左归降糖解郁方调控Glu-mGluR_(2/3)-PI3K信号通路在糖尿病并发抑郁症海马NVU神经元凋亡中的保护作用  

Protective Effects of Zuogui Jiangtang Jieyu Formulation on the Neuron Apoptosis of Hippocampal NVU via Regulating Glu-mGluR-PI3K Pathway in Diabetes Mellitus with Depression

作　　者：刘检[1] 刘林[1] 韩远山[1] 唐林[1] 杨蕙[1] 赵洪庆 蔺晓源[1] 王宇红[2,3,4] Liu Jian;Liu Lin;Han Yuanshan;Tang Lin;Yang Hui;Zhao Hongqin;Lin Xiaoyuan;Wang Yuhong(The first Hospital of Hunan University of Chinese Medicine,Changsha 410007;Hunan Key Laboratory of Power and Innovative Drugs State Key Laboratory of Ministry Training Bases,Changsha 410208;Institute of innovation and applied research,Hunan University of Chinese Medicine,Changsha 410208;The domestic first class construction discipline of Chinese Medicine in Hunan University of Chinese Medicine,Changsha 410208)

机构地区：[1]湖南中医药大学第一附属医院,长沙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,长沙410208 [3]湖南中医药大学科技创新中心,长沙410208 [4]湖南中医药大学中医学国内一流建设学科,长沙410208

出　　处：《中药药理与临床》2021年第1期136-142,共7页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金项目(编号:81573965、81874464);国家重大新药创制科技重大专项(编号:2017ZX09309026);湖南省卫健委科研项目(编号:20200165);湖南中医药大学中医学一流学科开放基金(编号:2018ZYX47);湖南省研究生创新项目(编号:CX20200752)。

摘　　要：目的:探讨左归降糖解郁方调控Glu-mGluR_(2/3)-PI3K信号通路在糖尿病并发抑郁症(DD)海马神经血管单元(NVU)中神经元凋亡的保护作用及其机制。方法:原代分离、纯化和培养SD大鼠海马神经元(Ne)、星形胶质细胞(As)与脑微血管内皮细胞(Bm),并对其进行免疫细胞化学染色鉴定;采用Transwell小室及高糖联合皮质酮干预构建DD海马NVU细胞模型;实验设正常对照组、模型对照组、10%空白血清对照组、mGluR_(2/3)激动剂5μmol/L组、PI3K阻断剂2μmol/L组、左归降糖解郁方32.8 g/kg 10%含药血清组。造模及给药18 h后,采用CCK8法检测NVU中海马神经元细胞存活力,采用ELISA法检测NVU中海马神经元细胞上清液谷氨酸(Glu)含量;采用免疫荧光联合高内涵细胞成像(HCA)分别检测NVU中AS细胞内代谢型谷氨酸受体2/3(mGluR_(2/3))蛋白表达水平和海马神经元细胞内的细胞外调节蛋白激酶(ERK)、磷脂酰肌醇3-激酶(PI3K)、半胱天冬酶-9(Caspase-9)蛋白表达水平;采用Tunel染色NVU中海马神经元细胞凋亡水平。结果:与正常对照组比较,模型对照组海马神经元细胞存活率显著降低(P<0.01),细胞上清液中Glu含量显著升高(P<0.01),AS细胞内mGluR_(2/3)蛋白表达显著上调(P<0.01),Ne细胞内ERK、Casepase-9蛋白表达明显上调、PI3K蛋白表达明显下调(P<0.05或P<0.01),海马神经元细胞凋亡显著增加(P<0.01);与模型对照组比较,左归降糖解郁方32.8 g/kg 10%含药血清组海马神经元细胞存活力显著增加(P<0.05),Glu含量显著降低(P<0.05),mGluR_(2/3)、ERK、Casepase-9蛋白表达显著下调,PI3K蛋白表达明显上调(P<0.05或P<0.01),海马神经元细胞凋亡数量显著下降,且核固缩、染色质浓染等阳性特征明显减轻。结论:左归降糖解郁方可调控mGluR_(2/3)活化介导的Glu-mGluR_(2/3)-PI3K信号通路异常,这可能是其抑制糖尿病并发抑郁症海马神经元凋亡的重要机制。Objective: To investigate the Protective effects of Zuogui Jiangtang Jieyu Formulation on the neuron apoptosis of hippocampal NVU via regulating Glu-mGluR_(2/3)-PI3 K pathway in diabetes mellitus with depression, and to explore its mechanism. Methods: Hippocampal neurons, astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated, purified and cultured, and then immunocytochemistry staining was used to identify cells. The DD model of NVU was established by applying intervention of high glucose combined with cortisone after hippocampal NVU co-culture system was builded by Transwell in vitro. The cultured cells were randomly divided into the control group, the model group, 10% serum group, 5 μmol/L mGluR_(2/3) agonist group, 2 μmol/L PI3 K blocking group, and 10% drug-containing serum of ZGF 32.8 g/kg group. After modeling and administration 18 h, the cell survival rate of hippocampal neurons was detected by CCK-8 assay. The content of glutamate(Glu) in neurons of hippocampal NVU was detected by ELISA. The expression of mGluR_(2/3) in astrocytes and ERK, PI3 K, Casepase-9 in neurons of hippocampal NVU were detected by immunofluorescent staining and high content analysis(HCA). The apoptosis of hippocampal neurons was detected by TUNEL staining. Results: Compared with the control group, the cell survival rate was obviously decreased in the model group(P<0.01),the content of glutamate in neurons of hippocampal NVU was obviously increased(P<0.01),the expression of mGluR2/3 in astrocytes, and ERK, Casepase-9 in hippocampal neurons were markedly up-regulated, while PI3 K was down-regulated(P<0.05 or P<0.01), the apoptosis of hippocampal neurous was significantly increased(P<0.01). Compared with the model group, the cell survival rate was obviously increased in 10% drug-containing serum of ZGF 32.8 g/kg group(P<0.05), the Glu content was decreased(P<0.05), the protein expressions of mGluR_(2/3)、ERK and Casepase-9 were significantly down regulated, the PI3 K expression was up regulated(

关 键 词：左归降糖解郁方 糖尿病并发抑郁症 神经血管单元 谷氨酸/代谢型谷氨酸受体2/3/磷脂酰肌醇3-激酶信号通路 凋亡 

分 类 号：R285.5[医药卫生—中药学]