海马反应性星形胶质细胞增加抑郁复发易感性的机制及芍药苷的治疗作用研究

Following the protection of reactive astrocytes (RA) against CNS injury, in an apparently counterintuitive response, RA inhibits neuronal regeneration. Studies from our laboratory have shown that RA played an important role in neuroprotection of imipramine and improved the vulnerability for recurrence of depression. So we hypothesize the RA improves the recurrence of depression by inhibition of neurogenesis in the period of remitted depression. Firstly, in our work, the model of recurrent depression in rats was established by the following protocol: the CUMS-treated rats recover from depression with the treatment of imipramine and then exposed to CUMS again. Specific enzymes (ChABC) were injected into brain of the rats in remitted depression to degrade CSPGs in RA. The neurogenesis, level of GFAP, BDNF and ProBDNF protein and mRNA and level and structure in different regions of RA in hippocampus were evaluated for the mechanism. Secondly, the effects of RA on neurogenesis were further detected by the model of RA and neural stem cells co-cultured in Transwell in vitro. Finally, the effect of paeoniflorin and ChABC were administrated together as combined treatment to reduce the RA and induce neurogenesis. This approach may stimulate the discovery of new biomarkers and mechanism underlying the vulnerability for recurrence of depression in neurobiological domain. Furthermore, it may provide insights that help prevent the recurrence of depression and to develop new therapies for it.

反应性星形胶质细胞（RA）胶质化在不同时期具有促脑组织损伤修复和抑制神经再生的“双刃”效应。课题组前期研究发现RA参与了盐酸丙咪嗪的神经修复作用，且能增加抑郁复发易感性。我们推测：RA在康复期通过对神经再生抑制作用增加抑郁复发易感性。本课题拟利用慢性不可预知应激制备首发抑郁模型，经盐酸丙咪嗪治疗康复后再次应激制备复发抑郁模型，考察检测海马RA空间结构分布、GFAP、BDNF及其前体基因和蛋白表达与海马神经再生相关性，阐明RA介导海马神经再生损伤而增加抑郁复发易感性的作用机制；利用RA和神经干细胞Transwell共培养细胞模型，进一步验证RA对神经再生的作用机制，并探索RA介导抑郁复发的可能信号通路。在此基础上，考察芍药苷联合ChABC（RA降解剂）抗复发抑郁作用的治疗时间窗，寻求联合治疗方案。研究将有助于从细胞分子水平发现抑郁症易复发的神经病理学机制，为有效防治抑郁症复发提供参考。

实验一：复发抑郁模型大鼠神经内分泌改变。制备大鼠复发抑郁模型，利用糖水消耗实验、游泳实验、体重测量等方法考察复发抑郁模型大鼠的行为学改变，利用Y迷宫新奇事物探索实验和敞箱实验考察复发抑郁模型大鼠的认知能力。利用透射电镜法观察复发抑郁模型大鼠海马区超微结构改变；运用ELISA和Western blot技术检测大鼠血清CORT、ACTH、cAMP和海马DG区CREB、BDNF和FGF2蛋白表达，考察复发抑郁模型大鼠下丘脑-垂体-肾上腺轴、脑神经营养因子及相关蛋白表达。结果发现，与正常组比较，复发模型组大鼠体重减轻、糖水消耗指数和游泳不动时间明显减少，存在明显抑郁样行为，且和首发抑郁模型组比较其造模时间缩短了一周。复发抑郁模型大鼠在Y迷宫新奇事物探索实验中在新臂的时间较正常大鼠明显缩短，说明其存在认知功能损伤。超微结构观察表明复发抑郁模型大鼠海马区存在一定神经损伤现象。复发模型组大鼠血清CORT、ACTH显著升高，cAMP、CREB和BDNF蛋白表达显著降低，但是FGF2蛋白表达未见明显改变。结果说明，复发交首发模型更容易成模；复发模型大鼠具有明显的抑郁样行为、存在认知能力和海马神经损伤；HPA轴内分泌明显上调，cAMP-CREB-BDNF信号通路相关蛋白表达因子明显下降。实验二，ChABC降低星型胶质细胞促抑郁复发作用。制备复发抑郁模型，并在康复期初始期给予ChABC，其余同上。利用利用糖水消耗实验、体重测量等方法考察ChABC对复发抑郁模型大鼠的行为学改变，考察ChABC对复发抑郁模型大鼠的影响。结果表明与复发模型组相比，ChABC组大鼠体重没有显著差异；糖水偏好指数在复发期具有显著差，结果表明星型胶质细胞介导抑郁复发。实验三，芍药苷联合ChABC对复发抑郁模型大鼠的治疗时间点考察。制备复发抑郁模型，并在再应激期分别提前、同时和退后一周给予芍药苷，通过行为学实验考察芍药苷联合ChABC对复发抑郁模型大鼠的最佳治疗作用时间窗。结果表明联合ChABC在应激期提早一周给予芍药苷对复发抑郁模型治疗作用较为明显。