生物钟基因BMAL1通过mTOR信号通路影响大鼠颞叶癫痫耐药性研究

Temporal epilepsy is common in drug resistant epilepsy. Besides, sleep deprivation can induce seizures and affect the prognosis of epilepsy. Our preliminary experiments show that sleep deprivation is significantly associated with pharmacoresistance in patients with epilepsy, with the down-regulation of the circadian clock gene BMAL1 and Per2, but the mechanism of sleep deprivation in the pharmacoresistance of epilepsy still needs further investigation. Recent studies showed that the circadian protein Per2 regulates the phosphorylation of S6K, a key protein of mTOR signaling pathway. Besides, our previous study confirmed that mTOR pathway is involved in the pharmacoresistance of epilepsy by regulating the expression of P-gp. Therefore, we hypothesize that the circadian clock gene BMAL1 may regulate seizure occurrence and pharmacoresistance of epilepsy through the mTOR pathway. To validate this hypothesis, we will establish a pharmacoresistant epilepsy rat model kindled by coriaria lactone, to explore the correlation of the circadian clock gene BMAL1 and mTOR pathway in pharmacoresistant epilepsy. Seizure frequencies and pharmacoresistance of epilepsy will be evaluated between sleep deprivation rats, normal sleep rats and BMAL1 knockout rats by video electroencephalography. In addition, RNA interference and rapamycin inhibition will be used to investigatethe molecular mechanism of BMAL1 in pharmacoresistant epilepsy. Expression of BMAL1, Per2, P-S6 and P-gp will be detected by western blot and immunohistochemistry. This study may provide new drug targets for the treatment of pharmacoresistant epilepsy.

耐药性癫痫以颞叶癫痫为主，且睡眠剥夺可诱发颞叶癫痫发作并影响药物治疗效果。我们的预实验显示睡眠剥夺增加癫痫耐药性，并伴有生物钟基因BMAL1、Per2的下调，但其参与癫痫耐药的作用机制仍待进一步研究。近期研究表明Per2可抑制mTOR信号通路关键蛋白S6K的磷酸化，而我们前期研究证实mTOR信号通路通过调控耐药蛋白P-gp表达参与癫痫耐药性；为此，我们提出生物钟基因BMAL1可能通过mTOR信号通路影响颞叶癫痫发作及耐药性。 为验证这一假说，我们将基于前期成功建立的马桑内酯点燃大鼠耐药癫痫模型，采用行为学观察、视频脑电、Western blot、免疫组化、RNA干扰等手段，从分子、细胞及动物水平比较睡眠剥夺、BMAL1敲基因和雷帕霉素对耐药性癫痫的治疗效果影响，并检测相关分子表达，探究BMAL1是否通过mTOR通路调控大鼠癫痫耐药性及其分子机制。本研究可能为耐药性癫痫的治疗提供新思路。

耐药性癫痫的治疗仍为癫痫治疗的难点，睡眠剥夺可诱发颞叶癫痫发作并影响药物治疗效果。本项目前期发现睡眠剥夺癫痫患者耐药率更高，且相比于正常睡眠大鼠，睡眠剥夺大鼠BMAL1下调伴有mTOR信号通路活化。因而我们提出假说，生物钟基因BMAL1可能通过mTOR信号通路影响颞叶癫痫发作及耐药性。通过siRNA下调人血管内皮细胞系HUVEC、小鼠血管内皮细胞BV2的 BMAL1表达， 发现敲低BMAL1后PER2 的基因及蛋白表达水平均下调，而P-S6、P-gp表达上调。而慢病毒转染上调BMAL1后，PER2表达上调，而P-S6、P-gp表达下降。健康成年SD大鼠分为睡眠剥夺组及正常睡眠组，采用戊四氮点燃癫痫模型并以苯妥英钠对大鼠耐药性进行筛选，睡眠剥夺组大鼠癫痫发作频率、发作等级及癫痫耐药率均高于正常睡眠组。对两组大鼠海马相关蛋白检测发现，睡眠剥夺癫痫大鼠BMAL1、PER2表达下调，P-S6、P-gp表达上调。在且通过下调BMAL1，导致mTOR信号通路活化及多耐药蛋白P-gp表达上调，增加癫痫耐药性。选取健康大鼠分别进行 BMAL1-shRNA及无义shRNA侧脑室注射，待病毒完全表达后进行戊四氮癫痫造模及耐药性筛选。结果发现病毒干预组BMAL1、PER2表达显著低于癫痫空载对照组，而P-S6、P-gp表达显著高于癫痫空载对照组。以上实验结果表明睡眠剥夺导致BMAL1下调， mTOR信号通路活化及其下游多耐药蛋白P-gp表达，增加癫痫耐药性。该机制的发现初步阐明了睡眠剥夺导致耐药性癫痫的机制，为耐药性癫痫治疗提供新的药物靶点，有助于新药研发。此外，该项目也表明癫痫患者的睡眠因素也是影响其癫痫预后的重要因素，后续将继续开发患者睡眠管理及监测软件，以改善患者预后。