针刺百会、印堂对脑卒中抑郁大鼠神经递质和蛋白组学作用的机制研究

Stroke and depression are hotspots of current research. The incidence rate of post-stroke depression is high, and life quality and prognosis of the patients is significantly affected. In this study, acupuncture is used as rehabilitation methods, and Baihui and Yintang acupoint which have definite therapeutic effects on post-stroke depression are chosen as major acupuncture points. Sucrose consumption test, open field test and forced swimming test are performed to evaluate effects of acupuncture on behavior of PSD rats. 5-HT and its metabolites 5-HIAA, DA and its precursor L-DOPA is evaluated with HPLC-ECD to investigate effects of oacupuncture on monoamine neurotransmitters of PSD rats. Quantitative relationship of excitatory neurotransmitter Glu and inhibitory neurotransmitter GABA is detected by microdialysis technique, in order to clear effects of acupuncture on amino acid neurotransmitters of PSD rats. Simultaneously, iTRAQ proteomics technology is applied to detect proteins in hippocampus of PSD rats. Differentially expressed proteins are screened, and associated pathways are identified. Related proteins in the pathways are verified by Western-blot, in order to investigate the mechanism of proteomics. This study provides an experimental basis for clinical mechanism of acupuncture at Baihui and Yintang in the treatment of post-stroke depression.

脑卒中和抑郁症均是目前研究的热点，脑卒中后抑郁发病率高，对罹患者的生活质量和预后有重要影响。本研究以针刺为康复手段，并选择临床有确切疗效的百会、印堂穴，考察针刺百会、印堂穴对PSD大鼠行为学方面糖水偏好实验、旷场实验、强迫游泳实验的影响；应用HPLC-ECD系统检测5-HT及其代谢产物5-HIAA、DA及其前体L-DOPA，考察针刺百会、印堂对PSD大鼠单胺类神经递质的影响；引入微透析技术，检测兴奋性神经递质Glu及抑制性神经递质GABA的量化关系，以明确针刺百会、印堂对PSD大鼠氨基酸类神经递质的影响；同时应用iTRAQ蛋白组学技术，通过PSD大鼠海马蛋白组学检测，筛选出差异表达蛋白，并鉴定出相关的通路，通过western-blot验证蛋白通路上的相关蛋白，以考察其蛋白组学作用机制。此研究为揭示针刺百会、印堂治疗脑卒中后抑郁的临床机制奠定实验基础。

卒中后抑郁是脑卒中患者中最常见的精神疾病并发症，至少影响三分之一脑卒中患者的生活质量。电针选择百会、印堂穴进行治疗，可使脑卒中患者抑郁症状明显缓解，生活显著质量提高。本项目通过大脑中动脉线栓阻塞法和慢性不可预知温和应激复制脑卒中抑郁（PSD）大鼠模型，考察针刺百会、印堂穴对PSD大鼠行为学方面糖水偏好实验、旷场实验、强迫游泳实验的影响，并运用ELISA法检测其血浆5-HT、DA、Glu和GABA水平，结合透射电镜考察电针对大鼠海马超微结构的影响，同时应用iTRAQ蛋白组学技术，通过PSD大鼠海马蛋白组学检测，筛选出差异表达蛋白，并鉴定出相关的通路，通过western-blot验证蛋白通路上的相关蛋白，以考察其蛋白组学作用机制。结果表明：电针百会、印堂能显著提高PSD大鼠对蔗糖的偏好，减少强迫游泳实验中的静止时间，并能提高旷场实验穿越距离和进入中央格次数；电针能提高单胺类神经递质5-HT和DA水平，并调整氨基酸类神经递质Glu和GABA水平；在透射电镜超微结构观察中，电针组突触数量多，突触间隙较清晰；而iTRAQ蛋白组学结果表明，在KEGG通路富集分析中，前10个上调通路多与抑郁障碍密切相关，如谷氨酸能突触、昼夜节律同步、多巴胺能突触、钙信号通路、长时程增强和柠檬酸循环（TCA循环）。前10个下调途径与炎症反应密切相关，如局灶性粘附、肌动蛋白细胞骨架调节、白细胞跨内膜迁移。Western blot验证结果表明电针百会、印堂使下调的大鼠海马谷氨酸受体Gria1活性增加，使上调的整合素相关蛋白Itgb1和Itgb2降低，从而发挥抗抑郁和抗炎作用。说明电针百会、印堂穴具有确切的改善PSD作用，其作用机制与调节神经递质水平、改变海马突触可塑性以及调节海马抑郁和神经炎症相关通路等密切相关，课题基于蛋白组学阐释了电针百会、印堂穴改善PSD的潜在作用机制，为深入揭示电针百会、印堂穴改善PSD的现代医学内涵奠定实验基础。