PI3K-AKT-mTOR介导癫痫海马神经元自噬与凋亡及草果知母汤的效应机制

It is been focusing on the damage caused by epilepsy such as the function of intelligence, consciousness and movement . Brain neuronal cells unusual discharge causes seizures repeatedly led to a substantial reduction in neurons, according to a new study jointly involved in the pathological process of autophagy and apoptosis of neurons, and autophagy in early apoptosis and antiapoptotic effect, promote the neuron survival; PI3K-AKT-mTOR signaling pathway may be more way to regulate the development of epilepsy.The early clinical confirmed Caoguozhimu decoction of epilepsy has a good clinical curative effect, have anticonvulsants, antiapoptotic effect, thus, our hypothesis: Caoguozhimu decoction could antiepileptic effect by regulating the mTOR signaling pathways. This project intends to replicate epilepsy rat models,transmission electron microscope observats autophagy body tem of hippocampal neurons, RT-PCR, immunohisto- chemistry and Western Blot detects such as PI3K-AKT - mTOR and downstream mTORC1, S6K, p70S6k, 4 EBP1 and p-4 EBP1, TFEB, LC3, Beclin1, Bcl - 2 gene transcription and protein expression, such as Co immunoprecipitation assay detects Beclin1 coprecipitation interact with Bcl - 2, discuss epilepsy mechanism of autophagy and apoptosis of neurons, for Caoguozhimu decoction antiepileptic neurons injury to provide the scientific basis.

因癫痫对智能、意识和运动等功能造成损害而倍受关注，脑神经元异常放电引起反复痫性发作导致神经元大量减少，最新研究表明神经元自噬和凋亡共同参与此病理过程，且自噬早发于凋亡且起到抗凋亡的效应，促进神经元存活；PI3K-AKT-mTOR信号通路可多途径调控癫痫的发生发展。前期临床证实草果知母汤对癫痫具有良好的临床疗效、具有抗惊厥、抗凋亡作用，由此，我们假说：草果知母汤可能通过调控mTOR信号通路而抗癫痫作用。本项目拟复制癫痫大鼠模型，透射电镜观察海马神经元自噬体、RT-PCR、免疫组化、Western Blot等检测PI3K-AKT-mTOR及下游mTORC1、S6K、p70S6k、4EBP1与p-4EBP1、TFEB、LC3、Beclin1、Bcl-2等基因转录与蛋白表达，免疫共沉淀检测Beclin1与Bcl-2相互作用，探讨癫痫神经元自噬与凋亡机制，为草果知母汤抗癫痫神经元损伤提供科学依据。

背景.癫痫的反复发作对患者的智能、意识和运动等功能造成损害,甚至导致死亡。脑神经元异常放电引起反复痫性发作导致神经元大量减少, 最新研究表明神经元自噬和凋亡共同参与此病理过程，且自噬早发于凋亡且起到抗凋亡的效应，促进神经元存活；PI3K-AKT-mTOR信号通路可多途径调控癫痫的发生发展。前期临床证实草果知母汤对癫痫具有良好的临床疗效、具有抗惊厥、抗凋亡作用。草果知母汤可能通过调控mTOR信号通路而抗癫痫作用。.目的.探讨草果知母汤通过mTOR信号通路而调控癫痫神经元自噬与凋亡的作用及机制，为草果知母汤抗癫痫神经元损伤提供科学依据。.方法.戊四氮(PTZ)点燃法建立慢性癫痫大鼠模型；正常组和慢性癫痫模型对照组给予双蒸水灌胃 2 mL/kg/天；中药草果知母汤给药组： 低剂量组给药1.25g/kg，中剂量组给药 2.5g/kg，高剂量组以5g/kg给药。正常、模型对照及中药给药每天1次，持续5周。西药组对照组每天给予丙戊酸钠缓释片(商品名：德巴金)混悬液 1mg/kg灌胃，每日 2 次，按时间点灌胃，持续5周。.给药结束后取海马组织：HE、Nissl 染色检测海马神经元损伤程度；电镜观察自噬体；qRT-PCR、Western Blot检测 β-actin、PI3K、Akt、mTOR、mTORC1、S6K、4EBP1、TFEB、Bcl-2、Beclin1、LC3 mRNA和蛋白；免疫组化检测PI3K、Akt、mTOR、mTORC1、S6K、p70S6k、4EBP1、p4EBP1、TFEB、bcl-2、Beclin1、LC3 蛋白表达；免疫共沉淀检测 Beclin1 与 Bcl-2 相互作用。.结果.草果知母汤可改善癫痫模型动物海马神经元损伤；促进Bcl-2、 Beclin1、 p70S6k和S6K表达，抑制mTOR、PI3K、4EBP1、TFEB和LC3表达来调控PI3K-AKT-mTOR 信号通路，促进癫痫模型的早期自噬，抑制晚期自噬，抑制凋亡，起到抗癫痫作用。.结论：.早期的自噬可能为Beclin1依赖性的，且起到抗凋亡作用；晚期的自噬与LC3相关，与凋亡具有协同作用。草果知母汤可通过调控PI3K-AKT-mTOR 信号通路调控自噬，抑制凋亡，起到抗癫痫作用。