基于肠道菌群及神经肽探讨“调神畅情”法不同电针干预帕金森病抑郁的脑肠相关机制

In this research, according to the pathogenesis of stagnation due to deficiency of PDD belonging to tremor syndrome combined with depression in Chinese medicine theory, we promote “TiaoShenChangQing” as the method of treatment. Models with PD were made by two targets injecting 6-OHDA in one side of striatum, and based on it, setting up PDD model by CUMS combined with separated living method. We take ERIC-PCR fingerprinting technique, ELISA, immunohistochemistry, and RT-PCR as the detection method.We take the feature of change of the intestinal microbiota as the beginning point to verifying the adjustment by “TiaoShenChangQing” acupuncture therapy to the intestinal brain of PDD rats, and take the brain–gut peptides including VIP, SP, and Nesfastin-1 as the observation point to ensuring the intervention effect by “TiaoShenChangQing” acupuncture therapy to the brain–gut peptides in the blood or brain of PDD rats, while take the expression of 5-HT、NE、CRF、CRFR2, TH positive neurons, and caspase-3,bax,bcl-2 in different brain area as the breakthrough point to prove that the final target of “TiaoShenChangQing” acupuncture therapy is to adjusting the unbalance of the system of 5-HT and NE while protecting the DA neurons. All of the above, it will provide the experimental baed for research of the treatment method of neurodegenerative disease with mental disorder through the double ways of brain and gut by different electro-acupuncture, and further more, it will provide powerful evidence for the comparative advantage for acupuncture method.

本项目针对本病在中医理论上属颤证与郁证合病的因虚致郁病机，提出“调神畅情”治法，采用6-OHDA纹状体双靶点注射法复制PD模型，在此基础上结合CUMS法与孤养法复制PDD模型，采用ERIC-PCR指纹图谱分析、ELISA法、免疫组织化学法、RT-PCR法等方法，以肠道菌群的特征变化为入手点，验证“调神畅情”针法对肠脑的干预作用；以VIP、SP、Nesfastin-1等脑肠肽含量的变化为观察点，明确其对相关脑肠肽的干预作用；以相关脑区5-HT、NE、CRF、CRFR2表达的变化，TH阳性神经元表达的变化，及caspase-3,bax,bcl-2等神经元凋亡影响因子表达的变化为切入点，明确其最终作用靶点为调整5-羟色胺、去甲肾上腺素系统失调及对多巴胺能神经元的保护作用，为阐明不同电针通过脑肠双重途径来实现对神经退行性疾病伴发精神障碍的治疗奠定实验基础，同时为优化出针刺的优效性方法提供有力证据。

本项目采用6-OHDA纹状体双靶点注射法结合CUMS法与孤养法复制PDD模型，大鼠随机分为6组：正常组、假手术组、模型组、药物组、音乐电针组、脉冲电针组，采用ERIC-PCR指纹图谱分析、16SrDNA高通量测序分析，ELISA法，免疫组织化学法，RT-PCR法等方法，观察了各组大鼠干预28天后（部分指标连续观察42天）肠道菌群的丰富度、多样性、结构比例；下丘脑及结肠中VIP、SP、Nesfastin-1等脑肠肽含量的变化；中缝背核5-HT、NE、CRF、CRFR2、TPH2表达的变化；黑质TH阳性神经元表达的变化，及caspase-3,bax,bcl-2等神经元凋亡影响因子表达的变化。结果表明：1“调神畅情”音乐电针干预PDD能够直接作用于肠脑，通过增加大鼠肠道菌群活性、多样性以及丰富度，调整拟杆菌与厚壁菌比例，使拟杆菌比例升高，厚壁菌比例减低，从而改善肠道微生物的菌群分布。2“调神畅情”音乐电针干预PDD可以双向调节脑肠轴通路中脑肠肽含量，通过同步上调脑肠中VIP的表达，下调SP、Nesfatin-1的表达来增强中枢对应激的调节能力，同时降低肠神经敏感性促进肠动力，起到抗帕金森抑郁和改善胃肠功能的作用。3“调神畅情”音乐电针干预PDD可以从整体上恢复五羟色胺能神经元系统的平衡，增加缺失的5-HT、NE神经元，提高5-HT合成限速酶TPH2的表达，抑制应激调节关键因子CRF及其受体CRFR2的表达，促进对五羟色胺能神经元的保护作用。4“调神畅情”音乐电针干预PDD可能是从抗多巴胺神经元凋亡的角度，增加抑凋亡因子bcl-2的表达，减少促凋亡因子bax的表达，以及抑制凋亡启动因子Caspase-3的表达，促进对多巴胺能神经元的保护作用。5“调神畅情”音乐电针干预PDD作用的途径之一可能为抑制脑组织cAMP/PKA信号通路的激活，从而发挥抗PDD作用。6音乐电针干预PDD作用效果不随时间延长而衰减，是提高针灸临床疗效的有效方法之一。本项目明确了电针干预PDD的作用靶点为调整5-羟色胺能神经系统失调及对多巴胺能神经元的保护作用，为阐明针刺作用于脑肠双重途径来实现对神经退行性疾病伴发精神障碍的治疗奠定实验基础，同时为优化针刺方法提供了有力证据。