刘慧, 问黎敏, 乔卉, 安书成
陕西师范大学生命科学学院, 西安 710062
本文旨在探讨应激性抑郁样行为发生中海马胶质细胞释放高半胱氨酸(homocysteine, Hcy)的作用，以及与谷氨酸(glutamic acid, Glu)及其受体的关系。建立慢性不可预见性温和应激(chronic unpredictable mild stress, CUMS)性抑郁模型，海马单侧微量注射Hcy、N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid, NMDA)受体拮抗剂MK-801和α-氨基羟甲基异恶唑丙酸(α-amino-3-hudroxy-5-methylisoxazole-4-propionic acid, AMPA)受体拮抗剂NBQX，观察大鼠体重变化率，并通过糖水偏爱测试、旷场实验和悬尾实验等检测行为表现，采用高效液相色谱法(high-performance liquid chromatography, HPLC)和Western blot方法检测海马内Glu水平及其NMDA受体和AMPA受体关键亚基的变化，运用酶联免疫吸附(enzyme linked immunosorbent assay, ELISA)法测定谷氨酸NMDA受体和AMPA受体的磷酸化水平和Hcy含量。结果显示，CUMS诱发大鼠表现出明显的抑郁样行为，且海马Glu和Hcy含量、NMDA受体的NR1亚基和NR2B亚基表达及其磷酸化水平显著升高，而AMPA受体的GluR2/3亚基含量及其磷酸化水平均明显降低；正常大鼠海马微量注射Hcy，也表现出抑郁样行为，且Glu含量明显升高，NMDA受体NR1亚基和NR2B亚基及其磷酸化水平均显著升高，AMPA受体的GluR2/3亚基含量明显升高，但其磷酸化水平明显降低；CUMS和海马注射Hcy引起的抑郁样行为可被海马注射NMDA受体拮抗剂 MK-801显著改善，同时MK-801可以显著抑制由Hcy引起的Glu升高，而AMPA受体拮抗剂NBQX不能改善其抑郁样行为，但同样可以使Hcy引起的Glu升高显著降低。以上结果表明，CUMS引起海马星形胶质细胞产生和释放Hcy增加，经NMDA受体和AMPA受体，引起海马Glu升高的同时，提高NMDA受体的NR1亚基和NR2B亚基表达，并促进其磷酸化，提高AMPA受体的GluR2/3亚基表达而降低其磷酸化水平，导致抑郁样行为发生。可见，星形胶质细胞释放Hcy在应激引起海马Glu升高及NMDA受体和AMPA受体变化中具有重要作用。
[The modulation of hippocampal glutamate and NMDA/AMPA receptor by homocysteine in chronic unpredictable mild stress-induced rat depression.] [Article in Chinese]
Liu Hui, WEN Li-Min, QIAO Hui, AN Shu-Cheng
College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China
The study was to investigate the role of homocysteine (Hcy) which was released by hippocampal glial cells and its relationship with NMDA receptor and AMPA receptor in depression induced by chronic unpredictable mild stress (CUMS), and explore the mechanism of changes of Glu/Glu receptor in glial cells and neurons. CUMS-induced depression model was established. The body weight of rats was weighed on the 1st, 7th, 14th, and 21st days during the experiment. The behavioral performances were observed by means of sucrose consumption test, open field test and tail suspension test. Intrahippocampal microinjection of Hcy, NMDA receptor antagonist MK-801 and AMPA receptor antagonist NBQX was performed under stereotaxic guide cannula. The concentration of Glu and the expression of its receptors' subunits were detected respectively by high performance liquid chromatography (HPLC) and Western blot. The Hcy content and the levels of phosphorylation of NMDA receptor and AMPA receptor in hippocampus were separately determined by enzyme linked immunosorbent assay (ELISA). The results showed that CUMS significantly induced the depression-like behaviors in rats, and the content of Glu and Hcy, the expression of NMDA receptors' subunits NR1/NR2B and the level of phosphorylation of NMDA receptor (p-NMDAR) in hippocampus increased significantly, while the expression of AMPA receptors' subunits GluR2/3 and the level of phosphorylation of AMPA receptor (p-AMPAR) decreased significantly. Microinjection of Hcy into hippocampus resulted in similar animal depression-like behaviors and increased Glu content compared to the CON/SAL group, the expression of NR1/NR2B/GluR2/3 and the level of p-NMDAR increased significantly, but the level of p-AMPAR reduced observably. Intrahippocampal injections of MK-801 effectively improved the depression-like behaviors induced by CUMS and Hcy, and attenuated the elevation of Glu content induced by Hcy in hippocampus, whereas NBQX could not improve the depression-like behaviors, but also decreased the Glu content induced by Hcy remarkably. These results suggest that CUMS may contribute to the production and release of Hcy via hippocampal astrocytes. Through the increase of expression of NR1/NR2B/GluR2/3 and level of p-NMDAR, and the decrease of level of p-AMPAR, Hcy results in elevation of Glu level, which leads to depression-like behaviors in the end. In a word, the Hcy released by astrocytes plays an important role in stress-induced elevation of Glu content and variation of NMDA/AMPA receptors in hippocampus.
刘慧, 问黎敏, 乔卉, 安书成. 高半胱氨酸对慢性应激性抑郁大鼠海马谷氨酸及其受体的调节[J]. 生理学报 2013; 65 (1): 61-71.
Liu Hui, WEN Li-Min, QIAO Hui, AN Shu-Cheng. [The modulation of hippocampal glutamate and NMDA/AMPA receptor by homocysteine in chronic unpredictable mild stress-induced rat depression.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (1): 61-71 (in Chinese with English abstract).