涤痰化瘀法干预CREB信号通路治疗PSD的机制研究

Post-stroke Depression (PSD) is a complication which will seriously influence the curative effect and prognosis of stroke. At present, four kinds of antidepressant drugs have been used frequently, but which have been regarded unsatisfying therapeutic, detrimental to the primary disease and with many side effects in some clinical documents. Eliminating phlegm and dissolving stasis is the basic therapy treating PSD, which is set by Zhang Xuewen (one of the greatest master of Chinese medicine) and is confirmed good curative effect through clinical research. Based on the eliminating phlegm and dissolving stasis, and focused on the key factor CREB of antidepressant drugs' receptor intracellular signaling transduction, we will research the influence of eliminating phlegm and dissolving stasis on expression of CREB, activation levels and downstream BDNF through cell model in vitro. And we'll analyze the regulating function of the eliminating phlegm and dissolving stasis to PSD model - animal hippocampus' upstream CREB signal transduction (CaMK、TrKB、PKA,etc.)and CREB-BDNF axis through PSD rat model, in order to prove the hypothesis of "the molecular mechanism is the eliminating phlegm and dissolving stasis' promoting the process of CREB phosphorylation, and furthermore regulating the target gene BDNF after CREB phosphorylation, and promoting neural regeneration, based on the CREB-BDNF axis", meanwhile to clarify the molecular mechanism of the eliminating phlegm and dissolving stasis' treating PSD, at the same time to supply the theoretical and experimental proof of TCM theory treating the same disease with different methods.

卒中后抑郁症（PSD）是严重影响脑卒中疗效和预后的并发症，临床多采用抗抑郁药物同期治疗，大量临床研究表明此类药物存在疗效不佳、对原发病不利和药物毒副作用等诸多问题。"涤痰化瘀"法是国医大师张学文教授长期治疗PSD的临床证治经验总结，本项目拟在前期研究的基础上，围绕抗抑郁药物受体细胞内信号转导通路的关键分子CREB，通过体外细胞模型，研究涤痰化瘀法对CREB的表达、活性水平及下游BDNF的影响；通过卒中后抑郁的大鼠模型，分析涤痰化瘀法对PSD模型动物海马CREB上游CaMK、TrKB、PKA等信号转导通路及CREB-BDNF信号轴的调节作用，以期验证"基于CREB-BDNF轴，涤痰化瘀法促进CREB磷酸化的过程、进一步调节CREB磷酸化后的靶基因BDNF、促进神经再生是其治疗卒中后抑郁症的分子机制"的理论假说，阐明涤痰化瘀法治疗PSD的分子机理，并为中医异病同治理论提供理论和实验依据。

卒中后抑郁症（Post-stroke depression,PSD）是脑卒中最常见的并发症之一，严重影响卒中的康复。最新研究发现脑内cAMP 反应元件结合蛋白（CREB） 及其靶基因脑源性神经营养因子（BDNF）表达下降是PSD 发生发展的关键。然而，针对该机制的有效治疗尚无突破性进展。本项目基于国医大师张学文教授临床经验，针对PSD 卒中和抑郁并存的病机特点，确立了涤痰化瘀为PSD 的基本治法并依此法立醒脑解郁方。该方临床应用十余年，疗效确切，且无明显副作用。前期研究发现，醒脑解郁方能上调BDNF 的表达，调节CREB 的活性。本课题我们通过大脑中动脉栓塞联合利血平皮下注射建立卒中后抑郁大鼠模型，运用醒脑解郁方进行早期干预和治疗。采用行为学检测评估抑郁状态，qRT-PCR检测p-CREB、ERK1/2、CaM mRNA水平，免疫组化法检测p-CREB、ERK1/2表达水平，Elisa检测海马cAMP、BDNF含量，蛋白印迹实验检测PKA蛋白水平。结果显示，模型组大鼠呈现明显抑郁状态，醒脑解郁方早期干预及治疗能有效改善大鼠体重、蔗糖水消耗量、增加运动评分，此外能提高p-CREB、ERK1/2 mRNA与蛋白水平，增加海马CaM mRNA、PKA与cAMP含量，促进神经元存活与再生。本项目明确了醒脑解郁方通过CREB/ERK/BDNF通路促进神经元的存活与再生，维持神经递质水平，进而发挥抗抑郁作用。为醒脑解郁方治疗PSD 提供了实验依据，有助于深化和拓展中医“异病同治”理论在临床复杂病机病证中的应用。