinoids. .OX1R and CB1R are all belong to the G-protein coupled receptors (GPCRs) superfamily. The distribution of these two GPCRs overlaps in the brain, especially in hippocampal, a region of key importance in epileptogenesis. In addition, electron microscopy experiments revealed that CB1 and OX1 are closely apposed at the plasma membrane level; they are close enough to form heterodimer. Although have indicated that OX1-CB1 receptor can existed as heterodimer, and have the "cross-talk" interaction in modulating the pain and feeding, there are no study about the effect of OX1/CB1 GPCRs on epilepsy..With this in mind, we observe the potential "cross-talk" of CB1 with OX1 in modulating the epilepticus-like activity, which inhibits the glutamatergic or GABAergic transmission mediated by retrograde endocannabinoid acting on presynaptic CB1 receptors. In this study, aimed to elucidate the possibility of intracellular interactions between CB1 and OX1, and the mechanism of OX1/CB1 GPCR signaling inhibits the hippocampal neuronal status-like activity in vitro and in vivo. These results will open new avenues to understand the mechanism by which the molecule may prevent status epilepticus through functional interaction between CB1 and OX1. As such, OX1/CB1 GPCR heterodimers may offer entirely novel sets of potential therapeutic targets.

癫痫持续状态（Status epilepticus，SE）属急重症，若不能即早给药，苯二氮卓类一线抗惊厥效果显著下降。研究显示大麻素通过大麻素受体1（CB1）逆行信使在SE延迟给药仍有强抗癫痫作用。大麻素作用位点尚存争议，因成瘾性限制临床应用。同为G蛋白偶联受体家族的CB1和食欲素受体1（OX1）在脑内分布高度重叠，作为异聚体在镇痛、食欲等方面存在"cross-talk"相互作用，但癫痫方面国内外尚未见报道。.因此，本研究在观察SE后海马OX1/CB1分布特点和CB1作用位点基础上，探索OX1/CB1受体交叉激活对神经元持续放电的影响。设计了在体海人酸SE模型，评估OX1/CB1受体逆行信使对海马神经元放电及变性影响；建立离体海马细胞培养膜片钳技术，观察SE后不同时间给予安定、CB1受体激动剂/OX1受体拮抗剂等抑制放电作用及内在分子机制。有望构建二者异聚体，为SE寻找新的药物治疗靶点。

癫痫持续状态（Status epilepticus，SE）属急危重症，死亡率和致残率极高，若不能即早给药，一线治疗的苯二氮卓类药物效果将显著下降。研究显示大麻素通过大麻素受体1（cannabinoid receptor 1，CB1）逆行信使介导抑制神经递质释放，在SE延迟给药仍有强抗癫痫作用。大麻素作用位点尚存争议，因成瘾性限制临床应用。同为G蛋白偶联受体家族的CB1和食欲素受体1（orexin-1，OX1）在脑内分布高度重叠，尤其是在海马。电镜显示二者作用位点接近，作为异聚体在镇痛、食欲等方面研究显示存在“cross-talk”相互作用，但在癫痫方面尚未见报道。因此，本研究拟利用在体海人酸SE模型观察SE后海马OX1/CB1分布特点，同时建立离体海马细胞膜片钳技术，观察OX1/CB1作用位点及对海马神经元放电影响，并在不同时间点给予安定、CB1受体激动剂/OX1受体拮抗剂等,观察其抑制放电的作用及内在分子机制。. 在体实验部分，我们利用海人酸制作大鼠癫痫持续状态模型，用免疫组化和PCR方法观察癫痫持续状态后不同时间点海马CB1、OX1变化特点，结果显示在癫痫持续状态后1周，海马各区域的CB1受体较对照组明显增高，而OX1分布在两组间无差异，应用免疫荧光双染法发现CB1、OX1在海马同样存在重叠分布情况。同时我们发现，随着年龄的增长，OX1和CB1受体表达增多。. 离体实验部分，我们发现CA1区锥体神经元可能存在大麻素受体功能表达且不限于大麻素受体1 ；而激活大麻素受体可能通过不同的机制起到抑制CA1区锥体神经元的作用，部分是通过TEA敏感的钾通道而发挥作用。同样，CA1区可能存在多种食欲素受体的表达，激活食欲素受体可兴奋海马神经元，可能参与调节多种生理、病理过程。同时，我们发现OX1/CB1可能存在交叉作用，如CB1抑制剂可抑制由OX1激动剂引起的细胞兴奋作用，但尚需进一步验证。. 进一步深入研究将有望构建OX1/CB1异聚体，为SE寻找潜在的药物治疗靶点。