摘要
目的:探究黑逍遥散通过调控环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/N-甲基-D-天冬氨酸受体(NMDAR)信号通路干预APP/PS1小鼠突触可塑性的作用及其机制。方法:选取4月龄雄性C57BL/6J小鼠12只作空白组,同月龄雄性APP/PS1双转基因小鼠60只,随机分为模型组、KW-6002(腺苷受体的拮抗剂)组(3 mg·kg^(-1))及黑逍遥散高、中、低剂量组(22.10、11.05、5.53 g·kg^(-1)),每组12只,对应药物连续干预90 d。透射电镜观察海马神经元突触超微结构,高尔基染色观察海马CA1区神经元树突棘密度,蛋白免疫印迹法(Western blot)检测cAMP、PKA、N-甲基-D-天冬氨酸受体1(NR1)、N-甲基-D-天冬氨酸受体2A(NR2A)、N-甲基-D-天冬氨酸受体2B(NR2B)、突触后致密物质95(PSD95)、突触蛋白1(SYN1)的蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测cAMP、PKA、NR1的mRNA表达,酶联免疫吸附测定法(ELISA)检测小鼠海马白细胞介素-12(IL-12)、白细胞介素-4(IL-4)含量。结果:与空白组比较,模型组小鼠海马CA1区突触前膜与突触后膜分界线清晰程度欠佳,突触囊泡明显减少且分布散乱,突触后膜密度降低,海马CA1区神经元树突棘形态欠规整,排列不连续、密度减少(P<0.01),海马cAMP、PKA、NR1、NR2A、NR2B、PSD95、SYN1的蛋白表达降低(P<0.01),cAMP、PKA、NR1的mRNA表达下调(P<0.01),海马IL-12含量增多、IL-4含量减少(P<0.01);与模型组比较,各用药组小鼠海马CA1区突触前膜与后膜分界线清晰明了且结构完整,突触囊泡增多,排列密集,突触后膜密度增加,神经元树突棘形态较为规则,排列连续,成熟型树突棘密度增多(P<0.05,P<0.01),海马cAMP、PKA、NR1、NR2A、NR2B、PSD95、SYN1的蛋白表达增加(P<0.05,P<0.01),cAMP、PKA、NR1的mRNA表达提高(P<0.01),海马IL-12含量降低(P<0.01),IL-4含量增加(P<0.01)。结论:黑逍遥散能改善APP/PS1小鼠海马神经元结构形态,其作用机制可能与激活cAMP/PKA/NMDAR信号通路、修复突触可塑性有关。
Objective:To explore the effect and mechanism of Hei Xiaoyaosan in modulating the synaptic plasticity in APP/PS1 mice by regulating the cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/N-methyl-D-aspartate receptor(NMDAR)signaling pathway.Method:Twelve 4-month-old male C57BL/6J mice were selected as the blank control group,and 604-month-old male APP/PS1 double transgenic mice were randomized into model,KW-6002(adenosine receptor antagonist,3 mg·kg^(-1)),and high-,medium-,and low-dose(22.10,11.05,5.53 g·kg^(-1),respectively)Hei Xiaoyaosan groups,with 12 mice in each group.Mice were administrated with corresponding drugs for 90 days.Transmission electron microscopy was employed to observe the synaptic ultrastructure of hippocampal neurons,and Golgi staining was used to observe the dendritic spine density of neurons in hippocampal CA1 region.Western blot was employed to measure the protein levels of cAMP,PKA,N-methyl-D-aspartate receptors 1,2A,and 2B(NR1,NR2A,and NR2B,respectively),postsynaptic density protein 95(PSD95),and synapsin 1(SYN1).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was performed to determine the mRNA levels of cAMP,PKA,and NR1.Enzyme-linked immunosorbent assay was employed to determine the content of interleukin-12(IL-12)and interleukin-4(IL-4)in the hippocampus.Result:Compared with the blank group,the model group showed blurred boundaries between presynaptic membrane and postsynaptic membrane in hippocampal CA1 region,reduced and scattered synaptic vesicles,and decreased density of postsynaptic membrane,and irregular,disarranged,and loosened dendritic spines of neurons in hippocampal CA1 region(P<0.01).In addition,the model group presented down-regulated protein levels of cAMP,PKA,NR1,NR2A,NR2B,PSD95,and SYN1 and mRNA levels of cAMP,PKA,and NR1,elevated IL-12 level,and lowered IL-4 level in the hippocampus(P<0.01).Compared with the model group,the drug intervention groups showed clear and intact boundaries between presynaptic membrane and postsynaptic membrane in hippocampal CA1 region,increased synaptic vesicles with dense arrangement,increased density of postsynaptic membrane,and improved morphology,arrangement,and density of neuronal dendritic spines(P<0.05,P<0.01).In addition,the drug interventions up-regulated the protein levels of cAMP,PKA,NR1,NR2A,NR2B,PSD95,and SYN1(P<0.05,P<0.01)and mRNA levels of cAMP,PKA,and NR1(P<0.01),lowered the IL-12 level(P<0.01),and elevated the IL-4 level(P<0.01)in the hippocampus.Conclusion:Hei Xiaoyaosan can improve the structure and morphology of hippocampal neurons in APP/PS1 mice by activating the cAMP/PKA/NMDAR signaling pathway and repairing synaptic plasticity.
作者
胡韵韵
吕育洁
孟志鹏
杨娇
陈怡琴
王虎平
HU Yunyun;LYU Yujie;MENG Zhipeng;YANG Jiao;CHEN Yiqin;WANG Huping(Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Key Laboratory of Traditional Chinese Medicine(TCM)Excavation and Innovative Transformation,Lanzhou 730000,China;Gansu Engineering Laboratory for New Products of TCM,Lanzhou 730000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第17期27-34,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81960828,82160862)
甘肃省自然科学基金项目(22JR11RA113)
第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕239号)
首批陇原青年英才项目(中共甘肃省委人才工作领导小组〔2022〕5号)。
