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J Zhejiang Univ (Med Sci)  2018, Vol. 47 Issue (5): 457-464    DOI: 10.3785/j.issn.1008-9292.2018.10.03
Efficacy of brain-targeted rapamycin for treatment of epilepsy in rats
ZHANG Yuanyuan(),Wang Qingmei,DONG Jingyin,ZHANG Binbin,LIU Luna,ZHU Feng,ZENG Linghui*()
School of Medicine, Zhejiang University City College, Hangzhou 310015, China
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Objective: To investigate the efficacy of brain-targeted rapamycin (T-Rap) in treatment of epilepsy in rats. Methods: Rapamycin nanoparticles targeting brain were prepared. The epilepsy model was induced by injection of pilocarpine in rats. The rats with pilocarpine-induced epilepsy were treated with rapamycin (Rap group) or brain-targeted rapamycin (T-Rap group). Seizure activity was observed by electroencephalography; the effect on mTOR signaling pathway was detected by Western blot; neuronal death and moss fiber sprouting were analyzed by Fluoro-Jade B (FJB) and Timm's staining, respectively. Results: Electroencephalography showed that both preparation of rapamycin significantly reduced the frequency of spontaneous seizures in rats, and the effect of T-Rap was stronger than that of conventional rapamycin (P < 0.05). Western blot showed that the phosphorylation levels of S6K and S6 in T-Rap group were lower than those in Rap group (all P < 0.05), indicating that T-Rap had a stronger inhibitory effect on mTOR signaling pathway. FJB staining showed that T-Rap significantly decreased neuronal death, but there was no significant difference as compared with Rap group. Timm's staining showed that both preparations of rapamycin significantly reduced the germination of mossy fibers, while the effect of T-Rap was more pronounced than Rap group (P < 0.05). The inhibition of body weight gain of T-Rap group was less than that of Rap group (P < 0.05). Conclusion: T-Rap has a better therapeutic effect on epilepsy than conventional rapamycin with a less adverse effects in rats.

Key wordsEpilepsy/drug therapy      Sirolimus/therapeutic use      Nanostructures      Brain/drug effects      Pilocarpine/pharmacology      Disease models, animal      Comp study     
Received: 20 August 2018      Published: 23 January 2019
CLC:  R742.1  
Corresponding Authors: ZENG Linghui     E-mail:;
Cite this article:

ZHANG Yuanyuan,Wang Qingmei,DONG Jingyin,ZHANG Binbin,LIU Luna,ZHU Feng,ZENG Linghui. Efficacy of brain-targeted rapamycin for treatment of epilepsy in rats. J Zhejiang Univ (Med Sci), 2018, 47(5): 457-464.

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目的: 比较雷帕霉素靶向制剂与雷帕霉素普通制剂治疗癫痫的差异。方法: 制备脑靶向的雷帕霉素纳米粒。采用毛果芸香碱诱发大鼠癫痫发作。脑电描记术监测自发性癫痫的发作;蛋白质印迹法检测不同剂型雷帕霉素对mTOR信号通路的影响;Fluoro-Jade B染色观察海马区神经元活性;Timm染色观察海马区苔藓纤维出芽情况。结果: 脑靶向制剂可降低大鼠自发性癫痫发作频率,作用效果较普通制剂明显(P < 0.05);以磷酸化S6K和S6为指征,脑靶向制剂比普通制剂雷帕霉素对mTOR信号通路异常激活的抑制作用更强(均P < 0.05);脑靶向制剂可显著改善神经元凋亡,但与普通制剂无差异(P > 0.05);脑靶向制剂可减少海马区苔藓纤维出芽,作用效果较普通制剂明显(P < 0.05);脑靶向制剂对大鼠体质量增长的抑制作用较普通制剂减弱(P < 0.05)。结论: 雷帕霉素脑靶向制剂对癫痫的治疗作用优于普通制剂,且其不良反应小于普通制剂。

关键词: 癫痫/药物治疗,  西罗莫司/治疗应用,  纳米结构,  脑/药物作用,  毛果芸香碱/药理学,  疾病模型, 动物,  对比研究 
[n(%)或$\bar x \pm s$]
组别 n 造模成功率 癫痫发作后死亡率 癫痫发作潜伏期(min) 癫痫发作程度 发作持续时间(h)
模型对照组 40 28(70.0) 6(21.4) 49±11 4.31±0.12 5.2±0.8
普通制剂组 40 30(75.0) 7(23.3) 51±10 4.23±0.29 4.9±1.1
靶向制剂组 40 29(72.5) 6(20.7) 50±12 4.31±0.16 5.1±0.9
Tab 1 Statistic analysis of modeling in each group
Fig 1 Effect of two different preparations of rapamycin on spontaneous seizures in rats (n=10-11)
Fig 2 The expression of S6K and S6 proteins in the cerebral cortex and hippocampus of rats
Fig 3 Quantification of expression of phosphorylated S6K and S6 proteins in the cerebral cortex and hippocampus of rats (n=6)
Fig 4 Representative images of Fluoro-Jade B (FJB) staining in hippocampus of rats
Fig 5 Representative images of Timm's staining in hippocampus of rats
Fig 6 Effect of two different preparations of rapamycin on weight growth in rats (n=10-11)
[1]   BRODIE M J , BESAG F , ETTINGER A B et al. Epilepsy, antiepileptic drugs, and aggression:an evidence-based review[J]. Pharmacol Rev, 2016, 68 (3): 563- 602
[2]   SCULIER C , GAíNZA-LEIN M , SáNCHEZF I et al. Long-term outcomes of status epilepticus:a critical assessment[J]. Epilepsia, 2018, 59 (Suppl 2): 155- 169
[3]   RYAN S G . Ion channels and the genetic contribution to epilepsy[J]. J Child Neurol, 1999, 14 (1): 58- 66
[4]   MARWICK K , SKEHEL P , HARDINGHAM G et al. Effect of a GRIN2A de novo mutation associated with epilepsy and intellectual disability on NMDA receptor currents and Mg2+ block in cultured primary cortical neurons[J]. Lancet, 2015, 385 (Suppl 1): S65
[5]   PENG W F , DING J , MAO L Y et al. Increased ratio of glutamate/glutamine to creatine in the right hippocampus contributes to depressive symptoms in patients with epilepsy[J]. Epilepsy Behav, 2013, 29 (1): 144- 149
doi: 10.1016/j.yebeh.2013.07.004
[6]   ROMERO-LEGUIZAMON C R , RAMIREZ-LATORRE J A , MORA-MUNOZ L et al. Signaling pathways mTOR and AKT in epilepsy[J]. Rev Neurol, 2016, 63 (1): 33- 41
[7]   CRINO P B . The mTOR signalling cascade:paving new roads to cure neurological disease[J]. Nat Rev Neurol, 2016, 12 (7): 379- 392
doi: 10.1038/nrneurol.2016.81
[8]   BRAKEMEIER S, VOGT L, ADAMS L, et al. Treatment effect of mTOR-inhibition on tissue composition of renal angiomyolipomas in tuberous sclerosis complex (TSC)[J/OL]. PLoS One, 2017, 12(12): e0189132.
[9]   ZENG L , XU L , GUTMANN D H et al. Rapamycin prevents epilepsy in a mouse model of tuberous sclerosis complex[J]. Ann of Neurol, 2008, 63 (4): 444- 453
doi: 10.1002/ana.v63:4
[10]   ZENG L H , RENSING N R , WONG M . The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy[J]. J Neuroscience Offic, 2009, 29 (21): 6964- 6972
doi: 10.1523/JNEUROSCI.0066-09.2009
[11]   GUO D, ZENG L, BRODY D L, et al. Rapamycin attenuates the development of posttraumatic epilepsy in a mouse model of traumatic brain injury[J/OL]. PLoS One, 2013, 8(5): e64078.
[12]   CRINO P B . mTOR signaling in epilepsy:insights from malformations of cortical development[J]. Cold Spring Harb Perspect Med, 2015, 5 (4): a022442
doi: 10.1101/cshperspect.a022442
[13]   MROSKE C , RASMUSSEN K , SHINDE D N et al. Germline activating MTOR mutation arising through gonadal mosaicism in two brothers with megalencephaly and neurodevelopmental abnormalities[J]. BMC Med Genet, 2015, 16:102
[14]   CHAMBERLAIN M C . Recurrent ganglioglioma in adults treated with BRAF inhibitors[J]. CNS Oncol, 2016, 5 (1): 27- 29
doi: 10.2217/cns.15.40
[15]   YAO J , TAVEIRA-DASILVA A M , JONES A M et al. Sustained effects of sirolimus on lung function and cystic lung lesions in lymphangioleiomyomatosis[J]. Am J Respir Crit Care Med, 2014, 190 (11): 1273- 1282
doi: 10.1164/rccm.201405-0918OC
[16]   LU Z, LIU F, CHEN L, et al. Effect of chronic administration of low dose rapamycin on development and immunity in young rats[J/OL]. PLoS One, 2015, 10(8): e0135256.
[17]   林晓宁, 田新华, 魏峰 et al. 载替莫唑胺制备方法比较研究[J]. 中国现代医学杂志, 2012, 22 (26): 1- 4
LIN Xiaoning , TIAN Xinhua , WEI Feng et al. Comparison of the preparation methods of Temozolomide-loaded nanoparticles[J]. China Journal of Modem Medicine, 2012, 22 (26): 1- 4
doi: 10.3969/j.issn.1005-8982.2012.26.001
[18]   RACINE R J . Modification of seizure activity by electrical stimulation. Ⅱ. Motor seizure[J]. Electroencephalogr Clin Neurophysiol, 1972, 32 (3): 281- 294
doi: 10.1016/0013-4694(72)90177-0
[19]   CAVAZOS J E , GOLARAI G , SUTULAT P . Mossy fiber synaptic reorganization induced by kindling:time course of development, progression, and permanence[J]. J Neurosci, 1991, 11 (9): 2795- 2803
doi: 10.1523/JNEUROSCI.11-09-02795.1991
[20]   LIM J S , KIM W I , KANG H C et al. Brain somatic mutations in MTOR cause focal cortical dysplasia type Ⅱ leading to intractable epilepsy[J]. Nat Med, 2015, 21 (4): 395- 400
doi: 10.1038/nm.3824
[21]   FRENCH J A , LAWSON J A , YAPICI Z et al. Adjunctive everolimus therapy for treatment-resistant focal-onset seizures associated with tuberous sclerosis (EXIST-3):a phase 3, randomised, double-blind, placebo-controlled study[J]. Lancet, 2016, 388 (10056): 2153- 2163
doi: 10.1016/S0140-6736(16)31419-2
[22]   MANNING B D . Game of TOR-the target of rapamycin rules four kingdoms[J]. N Engl J Med, 2017, 377 (13): 1297- 1299
doi: 10.1056/NEJMcibr1709384
[23]   MACKEIGAN J P , KRUEGER D A . Differentiating the mTOR inhibitors everolimus and sirolimus in the treatment of tuberous sclerosis complex[J]. Neuro Oncol, 2015, 17 (12): 1550- 1559
doi: 10.1093/neuonc/nov152
[24]   SATTLER M , GUENGERICH F P , YUN C H et al. Cytochrome P-4503A enzymes are responsible for biotransformation of FK506 and rapamycin in man and rat[J]. Drug Metab Dispos, 1992, 20 (5): 753- 761
[25]   SHAYANI S , PALMER J M , STILLER T et al. Aprepitant (Emend) significantly increases sirolimus levels in patients undergoing allogeneic hematopoietic SCT[J]. Bone Marrow Transplant, 2012, 47 (2): 291- 293
doi: 10.1038/bmt.2011.42
[26]   张哲明, 陈丽青, 辛欣 et al. 脑靶向纳米给药系统研究进展[J]. 国际药学研究杂志, 2016, 43 (5): 887- 892
ZHANG Zheming , CHEN Liqing , XIN Xin et al. Brain targeted nano-drug delivery systems:research advances[J]. Journal of International Pharmaceutical Research, 2016, 43 (5): 887- 892
[27]   SONI S , RUHELA R K , MEDHI B . Nanomedicine in central nervous system (CNS) disorders:a present and future prospective[J]. Adv Pharm Bull, 2016, 6 (3): 319- 335
doi: 10.15171/apb.2016.044
[28]   CHUNG C Y, LIN M H, LEE I N, et al. Brain-derived neurotrophic factor loaded PS80 PBCA nanocarrier for in vitro neural differentiation of mouse induced pluripotent stem cells[J/OL]. Int J Mol Sci, 2017, 18(3): E663.
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