基于RhoA/ROCK通路的“三焦”针法改善阿尔海默病海马突触可塑性研究

Alzheimer's disease (AD) is one of neurodegenerative diseases in the elderly people, and its incidence and mortality is increasing with the aging of the population rencently, but an effective cure is still lack. The project builds on the clinically effects of acupuncture on AD and the experimental research progression in the past years. Preliminary studies showed acupuncture had played a benign regulatory role of damaged dendrites and RhoA/ROCK pathway imbalance by neuropathology and proteomics. Thus, the working hypothesis is generated that acupuncture may improve hippocampal synaptic plasticity of dementia model mouse by regulating RhoA/ROCK signaling pathway. SAMP8 is the subject of this study, and hippocampal synaptic plasticity is a suitable solution. RhoA/ROCK signaling pathway which regulates synaptic plasticity is the cardinal line. LTP in vivo, Golgi staining, qPCR and Western techniques will be used, hippocampal synaptic plasticity related parameters (LTP, dendritic spine morphology and density) and the expression and activity of RhoA/ROCK pathway-related factors (RhoA, ROCKⅡ, MLC2, LIMK2 and CRAMP2) will be detected. Intervention effects of acupuncture on these indicators will be evaluated. So that we can explore the mechanism of acupuncture prevention of dementia and provide experimental evidence for acupuncture clinical application and generalization.

阿尔海默病（AD）是一种常见的老年性神经退行性疾病，伴随人口老龄化，其发病率和死亡率逐年增加，但仍缺乏有效疗法。本项目建立在针刺治疗AD临床有效，并经多年实验研究有所发现基础之上。我们前期研究发现针刺对SAMP8神经树突受损及RhoA/ROCK通路的相关因子失衡有良性调节作用，从而提出“针刺通过调节RhoA/ROCK通路改善痴呆模型鼠海马突触可塑性”的工作假说。课题以痴呆模型鼠SAMP8为研究对象，以海马突触可塑性为切入点，以调控突触可塑性的RhoA/ROCK信号通路为主线，应用在体LTP、高尔基染色、qPCR和Western等技术观测海马突触可塑性相关指标(LTP、树突棘形态和密度)的改变和RhoA/ROCK通路中相关因子（RhoA、ROCKⅡ、MLC2、LIMK2和CRAMP2）表达和活性的变化，以及针刺干预效应，探讨针刺防治AD的机制，为针刺AD的临床应用和推广提供实验依据。

阿尔茨海默病（AD）是一种常见的老年性神经退行性疾病，伴随社会老龄化进程，AD的发病率和死亡率呈逐年递增趋势。针刺作为祖国医学的重要组成部分，在治疗神经系统性疾病方面显示出巨大潜能。三焦针法是基于“三焦失司导致AD”的中医理论所设，在临床治疗痴呆中疗效确切。我们基于文献报道和前期实验研究提出了“针刺通过调节RhoA/ROCK通路促进海马突触重塑改善痴呆状态”的假说。在本研究中发现SAMP8小鼠海马CA1区树突的长度和分支数量明显减少，在距胞体40-160um处的交点数目明显减少，从而导致树突的复杂度明显减低。三焦针法对上述损伤有较好的改善作用。顶树突二级分支单位长度上棘的数目明显减少，进一步分析发现主要是细长型棘的减少。三焦针法对树突棘的作用并不显著。对RhoA/ROCK信号途径的研究发现，与SAMR1组相比，SAMP8组中存在RhoA/ROCK信号通路的紊乱，主要表现于相关因子的蛋白活性（RhoA）和磷酸化水平（CRMP2、LIMK2和MLC2）的增高，除MLC2 mRNA降低外，其他因子的转录和蛋白表达水平并未发生显著改变。针刺对SAMP8小鼠RhoA/ROCK信号途径有抑制作用。在针刺机制的研究中发现，RhoA抑制剂C3可改善SAMP8小鼠的空间学习能力和树突形态，增加突触标记性蛋白SYP的表达，针刺具有与之类似的效应。表明针刺可能通过抑制RhoA/ROCK信号途径发挥治疗AD的作用。本研究为针刺治疗AD提供了实验依据，丰富了针灸的科学内涵，有利于针灸治疗AD的推广。