新型镇静催眠化合物YZG-331对谷氨酸及其受体的调节及机制研究

Regulatory effect and mechanism of novel sedative hypnotic compound YZG-331 on glutamate and its receptor

本研究主要探究了新型腺苷类衍生物YZG-331对小鼠额叶皮层谷氨酸(glutamate, Glu)含量及对其受体N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor, NMDAR)的影响。动物实验均获得中国医学科学院药物研究所伦理委员会批准。采用脑微透析取样技术和高效液相色谱(high performance liquid chromatography, HPLC)检测小鼠额叶皮层组织匀浆和细胞外液中的Glu含量;Western blot和蛋白质免疫共沉淀技术检测小鼠额叶皮层中NMDAR在细胞膜上和内体中的表达量,以及引起NMDAR内吞相关蛋白的表达量及相互作用。研究结果显示,在灌胃给予YZG-331 (40 mg·kg^-1)后小鼠额叶皮层组织匀浆中Glu含量与溶剂对照组相比无统计学差异,在0~0.5 h和0.5~1 h两个时间段的小鼠额叶皮层透析液中Glu含量也无明显变化。给予YZG-331 15 min后小鼠额叶皮层细胞膜上NMDAR亚基NR1与NR2B的表达量均下调,额叶皮层细胞内体的NR1和NR2B蛋白表达量显著增加,额叶皮层组织中NMDAR亚基NR2B磷酸化水平显著上升;同时用NR2B作为诱饵蛋白的免疫共沉淀结果显示小鼠额叶皮层组织的NR2B和突触后致密蛋白95 (postsynaptic density-95, PSD95)免疫沉淀复合物中PSD95表达量显著降低。以上研究结果表明,化合物YZG-331对额叶皮层组织和细胞外液中Glu含量均无影响,但通过上调额叶皮层组织NR2B的磷酸化水平,减弱了NR2B与突触后致密蛋白PSD95的相互作用,降低了NMDAR的膜稳定性,促进了NMDAR的内吞,导致兴奋信号传导减少。这可能是其发挥其镇静催眠作用的机制之一。

This study investigated the effect of a novel adenosine derivative YZG-331 on the glutamate(Glu)content and its receptor N-methyl-D-aspartate receptor(NMDAR) in mouse frontal cortex. All procedures in this research were approved by the Institutional Animal Care and Use Committee of the Institute of Materia Medica,Chinese Academy of Medical Sciences. High performance liquid chromatography(HPLC) was used to detect the Glu contents in the mouse frontal cortex tissue homogenate and extracellular fluid which were collected by brain microdialysis method. Western blot and co-immunoprecipitation methods were used to detect the expressions of NMDAR in cell membranes and endosomes, as well as the expression levels of endocytosis-related proteins and their interaction. The results showed that there was no significant change in Glu content in the dialysates from mouse frontal cortex within 0-0.5 h period and 0.5-1 h period after intragastric administration of YZG-331(40 mg·kg^-1). Compare to the control group, the Glu content in mouse frontal cortex homogenates has no significant statistical differences after 15 minutes of administration of compound YZG-331. YZG-331 significantly decreased the expressions of NMDAR subunits NR1 and NR2 B in the mouse frontal cortex cell membrane, meanwhile significantly increased the expressions of NR1 and NR2 B proteins in the frontal cortex endosomes. It also increased the phosphorylation levels of NMDAR subunit NR2 B in the frontal cortex. In addition, the result of co-immunoprecipitation which used NR2 B as bait protein showed that the expression of postsynaptic density-95(PSD95) in NR2 B and PSD95 immunoprecipitation complexes in mouse frontal cortex tissues was significantly reduced. These results indicate that YZG-331 does not affect the Glu content in mouse frontal cortex, but it weakens the interaction between NR2 B and PSD95 by increasing the phosphorylation level of NR2 B in the mouse frontal cortex. Therefore, it reduces the membrane stability of NMDAR and promotes NMDAR’s endocytosis, which leading to the decrease of excitotary transmission. It may be one of the mechanisms of YZG-331 to exert sedative and hypnotic effects.