主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

北京中医药大学学报 ›› 2018, Vol. 41 ›› Issue (9): 752-758.doi: 10.3969/j.issn.1006-2157.2018.09.009

• 科技之窗 • 上一篇    下一篇

左归降糖解郁方对糖尿病并发抑郁症海马NVU神经元的调控作用及其机制*

刘检1, 王宇红2#, 凌佳2, 刘林1, 孟盼2, 杨蕙1, 赵洪庆2   

  1. 1 湖南中医药大学第一附属医院 湖南 410007;
    2 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
  • 收稿日期:2018-04-04 出版日期:2018-09-30 发布日期:2018-09-30
  • 通讯作者: 王宇红,女,博士,教授,博士生导师,主要研究方向:中药神经药理,E-mail: wyh107@126.com
  • 作者简介:刘检,男,硕士,助理研究员
  • 基金资助:
    国家自然科学基金项目(No. 81573965、81603604), 湖南省自然科学基金项目(No. 2017JJ3241), 湖南省教育厅科学研究项目(No. 17C1229)

Regulating effect of Zuogui Jiangtang Jieyu Fang on hippocampal NVU: a study based on diabetes complicated by depression*

Liu Jian1, Wang Yuhong2#, Lin Jia2, Liu Lin1, Meng Pan2, Yang Hui1, Zhao Hongqing2   

  1. 1 First Affiliated Hospital of Hunan University of Chinese Medicine, Hunan 410007, China;
    2 State Key Laboratory Base of Powdered and Innovative Chinese Medicinal of Hunan Province and Ministry of Education, Hunan 410208, China
  • Received:2018-04-04 Online:2018-09-30 Published:2018-09-30
  • Supported by:
    National Natural Science Foundation of China (No. 81573965、81603604), Natural Science Foundation of Hunan Province (No. 2017JJ3241), Education Department Scientific Research Program of Hunan Province

摘要: 目的 探讨糖尿病并发抑郁症(DD)状态下谷氨酸(Glu)失衡对海马神经血管单元(neurovascular unit,NVU)中神经元GluR2、Beclin-1蛋白表达的影响,及左归降糖解郁方对其干预作用。方法 原代分离、纯化和培养SD大鼠海马神经元、星形胶质细胞及脑微血管内皮细胞,构建海马NVU体外共培养体系,采用免疫细胞化学染色法进行细胞鉴定;采用高糖(150 mmol/L)联合皮质酮(200 μmol/L)干预构建海马NVU细胞模型;将培养的细胞随机分组,未造模细胞分为正常组、空白血清对照组,造模细胞分为模型组、左归降糖解郁方(32.82 g/kg)含药血清组和阳性药(氟西汀0.18 g/kg+二甲双胍1.8 mg/kg)含药血清组,除正常组与模型组给予等量培养液,其余组给予相应体积分数10%的含药血清或空白血清;采用酶联免疫吸附法检测各组海马NVU神经元培养液上清中Glu含量;采用免疫荧光染色与高内涵细胞成像分析技术(HCA)检测各组海马神经元内GluR2、Beclin-1蛋白表达情况;采用Tunel染色检测各组海马神经元凋亡情况。结果 与正常组及空白血清对照组比较,模型组海马NVU培养液上清中Glu含量显著升高(P<0.01),GluR2表达明显减少(P<0.01),Beclin-1表达明显增加(P<0.01),细胞凋亡明显增多;与模型组比较,阳性药含药血清组与左归降糖解郁方含药血清组Glu含量显著降低(P<0.01),GluR2表达上调(P<0.01),Beclin-1表达下调(P<0.01或P<0.05),细胞凋亡显著减少。结论 左归降糖解郁方对海马NVU细胞模型中神经元GluR2、Beclin-1蛋白表达具有明显的调控作用,该作用可能与改善谷氨酸失衡有关,这可能是其抑制细胞自噬继而抗DD海马神经元凋亡的重要机制。

关键词: 糖尿病并发抑郁症, 神经血管单元, 谷氨酸, GluR2, Beclin-1, 自噬, 左归降糖解郁方, 大鼠

Abstract: Objective To discuss the influence of glutamic acid (Glu) imbalance on protein expressions of GluR2 and Beclin-1 in hippocampal neurovascular unit (NVU) in status of diabetes complicated by depression (DD), and interventional effect of Zuogui Jiangtang Jieyu Fang (Zuogui Blood Sugar-decreasing Depression-relieving Decoction, ZJJF). Methods The hippocampal neurons, astrocytes (Ast) and brain microvascular endothelial cells were primitively isolated, purified and cultured in SD rats to establish in vitro co-cultural system of hippocampal NVU. The immunocytochemistic staining method was used to identify cells, and high glucose (150 mmol/L) combined with corticosterone (CORT, 200 μmol/L) were used to intervene establishment of hippocampal NVU model. The non-modeled cells were randomly divided into normal group and blank group, and modeled cells were divided into model group, group of medicated serum of Zuogui Jiangtang Jieyu Fang (ZJJF group) and group of medicated serum of positive drugs [positive drug group, fluoxetine (0.18 g/kg)+metform (1.8 mg/kg)]. The normal group and model group were given the same amount of nutrient solution, and other groups were given medicated serum or blank serum in corresponding volume fraction of 10%. Glu content in supernatant of hippocampal NVU neurons was detected by using enzyme-linked immunosorbent assay (ELISA) in all groups. The protein expressions of GluR2 and Beclin-1 in hippocampal neurons were detected by using immunofluorescent staining and high content analysis (HCA) in all groups. The apoptosis of hippocampal neurons was detected by using Tunel staining in all groups. Results Compared with normal group and blank group, Glu content in supernatant of hippocampal NVU neurons increased significantly (P<0.01), GluR2 expression decreased significantly (P<0.01), Beclin-1 expression increased significantly (P<0.01), and apoptosis rate increased significantly in model group. Compared with model group, Glu content decreased significantly (P<0.01), GluR2 expression was up-regulated (P<0.01), Beclin-1 expression was down-regulated (P<0.01 or P<0.05), and apoptosis rate decreased significantly in positive drug group and ZJJF group. Conclusion ZJJF has significant regulation effect on the protein expressions of neuron GluR2 and Beclin-1 in hippocampal NVU model, which may be related to the relief of Glu imbalance. It may be an important mechanism of ZJJF inhibiting autophagy and antagonizing hippocampal neuron apoptosis of DD.

Key words: diabetes complicated by depression, neurovascular unit, glutamic acid, GluR2, Beclin-1, autophagy, Zuogui Jiangtang Jieyu Fang (Zuogui Blood Sugar-decreasing Depression-relieving Decoction), rats

中图分类号: 

  • R285.5