摘要
目的观察温阳解郁颗粒(Wenyang Jieyu granule,WYJY)对皮质酮(Corticosterone,CORT)诱导损伤型小鼠海马神经细胞(TH22 cell)的保护作用,基于脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)/酪氨酸激酶B(Tyrosine kinase B,TrkB)/细胞外信号调节蛋白激酶(Extra cellular regulated protein kinases,ERK)信号通路探讨WYJY保护海马神经细胞的作用机制。方法体外构建小鼠海马神经细胞皮质酮诱导损伤模型,以不同浓度的WYJY和氟西汀(Fluoxetine,FXT)含药血清作用于模型细胞,细胞增殖-毒性检测(Cell Counting Kit-8,CCK-8)法分析细胞活性,倒置显微镜下观察给药前后细胞形态结构的改变,采用蛋白免疫印迹法(Western Blot)、实时荧光定量PCR(Quantitative real-time PCR,qPCR)法检测神经细胞内凋亡因子(BCL2-Associated X,Bax)、抗凋亡因子(B-cell lymphoma-2,Bcl-2)、BDNF、Trkb、ERK以及丝氨酸/苏氨酸激酶(Phospho-p90RSK,RSK)、环磷腺苷效应元件结合蛋白(cAMP-response element binding protein,CREB)蛋白表达水平以及相关基因的表达水平。结果在浓度为459.5μmol·L^(-1)的CORT作用24 h后,HT22细胞的活性抑制率达到50%,在此条件作用下细胞形态结构损伤明显,凋亡程度严重,细胞上清中BDNF的含量显著减少(P<0.05),细胞内凋亡相关因子Bcl-2/Bax的比值明显降低(P<0.01),BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平明显降低(P<0.01);以5%的浓度为2.85 g·kg-1的WYJY和10%的FXT含药血清作用于受损的HT22细胞后,HT22细胞存活率明显提升(P<0.01),细胞结构的损伤明显改善,细胞凋亡程度减轻,细胞外BDNF的含量显著升高(P<0.05),细胞内Bcl-2/Bax比值显著上调(P<0.01),BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平显著提升(P<0.05,P<0.01)。结论温阳解郁颗粒可有效保护高浓度CORT造成的小鼠海马神经细胞损伤。调控BDNF/Trkb/ERK通路,放大CREB信号传导,影响Bcl-2、BDNF水平,�
Objective To observe the protective effect of Wenyang Jieyu granule(WYJY)on neurons in hippocampus in corticosterone(CORT)damaged mice,and to explore its mechanism based on the brain-derived neurotrophic factor(BDNF)/tyrosine kinase B(TrkB)/extracellular signaling regulatory protein kinase(ERK)signaling pathway.Methods To detect cell activity,hippocampus neuron cell(HT22)corticosterone injury model was constructed in vitro,different concentrations of containing Wenyang Jieyu granule serum and containing fluoxetine(FXT)serum were acted on the corticosterone damaged HT22 cells,cell activity was analyzed by the cell proliferation-toxicity detection(CCK-8)method.The cell morphological structure was observed under inverted microscope.The BDNF level of supernatant of each group were detected by enzyme-linked reaction,the expression levels of apoptotic protein(Bax),antiapoptotic factor(Bcl-2),BDNF,TrkB,ERKand Serine/Threonine Kinase(RSK),cyclophosphate adenosine effect element binding protein(CREB)were detectd by method of western blot and their mRNA expression level were detectd by method of qPCR.Results After CORT action at 24 h of 459.5μmol·L^(-1),the activity inhibition rate of HT22 cells reached 50%,under this condition,cell morphological and structural damage was obvious and apoptosis was severe.The content of BDNF in the cell episerum was significantly reduced(P<0.05),the ratio of the intracellular apoptosis-related factor Bcl-2/Bax was significantly lowered(P<0.01),the BDNF,Trkb,ERK,RSK,CREB phosphorylated protein expression levels and the mRNA expression levels were significantly reduced(P<0.01).After WYJY at 5%concentration of 2.85g·kg-1 and 10%FXT-containing serum acted on the damaged HT22 cells,HT22 cell survival increased significantly(P<0.01),cell structural damage has improved significantly and apoptosis has decreased,the extracellular BDNF content was increased significantly(P<0.05),the intracellular Bcl-2/Bax ratio was significantly increased(P<0.01),BDNF,Trkb,ERK,RSK,CREB phosphorylation protein exp
作者
马小娟
孟霜
冯振宇
史敏
王鑫鑫
周晓荣
赵杰
Ma Xiaojuan;Meng Shuang;Feng Zhenyu;Shi Min;Wang Xinxin;Zhou Xiaorong;Zhao Jie(Shanxi Provincial Integrated TCM And WM Hospital Central Lab,Taiyuan 030013,China;Shanxi University of TCM,Jinzhong 030600,China;Shanxi Provincial Integrated TCM And WM Hospital Famous Chinese Medicine Studio,Taiyuan 030013,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2022年第9期3504-3515,共12页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
山西省科学技术厅重点实验室项目(202104010910011):经方扶阳山西省重点实验室,负责人:赵杰
山西省科学技术厅应用基础研究计划面上青年基金项目(201901D211530):温阳解郁汤通过调控CG-GR-SGK1信号通路对肾阳虚型抑郁症大鼠海马神经可塑性的影响与机制研究,负责人:马小娟
山西省卫生健康委员会科研项目(2019085):温阳解郁汤对肾阳虚型抑郁症大鼠海马神经可塑性的影响机制研究,负责人:马小娟
