基于AMPK/ULK1通路介导的自噬作用探究分心木多糖防治抑郁症的作用及机制

Diaphragma juglandis fructus, which is the dry wood diaphragm tissue that grows inside the walnut, has attracted much attention in traditional Chinese medicine.It has been used as a kind of tea material in civil. Its decoction has been proved to treat insomnia and to relieve bad mood. In our previous study, we found that the polysaccharides of diaphragma juglandis fructus could improve depressive behavior in chronic unpredictable mild stress model (CUMS). After detecting the relative proteins in mouse hippocampal, we found that the autophagic marker LC3B-II was up-regulation and p62/SQSTM1 was down-regulation compared with model group，suggested that its antidepressant effect was closely related to autophagic regulation of nerve cells in hippocampal. After the intervention of polysaccharides, the results of the protein expression, such as AMPK, ULK1 and mTOR, showed an interesting relationship between AMPK and ULK1. We speculated that the autophagic regulation was related to AMPK/ULK1 pathway. Therefore this subject aims to build CUMS model and detect the autophagic effect in different times by using different strategies, including Transmission electron microscopy (SEM), Western blot, immunohistochemistry and immunofluorescence. We could summarize the autophagic characteristics and reveal the mechanism of AMPK/ULK1, which could provide new strategies and targets for depression and provide a scientific basis of Diaphragma juglandis fructus.

分心木系核桃中分离核桃肉的隔膜，民间验方证实其有治疗失眠、缓解不良情绪作用。申请者在前期研究中发现分心木多糖类成分对慢性温和应激模型小鼠（CUMS）行为学具有明显改善作用，通过对小鼠海马组织蛋白检测，发现相较于模型组，上调了自噬体标志分子LC3B-II的表达，下调了自噬选择性降解底物p62/SQSTM1的表达，提示其抗抑郁疗效与海马神经细胞的自噬调控紧密相连；通过进一步对AMPK、ULK1、mTOR蛋白的检测，发现给予分心木多糖后，AMPK与ULK1存在有趣的联系，推测该自噬调控与AMPK/ULK1通路密切相关。因此，本课题拟通过构建CUMS模型，采用透射电镜、蛋白质印迹法、免疫组化、免疫荧光等技术，动态检测给药后不同时间段分心木多糖诱导神经细胞自噬的作用，总结其自噬特征规律，揭示其调控AMPK/ULK1信号机制，为分心木多糖抗抑郁研究提供新的策略和靶点，为充分开发分心木资源提供科学依据。

抑郁症是一种由长期抑郁情绪所引起的神经精神疾病，是全球发病率最高的精神类疾病。越来越多的研究表明，自噬作为一把双刃剑，参与了抑郁症的病理过程。前期研究发现分心木多糖抗抑郁疗效可能与神经细胞的自噬调控AMPK通路紧密相连。首先，本项目针对市场分心木良莠不齐、缺乏质量标准的现状，采用指纹图谱结合化学计量学的方法，对12批次不同产地分心木质量进行了评价，进行了外观、水分、灰分、浸出物及含量测定研究，指纹图谱共确定了6个共有峰作为分心木品质优劣指标，建立了分心木的质量控制体系。其次，选用质优分心木进行了多糖组分的提取，采用水提醇沉的方式获得了来源稳定的粗多糖组分，进而采用离子交换柱层析对粗多糖进行了极性分离，共获得4个不同极性部位，采用凝胶色谱柱进行分子量测定。细胞筛选研究表明FXM-PC2部位对于皮质酮诱导的HT22小鼠海马神经细胞损伤具有较好的保护作用。在此基础上，对FXM-PC2部位进行了凝胶纯化，获得了均一化多糖组分PC2A；并采用离子色谱发测定了PC2A多糖的单糖组成，采用甲基化、核磁共振氢谱、核磁共振碳谱、二维核磁共振谱2D-NMR对PC2A多糖进行了结构解析。最后，项目构建了慢性不可预知刺激小鼠抑郁模型，观察PC2A多糖对抑郁模型的影响，结果表明给予分心木多糖组分后，可以改善小鼠糖水偏好、开放旷场、强迫游泳等行为学指标；HE及尼氏染色表明给药后可以有效保护小鼠海马细胞，减少海马神经元损伤；小鼠全脑核磁共振实验证明多糖的介入可以防止模型小鼠脑室增大，海马萎缩；Elisa测定了不同组别小鼠血清中5-羟色胺及皮质酮含量；免疫荧光实验发现给药后可以有效增加自噬标记物LC3B的阳性表达。Western blot实验对小鼠海马组织中LC3I、LC3II、P62、AMPKα、p-AMPKα、mTOR、p-mTOR、ULK1、p-ULK1等蛋白进行测定，结果表明分心木多糖组分可以有效解除抑郁模型小鼠的自噬抑制，通过增加自噬为神经细胞提供必要的养分，其机制可能与AMPK/mTOR/ULK1通路密切相关。通过本项目研究，分心木多糖抗抑郁研究提供新的策略，为充分开发分心木资源提供了科学依据。