钩藤碱抑制病理性突触可塑性改变治疗脑卒中神经功能损伤的机制研究

Relieving nerve impairment after stroke has been intensively investigated. Developing a theraputic agent for stroke neurological impairment and reduce the mortality and morbidity of this disease has been a focus in the field of neurology. previous studies have shown that rhynchophylline can inhibit the delayed neuronal death after cerebral ischemia-reperfusion by regulating the expression of ionotropic glutamate receptor NMDAR, however its specific mechanism remained to be investigated. Our preliminary study showed that rhynchophylline could significantly reduce the neurological impairment of MCAO mice. And rhynchophylline had a significant effect on inhibiting the loss of dendritic spines as well as promoting their remodeling after stroke. In view of this, our project would investigate the effects of rhynchophylline on loss and remodeling of dendritic spines by two-photon microscopy imaging and Golgi staining. Moreover, we would apply electrophysiological patch clamp techniques to investigate the regulating effects of rhynchophylline on pathological synaptic plasticity. Futhermore, we would combined CoIP and western-blot experiments to explore the effects of rhynchophylline on the expression of ionotropic glutamate receptor NMDAR and its downstream death signal transductional molecule DAPK-1. We commit to uncover the protective mechanism of rhynchophyllines on neurological impairment after brain ischemia, and provide new ideas for the treatment of clinical stroke, and lay the foundation for experimental research to promote the study of traditional Chinese medicine for major diseases in our country.

减轻脑卒中后的神经损伤是国内外研究热点，开发脑卒中神经损伤的治疗药物降低该病的致死率和致残率一直是神经病学领域亟待解决的焦点问题。相关研究表明钩藤碱可通过调节离子型谷氨酸受体NMDAR的表达有效抑制脑缺血再灌注后的延迟性神经元死亡，然而其具体机制有待进一步研究。申请人前期预实验发现钩藤碱能够显著减轻脑缺血模型小鼠的神经损伤并且该药物对于抑制脑卒中后的神经元树突棘丢失促进其重塑具有显著影响。为此本项目将通过双光子显微成像技术与Golgi染色联合分析钩藤碱对神经元树突棘丢失和重塑的影响；利用电生理脑片膜片钳技术探究钩藤碱对缺血后病理性突触可塑性改变的调节作用；结合CoIP和WB实验进一步明确钩藤碱对NMDAR及其下游死亡信号转导分子DAPK-1表达的影响，力争阐明钩藤碱对脑卒中神经损伤的保护机制，为临床脑卒中的治疗提供新思路，为推进我国重大疾病的中药研究奠定实验研究基础。

改善缺血性脑卒中后的微循环障碍阻止神经元进一步损伤是近年来脑卒中治疗领域的研究方向。前期有研究报道了卒中治疗后虽然一部分大血管已恢复再灌注，但仍然有一部分小动脉与毛细血管由于受到缺血性损伤的影响在相当长的一段时间内处于持续痉挛状态。因此，在溶栓治疗的基础上改善微循环障碍减轻神经损伤的治疗方法将进一步提升卒中患者的预后。我国传统道地药材钩藤，具有清热平肝、息风止痉的功效，钩藤碱是其主要活性成分。现代中药药理学研究报道了钩藤碱对于心脑血管系统和中枢神经系统的潜在影响，但钩藤碱对于缺血性脑卒中的治疗作用未被阐明。本研究采用缺血性脑卒中小鼠模型，发现钩藤碱具有缓解缺血性脑卒中后的神经功能损伤、减少脑梗死灶体积、改善脑卒中后小鼠认知记忆障碍的作用。对于药物作用机理的研究，本项目通过激光散斑血流成像以及双光子显微镜血管成像等技术发现钩藤碱具有调节微循环血流促进再灌注后微小血管再通从而对神经元以及同神经可塑性密切相关的树突复杂度与树突棘产生保护效应。综上所述，本研究从传统中医药出发，通过现代药理学研究方法进一步阐述了钩藤碱对卒中后脑部微循环的调节效应以及神经保护机制，这将为传统中医药应用于脑卒中的治疗提供坚实的实验研究基础。