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向韵,吴梦瑶,赵洪庆,刘检,雷昌,孟盼,张秀丽,凌佳,王宇红.左归降糖解郁方对糖尿病并发抑郁症胎鼠海马神经元NR及mEPSC的影响[J].湖南中医药大学学报,2020,40(3):274-279[点击复制] |
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| 左归降糖解郁方对糖尿病并发抑郁症胎鼠海马神经元NR及mEPSC的影响 |
| 向韵,吴梦瑶,赵洪庆,刘检,雷昌,孟盼,张秀丽,凌佳,王宇红 |
| (湖南中医药大学科技创新中心, 湖南 长沙 410208;湖南中医药大学湖南省中药粉体与创新药物省部共建国家重点实验室培育基地, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 探讨左归降糖解郁方对模拟糖尿病并发抑郁（diabetes mellitus with depression，DD）症状态下胎鼠海马神经元N-甲基-D-天冬氨酸受体（n-methyl-d-aspartic acid receptor，NR）和微小兴奋性突触后电流（miniature excitatory postsynaptic current，mEPSC）频率及幅度的干预作用。方法 取胎鼠（16~18 d）的海马，进行分离纯化和培养，用神经元特异性烯醇化酶（NSE）免疫细胞化学染色法鉴定。随机把海马原代神经元分6组：空白组、模型组、空白血清组、MK-801组、二甲双胍+氟西汀组、左归降糖解郁方组。根据分组，于空白组加10%的培养液；其他5组加15 mmol/L的高糖和50μmol/L的皮质酮予以造模；造模后，空白血清组、二甲双胍+氟西汀组、左归降糖解郁方组中分别加10%的空白血清、二甲双胍+氟西汀含药血清、左归降糖解郁方含药血清，MK-801组中加10μmol/L的MK-801，同时干预18 h。蛋白NR2A、NR2B的表达运用高内涵细胞成像技术（high content analysis，HCA）检测；海马神经元细胞的mEPSC采用全细胞膜片钳技术来记录，并对比不同组间神经元细胞的mEPSC频率和电流幅度。结果 经镜下观察及NSE鉴定，所培养的细胞是海马神经元细胞，阳性率在95%以上。经HCA检测，空白组细胞的胞体明显，形态完整，突触之间形成丰富的神经网络。与空白组比，模型组和空白血清组的海马神经元细胞出现萎缩、突触断裂或神经网络消失，蛋白NR2A、NR2B的荧光值增强（P<0.01）；与空白血清组比，MK-801组、二甲双胍+氟西汀组、左归降糖解郁方组细胞突触间的连接恢复，蛋白NR2A、NR2B荧光值减弱（P<0.01或P<0.05）。膜片钳结果显示，对比空白组，模型组、空白血清组的mEPSC频率和幅度上升（P<0.01）；与空白血清组比，MK-801组、二甲双胍+氟西汀组和左归降糖解郁方组的mEPSC频率和幅度下降（P<0.01）。结论 左归降糖解郁方抗DD大鼠海马神经元细胞受损的保护作用机制，可能在于其能够调控DD状态下的海马神经元蛋白NR2A、NR2B的表达及突触可塑性功能。 |
| 关键词: 糖尿病并发抑郁症 海马神经元 突触可塑性 膜片钳 N-甲基-D-天冬氨酸受体 兴奋性突触后电流 左归降糖解郁方 |
| DOI：10.3969/j.issn.1674-070X.2020.03.006 |
| 投稿时间：2018-09-07 |
| 基金项目:国家自然科学基金面上项目（81874464）；湖南省教育厅项目（17C1210）；湖南省自然科学基金青年项目（2018JJ3388）；湖南中医药大学中医内科学重大疾病防治及成果转化教育部重点实验室开放基金资助项目（ZYNK201608，ZYNK201708）；湖南中医药大学中医学国内一流建设学科开放基金资助项目（2018ZYX49）。 |
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| Effects of Zuogui Jiangtang Jieyu Fang on NR and mEPSC in Hippocampal Neurons of Fetal Rat with Diabetes Mellitus Complicated with Depression |
| XIANG Yun,WU Mengyao,ZHAO Hongqing,LIU Jian,LEI Chang,MENG Pan,ZHANG Xiuli,LING Jia,WANG Yuhong |
| (Hunan University of Chinese Medicine, Institute of Innovation and Applied Research, Changsha, Hunan 410208, China;Hunan University of Chinese Medicine, Breeding Base of Hunan Key Laboratory of Chinese Materia Medical Powder and Innovative Drugs Established by Province and Ministry, Changsha, Hunan 410208, China;The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To investigate the intervention effects of Zuogui Jiangtang Jieyu Fang on the frequency and amplitude n-methyl-d-aspartic acid receptor (NR) and miniature excitatory postsynaptic current (mEPSC) in hippocampal neurons of fetal rat under simulated diabetes mellitus complicated with depression(DD). Methods The hippocampus of fetal rats (16-18 d) was selected, isolated, purified and cultured. The cultured cells were identified by neuron-specific enolase (NSE) immunocytochemical staining. The hippocampal primary neurons were randomly divided into 6 groups of a normal group, a model group, a blank serum group, a MK-801 group, a metformin + fluoxetine group, a Zuogui Jiangtang Jieyu Fang group. According to the grouping situation, the blank group was added 10% culture medium; other five groups were added 15 mmol/L high glucose and 50 μmol/L corticosterone to establish model; After modeling, the blank serum group, the metformin + fluoxetine group and the Zuogui Jiangtang Jieyu Fang group were added 10% volume blank serum, 10% volume metformin + fluoxetine drug content serum, 10% volume Zuogui Jiangtang Jieyu Fang drug content serum, and the MK-801 group was added 10 μmol/L MK-801, intervention for 18 h. The expression of protein NR2A and NR2B were detected by high content analysis (HCA). The mEPSC of hippocampal neuronal cells was recorded using whole cell patch clamp technique, and the mEPSC frequency and current amplitude of neuronal cells in different groups were compared. Results Microscopic observation and NSE identification, the cultured cells were hippocampal neurons, and the positive rate was above 95%. HCA analysis showed that the cell bodies of the blank group were obvious, the morphology was intact, and synapses were interwoven into a rich neural network. Compared with the blank group, hippocampal neurons in the model group and the blank serum group showed atrophy, synaptic rupture or neural network disappeared, and the fluorescence intensity of protein NR2A and NR2B increased (P<0.01); Compared with the blank serum group, the synaptic connections in the MK-801 group, the metformin + fluoxetine group and the Zuogui Jiangtang Jieyu Fang group were recovered, and the fluorescence intensity of protein NR2A and NR2B decreased (P<0.01 or P<0.05). In whole cell patch clamp test, compared with the blank group, the mEPSC frequency and current amplitude of the model group and the blank serum group increased significantly (P<0.01); Compared with the blank serum group, the mEPSC frequency and current amplitude of the MK-801 group, the metformin + fluoxetine group and Zuogui Jiangtang Jieyu Fang group decreased significantly (P<0.01). Conclusion The mechanism of Zuogui Jiangtang Jieyu Fang in protecting hippocampal neurons damage of DD rats may be its regulation of protein NR2A, NR2B and synaptic plasticity in in hippocampal neurons under DD status. |
| Key words: diabetes mellitus with depression hippocampal neurons synapse plasticity patch clamp n-methyl-d-aspartic acid receptor miniature excitatory postsynaptic current Zuogui Jiangtang Jieyu Fang |
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