东南大学附属中大医院神经精神医学科，东南大学脑血管病研究所
郭怡菁 张志珺 王少华 隋毓秀 孙奕

【摘要】研究背景及目的：Notch信号通路是抑制性信号转导途径，在决定神经前体细胞（neural precursor cells, NPCs）增殖和分化方向上起着关键的作用。有研究提示缺血性卒中后大鼠海马区Notch信号系统可能参与了卒中后海马神经再生的调节，大量的研究表明慢性不可预见性应激（chronic unpredictable mild stress, CUMS）抑制海马神经再生。本研究拟探讨卒中后应激对海马神经再生及其可能分子调控机制-Notch信号系统的影响，以及抗抑郁药物氟西汀干预后的变化。方法：在建立大鼠MCAO局灶性缺血模型的基础上，予18天CUMS流程处理，建立拟卒中后抑郁（post-stroke depression, PSD）动物模型。实验分为假手术组（Sham）、卒中组（MCAO）、卒中后抑郁组（MCAO+CUMS）以及氟西汀药物干预组（MCAO+CUMS+Flu）。采用糖水试验、敞箱试验及强迫游泳试验评估实验动物抑郁行为。分别在MCAO术后第19天和第28天采用BrdU+免疫组织化学染色及BrdU+/NeuN+、BrdU+/GFAP+免疫荧光双标染色法，比较不同组间、不同时间点时海马齿状回NPCs的增殖及向神经元、星形胶质细胞分化的变化；并采用western blotting、real time RT-PCR测定各组不同时间点Notch1、及其配体Jagged1、下游靶点Hes1、Hes5蛋白及其mRNA表达。结果：与MCAO大鼠相比，给予CUMS处理的MCAO大鼠在术后19天出现抑郁症状的行为学改变，海马齿状回NPCs增殖减少（P<0.01），Hes1、Hes5的蛋白、mRNA表达降低（均P<0.05）。MCAO术后28天时，CUMS导致MCAO大鼠海马齿状回颗粒下层（SGZ）新生NPCs向神经元方向分化减少（P<0.01），而齿状回门区的新生NPCs向星形胶质细胞方向的分化增多（P<0.05），同期的Hes1、Hes5蛋白及其基因表达增加（均P<0.05）。但与MCAO组相比较，CUMS＋MCAO组的Notch1的蛋白、mRNA表达在MCAO术后19天和28天时均显著下降（P<0.05），而其配体Jaaged1的蛋白、mRNA表达在MCAO术后19天时无显著差异（P>0.05），但在MCAO术后28天显著增加（P<0.05）。氟西汀干预可部分逆转CUMS对MCAO大鼠海马神经再生及Notch系统活性的影响。结论：持续的慢性应激可逆转卒中后海马神经再生的上调，同时导致海马Notch信号系统功能的动态变化，抗抑郁剂氟西汀则可逆转CUMS导致的卒中后海马神经再生以及Notch信号系统功能的变化，提示Notch信号系统有可能参与了成年海马神经再生的调控，并且在卒中、慢性应激等病理状态下该通路的活性状态受到影响，而氟西汀对该通路的活性也可起到调节作用。其次，CUMS以及氟西汀对Notch信号系统活性的影响可能不仅仅是通过Jagged1激活途径，进一步深入研究Notch信号系统各成分及调节因素的作用，将有助于为调控海马神经再生进而为相关疾病的治疗找到新的靶点。
【关键词】 Notch信号，卒中后抑郁，神经再生，海马


Effect of Chronic Unpredictable Mild Stress on Notch Signal Activity in Hippocampus of Local Ischemic Rats

Yi-jing Guo, Zhi-jun Zhang, Shao-hua Wang, Yu-xiu Sui, Yi Sun
The Department of Neurology and Institution of Cerebral Vascular Disease, Affiliated ZhongDa Hospital of Southeast University, China.

【Abstract】Background and objectives Accumulating evidence indicates that the Notch signaling pathway fulfills important roles in ischemiastimulated neurogenesis, which may be regarded as an etiological factor in post-stroke depression. Here we explored Notch1 signaling, hippocampal neurogenesis and behavioral responses to chronic unpredicted mild stress (CUMS) in adult ischemic rats. Methods Animals were treated with permanent middle cerebral artery occlusion followed by an 18 day CUMS procedure. Proliferating cells in the hippocampus and their cell fate were investigated on days 19 and 28 after ischemic surgery. Additionally, expression of the Notch1, it ligand Jagged1 and its downstream targets Hes1 and Hes5 was examined. A sucrose preference test, open-field test and forced swim test were used to assess behavioral responses. Results CUMS produced depressive-like behaviors and decreased the number of proliferating cells on day 19 (both p<0.01), accompanied by a decreased protein and mRNA expression of both Hes1 and Hes5 in the hippocampus of ischemic animals (all p<0.05). On day 28, CUMS resulted in a decreased number of neurogenically-differentiating cells in the subgranular zone (p<0.01) while permitting differentiation into astrocytes in the hilus (p<0.05). Hes1 and Hes5 protein and mRNA expression levels were increased. The Notch1expression of MCAO+CUMS group was significantly decreased at both time-points in comparison with that of MCAO group (p<0.05). The Jagged1 expression of MCAO+CUMS group only increases on the day 28 (p<0.05). Fluoxetine treatment transverse almost all the changes induced by CUMS.Conclusions CUMS led to expression changes in the Notch signaling cascade in ischemic rats, most of which concerned hippocampal neurogenesis. This suggests that variation in Notch1 activity and subsequent expression of its downstream targets, including Hes1 and Hes5, may, at least in part, contribute to modulation of ischemiarelated hippocampal neurogenesis by CUMS. The regular of the expression changes of Notch1 and its ligand Jagged1 also demonstrates that CUMS does not regulate the activity of Notch signaling pathway only via ligand activating.
【Keywords】 Notch signaling, post-stroke depression, neurogenesis, hippcampus