电针干预慢性应激模型大鼠CREB-BDNF通路表观遗传学表达的机制研究

Electroacupuncture (EA) is recognized as an effective rapid-acting antidepressant treatment without significant side effects and drug toxicity. EA can give the depressive body an holistic adjustment to bring it back to its normal dynamic balance. Therefore,how to clarify its mechanism, develop the advantages, and improve its positive effectiveness, has been becoming a valuable and urgent researching topic. The environmental factor is strongly associated with the occurrence of antidepressant. This is consistent with epigenetic expression can be changed with environment, which is considered as a supplementation of the field of traditonal genetics. So more and more researches focus on the epigenetic alteration on the CREB-BDNF signaling pathway on neural plasticity in the development of depression and disclosing the mechanisms of antidepressant.According to this theory, a new research derection of depression is also raised in EA treatment. EA can stimulate CREB-BDNF pathway, increase expression of BDNF factor, and enhance the capability of neural plasticity in depression. But where EA exerts its action on CREB-BDNF pathway, whether on increasing CREB phosphorylation or turnning the BDNF genes itself directly, is still unknown. Thus, we are going to launch a study from this fresh perspective in antidepressant treatment with EA. Based on the preliminary works, we adopt CUMS model rats and stimulate them on Baihui, Yintang with EA. Compared with fluoxetine treatment group and CREB antagonist group in which CREB-BDNF signaling pathway is blocked, Radioimmunoassary， Western-blot，Real-time PCR and MSP methods are used to investigate the influence of EA on genetic expression which is expected to disclose the mechanism of antidepressant mechanisms by EA from a novel aspect. Our research analyzes these problems with a novel perspective, hastens the research of the acupuncture mechanism and provide a fresh and effective efficient way in this research field.

电针治疗抑郁症高效低毒副作用，是公认的绿色疗法。电针对抑郁症起整体调节作用，恢复机体固有动态平衡。抑郁症发病环境因素不容忽视，这与表观遗传学可随环境变化而变化的理论相吻合，弥补了经典遗传学理论不足。因此神经营养通路表观遗传学改变的系列研究成为揭示抑郁症发病机制的新热点，也是电针抗抑郁机制研究的新思路。电针激活CREB-BDNF通路增加BDNF表达提高神经可塑性，是通过激活CREB磷酸化，还是直接干预BDNF因子启动子，目前尚不明确。本研究以此为切入点，引入氟西汀对照组，CREB磷酸化拮抗组，采用放免法、Western-blot法、Real-time PCR法、甲基化PCR法，观察电针百会、印堂穴干预慢性应激模型大鼠CREB-BDNF通路相关因子蛋白、mRNA及表观遗传学表达改变，探讨电针抗抑郁机理。旨在从多层次、多角度揭示针刺抗抑郁途径，为针刺整体调节理论提供全新科学佐证，拓展学术空间。

1.研究目的: 抑郁症受基因、环境、社会心理等多因素交互作用控制而发病，针刺治疗抑郁症疗效肯定，整体调节机体，恢复机体原有平衡状态。表观遗传学的出现，弥补经典遗传学理论不足，解释了抑郁症受环境影响，且其发病可被逆转的病理机制。对神经营养假说核心CREB-BDNF通路上相关因子的表观遗传学改变的研究是抗抑郁治疗提供新方向，为针刺抗抑郁机制研究提供新思路。针刺到底是通过激活神经营养通路上游CREB 磷酸化，还是直接干预下游BDNF 因子启动子区增加BDNF 表达，起到抗抑郁作用目前尚不明确。本研究以此为切入点，观察针刺对CREB-BDNF 通路相关因子遗传学改变的研究，从表观遗传学改变角度揭示针刺抗抑郁可能的机制，为针刺整体调节理论拓展学术空间。.2.实验方法：设立西药氟西汀为对照组，引入CREB 拮抗剂H89阻断CREB-BDNF 通路，采用行为学法、Elisa法、RT-PCR法、Western-blot法、MassArray质谱法，观察针刺百会、风池、肾俞穴对慢性应激模型大鼠CREB-BDNF 通路相关因子表观遗传学改变的研究。.3.实验结果：针刺可显著增加慢性应激模型大鼠体重，明显改善慢性应激模型大鼠行为学，增加慢性应激模型大鼠蔗糖水消耗试验糖水消耗量。各阻断剂组体重、活动度、对新鲜环境好奇程度及对奖赏反应水平，均低于非阻断剂组，针刺可改善阻断剂组糖水摄入量。针刺可显著增加海马5-HT表达的含量，抑制HPA轴亢进，增加CREB-BDNF通路PKA、CREB、BDNF、TrkBmRNA含量，增加海马PKA、CREB、pCREB、BDNF蛋白表达含量，阻断剂阻断该通路，针刺失去对BDNF 因子的调整作用。经针刺干预未对BDNF island 1-17 CpG甲基化水平产生影响，但模型组大鼠BDNF-17_CpG6、BDNF-17_CpG13位点甲基化水平出现降低表现。.4.研究结论: 针刺对CREB-BDNF通路有调整作用，阻断这个通路可阻断针刺对BDNF 因子的调整作用，提高体内CREB磷酸化水平，增加BDNF 水平是针刺抗抑郁机制之一，针刺对BDNF 因子表观学表达表现出有调整作用的倾向。 .5.研究成果:发表核心期刊文章3篇，参加国内外会议交流各1次，参与培养硕士研究生2名（毕业），博士研究生1名（在读）