左归降糖解郁方对自噬介导的糖尿病并发抑郁症大鼠海马神经血管单元中神经元损伤的保护作用及其机制
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摘要
目的探讨左归降糖解郁方(ZGF)对自噬介导的糖尿病并发抑郁症(diabetes mellitus with depression,DD)大鼠海马神经血管单元(neurovascularunit,NVU)中神经元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、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,并进行免疫细胞化学染色鉴定。采用高糖(150mmol/L)联合皮质酮(200μmol/L)干预制备海马NVU细胞模型,实验设对照组、模型组、空白血清对照组和ZGF含药血清组。采用Furo-3/AM染色法检测海马NVU神经元细胞内钙水平,高内涵细胞成像分析技术(high content analysis,HCA)检测海马NVU神经元细胞内自噬标志物自噬相关蛋白1(Beclin-1)、人微管相关蛋白轻链3II(LC3-II)及谷氨酸受体2(Glu R2)/Ca2+/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路关键蛋白的表达,TUNEL染色法观察各组海马神经元细胞凋亡。结果与对照组比较,模型组及空白血清对照组海马NVU神经元细胞内钙水平显著升高,自噬标志物Beclin-1、LC3-II蛋白表达水平显著升高,Glu R2、m TOR蛋白表达水平显著降低,AMP依赖的蛋白激酶(AMPK)蛋白表达水平显著升高,且细胞凋亡明显增多;与模型组比较,ZGF含药血清组海马NVU神经元细胞内钙水平明显降低,自噬标志物Beclin-1、LC3-II蛋白表达水平降低,GluR2/Ca2+/mTOR信号通路中Glu R2、m TOR蛋白表达水平升高,AMPK蛋白表达水平降低,同时细胞凋亡显著减少。结论 ZGF对自噬介导的DD大鼠海马NVU神经元细胞损伤具有明显的保护作用,其作用机制与调控GluR2/Ca2+/mTOR信号通路及细胞凋亡有关。
Objective To investigate the protective effects and mechanism of Zuogui Jiangtang Jieyu Formulation(ZGF) on hippocampal neuron of neurovascular unit(NVU) induced by autophagy in diabetes mellitus with depression(DD). Methods Hippocampal neurons,astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated, purified and cultured, and then immunocytochemistry staining was employed to identify primitive cells. The DD model of NVU was established by applying intervention of high glucose(150 mmol/L) and cortisone(200 μmol/L) after co-culture system was built in vitro. The cultured cells were randomly divided into normal group, blank serum group, model group, and medicated serum of ZGF group. Intracellular calcium levels(Ca2+) on hippocampal neuron of NVU was detected by Furo-3/AM staining. The expression of autophagy marker Beclin-1,LC3-II, and GluR2/Ca2+/m TOR signaling pathway key proteins was detected by high content analysis. The apoptosis of hippocampal neurons was observed by Tunel staining. Results Compared with the normal group, Ca2+ levels was remarkably increased, expression of autophagy marker Beclin-1 and LC3-II was dramatically up-regulated, proteins expression of GluR2 and mTOR was obviously down-regulated while AMPK up-regulated, and the apoptosis in hippocampal neuron of NVU was significantly increased in model and blank serum group. Compared with the model group, intracellular calcium levels was obviously decreased, expression of autophagy marker Beclin-1 and LC3-II was down-regulated, GluR2/Ca2+/m TOR signaling pathway proteins expression of GluR2, mTOR was up-regulated while AMPK down-regulated, furthermore the apoptosis of hippocampal neurons was significantly decreased in ZGF group. Conclusion ZGF has significant protective effects on hippocampal neuron of NVU induced by autophagy in DD, and its mechanism is related to the GluR2/Ca2+/mTOR signaling pathway and apoptosis.
Objective To investigate the protective effects and mechanism of Zuogui Jiangtang Jieyu Formulation(ZGF) on hippocampal neuron of neurovascular unit(NVU) induced by autophagy in diabetes mellitus with depression(DD). Methods Hippocampal neurons,astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated, purified and cultured, and then immunocytochemistry staining was employed to identify primitive cells. The DD model of NVU was established by applying intervention of high glucose(150 mmol/L) and cortisone(200 μmol/L) after co-culture system was built in vitro. The cultured cells were randomly divided into normal group, blank serum group, model group, and medicated serum of ZGF group. Intracellular calcium levels(Ca2+) on hippocampal neuron of NVU was detected by Furo-3/AM staining. The expression of autophagy marker Beclin-1,LC3-II, and GluR2/Ca2+/m TOR signaling pathway key proteins was detected by high content analysis. The apoptosis of hippocampal neurons was observed by Tunel staining. Results Compared with the normal group, Ca2+ levels was remarkably increased, expression of autophagy marker Beclin-1 and LC3-II was dramatically up-regulated, proteins expression of GluR2 and mTOR was obviously down-regulated while AMPK up-regulated, and the apoptosis in hippocampal neuron of NVU was significantly increased in model and blank serum group. Compared with the model group, intracellular calcium levels was obviously decreased, expression of autophagy marker Beclin-1 and LC3-II was down-regulated, GluR2/Ca2+/m TOR signaling pathway proteins expression of GluR2, mTOR was up-regulated while AMPK down-regulated, furthermore the apoptosis of hippocampal neurons was significantly decreased in ZGF group. Conclusion ZGF has significant protective effects on hippocampal neuron of NVU induced by autophagy in DD, and its mechanism is related to the GluR2/Ca2+/mTOR signaling pathway and apoptosis.
引文
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[2]Islam S M,Ferrari U,Seissler J,et al.Association between depression and diabetes amongst adults in Bangladesh:A hospital based case-control study[J].JGlob Health,2015,doi:10.7189/jogh.05.020.
[3]刘检,王宇红,凌佳,等.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[J].北京中医药大学学报,2018,41(9):752-758.
[4]Man H Y.Glu R2-lacking,calcium-permeable AMPAreceptors-inducers of plasticity?[J].Curr Opin Neurobiol,2011,21(2):291-298.
[5]凌佳,杨琴,刘检,等.左归降糖解郁方对模拟糖尿病并发抑郁症环境下模型大鼠海马神经元凋亡相关蛋白的影响[J].中国中医药信息杂志,2017,24(10):35-39.
[6]赵洪庆,杜青,凌佳,等.左归降糖解郁方对糖尿病并发抑郁症大鼠海马神经元JNK/Elk-1/c-fos通路的调控作用[J].中国临床药理学与治疗学,2016,21(6):625-629.
[7]刘检,王宇红,徐雅岚,等.改良的胎鼠海马神经元原代培养及模拟糖尿病并发抑郁环境对其的损伤作用[J].神经解剖学杂志,2016,32(4):459-465.
[8]刘检,王宇红,徐雅岚,等.大鼠脑皮质星形胶质细胞的原代培养、纯化及采用HCA技术进行GFAP鉴定[J].沈阳药科大学学报,2016,33(7):581-587.
[9]Han H S,Suk K.The function and integrity of the neurovascular unit rests upon the integration of the vascular and inflammatory cell systems[J].Curr Neurovasc Res,2005,2(5):409-423.
[10]王宇红,谭小雯,柴上,等.左归降糖解郁方对糖尿病并发抑郁症大鼠海马谷氨酸及N-甲基-D-天冬氨酸受体2A、2B的影响[J].中国中医药信息杂志,2015,22(10):70-73.
[11]徐雅岚,赵洪庆,杜青,等.左归降糖解郁方对糖尿病并发抑郁症大鼠海马区星形胶质细胞谷氨酸转运体的影响[J].北京中医药大学学报,2016,39(6):470-475.
[12]Guma M,Firestein G S.c-Jun N-terminal kinase in inflammation and rheumatic diseases[J].Open Rheumatol J,2012,doi:10.2174/1874312901206010220.
[13]Su S H,Wu Y F,Lin Q,et al.Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 suppress chronic cerebral hypoperfusion-induced neuronal apoptosis by inhibiting c-Jun N-terminal kinase signaling[J].Neuroscience,2015,doi:10.1007/s00210-017-1417-9.
[14]Zhu J,Chen Y,Ji Y,et al.Gemcitabine induces apoptosis and autophagy via the AMPK/mTOR signaling pathway in pancreatic cancer cells[J].Biotechnol Appl Biochem,2018,65(5):665-671.
[15]佳娜提·达吾列提,姜楠,寇俊萍,等.调节细胞自噬的中药有效成分研究进展[J].中草药,2014,45(16):2283-2292.
[16]Maria H H,Marja J.AMP-activated protein kinase:Auniversal regulator of autophagy?[J].Autophagy,2007,3(4):381-383.
[17]Meng X F,Yu J T,Song J H,et al.Role of the m TORsignaling pathway in epilepsy[J].J Neurol Sci,2013,doi:10.1016/j.jns.2013.05.029.
[18]唐晓娟.Ca-A/K通道在缺氧缺血性脑损伤新生大鼠海马神经元自噬发生中的作用[D].苏州:苏州大学,2014.
[19]Bootman M D,Chehab T,Bultynck G,et al.The regulation of autophagy by calcium signals:Do we have a consensus?[J].Cell Calcium,2017,doi:10.1016/j.ceca.2017.08.005.
[20]蔺晓源,韩远山,孟盼,等.糖尿病并发抑郁症“虚、瘀、郁”的中医病机探讨[J].时珍国医国药,2016,27(8):1942-1943.