基于SIRT2/AMPAR研究电针干预CUMS抑郁模型动物快速起效的海马突触可塑性机制

Electroacupuncture therapy provides a less adverse treatment for depression. However, its mechanism remains largely unknown. Our previous RCT study shows that combination treatment of electroacupuncture (at GV20 and GV29) and paroxetine (a widely used SSRI) improves depressive symptoms within short period of time (less than 1 week). fMRI study shows that one of its targeting brain area is hippocampus. Later animal experiment remains consistent. Further ultrastructure study shows significant hippocampal postsynaptic density change, and proteomics study reveals that mechanism of electroacupuncture effect is tightly connected to hippocampal SIRT2-regulating post-translational acetylation of AMPAR. Therefore, based on our previous studies, we hypothesize that electroacupuncture works by improving AMPAR density related synaptic plasticity in hippocampus, through changing the enzymatic activity of SIRT2. Firstly， evaluate the efficacy of electroacupuncture, hippocampal synaptic plasticity change, its impact on metabolism, SIRT2 activity and SIRT2-AMPAR interaction using ethological tests, electrophysiology tests and immunoelectron microscopy, enzymatic activity testing and immunoprecipitation, immunoblotting and immunofluorescent assays. Then we are gonging to perform electroacupuncture on SIRT2 knockout mice to further prove the hypothesis.

电针治疗抑郁有效且副作用少，但作用机制仍不明确。本课题组临床研究表明，针药结合可快速起效，fMRI研究表明电针靶向作用的脑区之一为海马；动物实验与临床观察一致，且电针能逆转抑郁状态的海马突触结构可塑性变化；蛋白组学研究发现其分子机制与突触后膜AMPA受体（AMPAR）特异性去乙酰化酶SIRT2的活性相关。因此我们提出假说：电针通过影响海马SIRT2活性调控突触后膜AMPAR乙酰化水平，减少受体蛋白降解，影响突触可塑性，促进药物快速起效。基于假说，本研究拟对抑郁大鼠进行电针、针药结合干预，基于行为学指标评估干预效果；运用电生理及免疫电镜技术，观察海马突触可塑性的改变；并检测能量通路改变及SIRT2酶活性的变化；运用免疫沉淀技术、免疫荧光技术和免疫印迹法检测SIRT2与AMPAR相互作用的改变；最后，对SIRT2敲除动物进行干预，完成验证。为电针治疗抑郁症提供新的依据。

目的：探讨电针、针药结合快速起效通过影响海马SIRT2活性调控突触后膜AMPAR和突触可塑性机制。.方法：本研究对慢性不可预见性温和应激刺激（chronic unpredictable mild stress，CUMS）抑郁动物进行电针百会穴和印堂穴干预，电针结合选择性5-羟色胺再摄取抑制剂（selective serotonin reuptake inhibitor，SSRI）抗抑郁药进行针药结合干预；基于行为学指标和海马尼氏染色评估干预效果；运用高尔基染色和透射电镜技术、免疫印迹检测，观察DG神经元突触可塑性的改变，检测海马DG AMPAR水平；运用透射电镜技术、免疫印迹检测，观察海马DG神经元线粒体的改变，测量NAD+/NADH比值，评估SIRT2通路变化；运用免疫沉淀技术、免疫荧光技术检测SIRT2与AMPAR相互作用的改变；最后，对SIRT2敲除动物进行造模干预和评估，运用膜片钳技术检测小鼠海马区电生理变化，完成验证。.结果：电针和针药结合干预显著减少CUMS抑郁模型动物抑郁样行为，且尼氏染色表明海马DG为靶向脑区之一。同时电针和针药结合干预增加CUMS抑郁模型动物海马DG突触数量，改善超微结构，上调AMPAR亚基——GluA1水平，激活线粒体及SIRT2通路，促进SIRT2与GluA1相互作用。SIRT2敲除（knockout，KO）小鼠具有明显的抑郁易感性，海马DG突触可塑性和线粒体及相关通路的异常，且电针和针药结合对KO小鼠快感缺失的抑郁样核心行为改善不明显，对其海马DG的GluA1相关突触可塑性和线粒体能量通路的改变也不显著。电生理结果显示，KO小鼠造模后兴奋性和抑制性突触后自发电流均较野生型小鼠显著增加。.结论：上述实验结果表明，电针、针药结合干预抑郁起效机制与调控CUMS抑郁模型动物海马齿状回的SIRT2/AMAPR通路，增加突触可塑性有关。