摘要
目的:探讨温阳解郁方调节“母婴分离+束缚应激(MS+RS)”抑郁小鼠海马神经元凋亡改善突触可塑性的机制。方法:仔鼠出生第0天(PD0)随机分为空白组10只和造模组50只。造模采用MS+RS建立抑郁模型,PD21离乳后随机分为模型组、温阳组、解郁组、温阳解郁组、氟西汀组,每组10只,PD21~PD111分组给药。糖水偏好、开放旷场、O迷宫及新物体识别行为实验评估小鼠焦虑抑郁和学习记忆能力;免疫组化法(IHC)检测小鼠海马突触后致密物95(PSD95)表达情况;原位末端标记法(TUNEL)染色检测小鼠海马神经元凋亡情况;蛋白免疫印迹法(Western blot)检测海马脑源性神经营养因子(BDNF)、磷酸化酪氨酸蛋白激酶/酪氨酸蛋白激酶(p-TrkB/TrkB)、磷酸化蛋白激酶B/蛋白激酶B(p-Akt/Akt)、磷酸化哺乳动物雷帕霉素靶蛋白/哺乳动物雷帕霉素靶蛋白(p-mTOR/mTOR)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶-3(Caspase-3)、突触后致密蛋白95(PSD95)、突触素(Syn)蛋白表达情况。结果:与空白组比较,模型组小鼠糖水偏好程度、中央区停留时间、运动总距离、开臂停留时间和认知指数显著减少(P<0.01),IHC结果显示海马PSD95表达明显减少,海马神经元凋亡数量增加(P<0.01),BDNF、p-TrkB/TrkB、p-Akt/Akt、p-mTOR/mTOR、Bcl-2、p-mTOR/mTOR、PSD95、Syn蛋白表达显著减少(P<0.01),Bax、Caspase-3蛋白含量明显增加(P<0.05);与模型组比较,温阳解郁组和氟西汀组糖水偏好程度、5 min中央运动时间、5 min开臂停留时间和认知指数明显增加(P<0.05,P<0.01),海马PSD95表达明显增加、海马凋亡指数显著增加(P<0.01),BDNF、p-TrkB/TrkB、p-Akt/Akt、p-mTOR/mTOR、Bcl-2、PSD95、Syn蛋白含量显著增加(P<0.01),Bax、Caspase-3蛋白含量显著减少(P<0.01)。结论:温阳解郁方可以通过BDNF/Akt/mTOR信号通路,改善模型组小鼠神经元凋亡情况,保护海马突触结构和功能,缓解模型组小鼠抑郁样行为,其综合疗效及分子机制效果优于温阳方和解郁方。
Objective:To explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress.Method:The mice on postnatal day 0(PD0)were randomly assigned into a control group(n=10)and a modeling group(n=50).Maternal separation combined with restraint stress was adopted to establish the mouse model of depression,and the modeled mice were randomized into model,Wenyang prescription,Jieyu prescription,Wenyang Jieyu prescription,and fluoxetine groups(n=10)on the weaning day(PD21).From PD21 to PD111,the mice were fed with the diets mixed with corresponding medicines.The sucrose preference test,open field test,O-maze test,and novel object recognition test were then conducted to evaluate the depression,memory,and learning abilities of mice.Immunohistochemistry(IHC)was employed to measure the atomic absorbance(AA)of postsynaptic density protein 95(PSD95)in the hippocampus.Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL)was employed to detect the apoptosis of hippocampal neurons.Western blot was employed to determine the protein levels of brainderived neurotrophic factor(BDNF),phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B(p-TrkB/TrkB),phosphorylated protein kinase B/protein kinase B(p-Akt/Akt),phosphorylated mammalian target of rapamycin/mammalian target of rapamycin(p-mTOR/mTOR),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X(Bax),cysteinyl aspartate-specific proteinase-3(Caspase-3),synaptophysin(Syn),and PSD95.Result:Compared with the control group,the modeling decreased the sucrose preference rate,time spent in central zone within 5 min,total movement distance,time spent in the open arm,and cognition index(P<0.01).Furthermore,it decreased the expression of PSD95,increased the neuron apoptosis in the hippocampus(P<0.01),down-regulated the protein levels of BDNF,p-TrkB/TrkB,p-Akt/Akt,p-mTOR/mTOR,Bcl-2,PSD95,and Syn(P<0.01),and up-regulated the protein levels of Bax and Caspase-3(P<0.05)in the hippocampus.Compared with the model group,Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate,time spent in central zone within 5 min,total movement distance,time spent in the open arm,and cognition index(P<0.05,P<0.01).Moreover,the drugs increased the expression of PSD95,reduced the neuron apoptosis(P<0.01),up-regulated the protein levels of BDNF,p-TrkB/TrkB,p-Akt/Akt,p-mTOR/mTOR,Bcl-2,PSD95,and Syn(P<0.01),and down-regulated the protein levels of Bax and Caspase-3(P<0.01).Conclusion:Wenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice.It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.
作者
孟丹华
佘楷杰
孟晓莹
巩子汉
梁文青
王英
梁媛
岳广欣
MENG Danhua;SHE Kaijie;MENG Xiaoying;GONG Zihan;LIANG Wenqing;WANG Ying;LIANG Yuan;YUE Guangxin(Institute of Basic Theroy for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;School of Basic Medicine,Henan University of Chinese Medicine,Zhengzhou 450000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第6期48-57,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金面上项目(82174251,81573846)
中国中医科学院科技创新工程项目(CI2021A00607)
中央级公益性科研院所基本科研业务费专项(YZ-202107,YZ-202153,YZ-202216)。
