| Objective:To investigate the neuroprotective effect of Eucommia ulmoides on APP/PS1 double transgenic AD model mice and the related mechanisms.To screen the phytoestrogen component of baicalein in Eucommia ulmoides,and to explore its protective effect on AD model cells and its related mechanisms.Methods:1.Clean grade 3 month old APP/PS1 mice were selected as the AD model for the experiment.60 APP/PS1 mice were randomly divided into 4 groups(n=15),which were model group,donepezil group,kangnaoshuai capsule group and eucommia group,respectively.In addition,15 clean grade 3 month old C57BL/6 mice were selected as control group.The control group and model group were given equal volume physiological saline,donepezil group,kangnaoshuai capsule group and Eucommia group were given donepezil,kangnaoshuai capsule and Eucommia ulmoides extract with dosages of 0.001g·kg-1,0.584 g·kg-1 and 1.3 g·kg-1,respectively.After 3 months of continuous gavage,the experiment of new object recognition experiment,water maze experiment and step-through test were used to determine the learning and memory ability of mice.The morphological characteristics of hippocampus cells in mice were observed by electron microscopy.Immunohistochemistry was used to detect the expression of ER beta,ER alpha,FSHR,LHR in hippocampus.The expression of Aβ,acetylcholine system index(Ach,ChAT,AchE),SP,β-EP and GnRH was accomplished by Kit.Western blot was used to detect the relative expression of related protein in the hippocampus of the mice,including ER beta,p-JNK/JNK,A beta metabolism related protein(APP,BACE1),Tau protein phosphorylation related protein(p-Tau396/Tau,CDK5),glutamate receptor protein system(NMDAR1,GluR2,CAMK Ⅱ)and apoptosis related proteins(Bax,Bcl-2,Caspase-3).2.In the experiment,PC 12 cells were selected as the research objects,and AD model cells were established by 2×10-5mol/L Aβ25-35.MTT assay was used to screen the effective concentration of baicalein in AD cells.The groups were divided into four groups:control group,model group,estradiol group and baicalein group.The effects of baicalein on the morphology of PC 12 cells were observed by inverted microscope.The apoptosis rate of cells was detected by flow cytometry.The expression level of ER beta,p-JNK/JNK,glutamate receptor system NMDAR1,GluR2 and CaMK II were detected by western blot.Results:1.In the new object recognition experiment,Eucommia ulmoides can obviously increase the new object identification index(P<0.01)of APP/PS1 mice.The results of location test in the water maze test showed that Eucommia ulmoides Oliv significantly decreased the incubation period(P<0.01)of APP/PS1 mice,and it could significantly increase the number of platform crossing times and the target quadrant residence time(P<0.01)in space exploration experiments.In the step-through test,Eucommia ulmoides can obviously increase the dark latency(P<0.01)of APP/PS1 mice.2.The results of HE staining showed that,compared with Control Group,the number of neurons in the hippocampus and hypothalamus of Model Group mice decreased,with the degeneration and necrosis of the neurons.The pyramidal cells were arranged in disorder,the morphology was irregular,the cells were narrowed,and the nuclei were fixed and the glial cells proliferated.Compared with Model Group,the pyramidal nucleus of the Eucommia group was relatively neatly arranged,the number of neurons was more,the morphology was not obvious,the distribution was uniform,the color was not deep,no obvious nuclear condensation and dissolution.3.The experimental results of electron microscope show that,compared with Control group,there was some change in Model group,for example,neuronal nuclei pyknosis,nuclear membrane thickening and distortion,decrease of cytoplasmic Nissl body,increase of lipofuscin,dilatation of endoplasmic reticulum.In the nerve felt area,the nerve fibers were disorganized,synaptic membrane was dissolved,the number of synaptic vesicles decreased slightly,and the structure of the organelle in the end of the nerve was slightly membranous.Compared with Model group,the morphology of Eucommia group,KNSC group and Donepezil group cells has a certain degree of recovery,increase of Nissl body,mitochondrial structure and membrane structure clear,neural synaptic membrane synaptic structure clear,obvious gap,increase of synaptic vesicles.4.The extract of Eucommia ulmoides can obviously increase the expression of ER beta,ER alpha,FSHR and LHR in the hippocampus and hypothalamus of APP/PS1 mice(P<0.01),and can significantly reduce the expression of A beta in the hippocampus of mice(P<0.01).The extract of Eucommia ulmoides can also obviously increase the expression of ER beta,NMDAR1,GluR2,Bcl-2,Ach content and ChAT activity(P<0.01)in the hippocampus of APP/PS1 mice(P<0.01),and reduce the expression of p-JNK/JNK,A beta,APP,BACE1,p-Tau396/Tau,CDK5,CaMK Ⅱ,Bax and Caspase-3,and reduce the activity of AchE.In the same time,the extract of Eucommia ulmoides can increase the content of GnRH,β-EP,SP in the hypothalamus of APP/PS1 mice.5.The results of flow cytometry showed that baicalein could significantly reduce the apoptosis rate of A beta PC 12 cells(P<0.01).Western blot results showed that compared with the control group,the expression of ERβ,p-JNK/JNK,NMDAR1,GluR2 decreased significantly(P<0.01)in model group,CaMKII was significantly increased(P<0.01),baicalein can significantly increase the expression of ERβ,NMDAR1 and GluR2(P<0.01),lower the expression of p-JNK/JNK,CaMKII(P<0.01)in PC 12 cell injured by Aβ.Conclusion:1.Chinese medicine Eucommia ulmoides can reduce the expression of APP and BACE1 protein in the hippocampus of APP/PS1 mice and reduce the deposition of amyloid A beta in hippocampus,so as to reduce its toxic effect on neurons,and exert its neuroprotective effect on AD mice.2.Chinese medicine Eucommia ulmoides can reduce the expression of GSK-3βand CDK5 in the hippocampus of APP/PS1 mice and reduce the phosphorylation of Tau in hippocampus,so as to reduce the formation of nerve fiber tangles,and exert the neuroprotective effect on AD mice.3.Chinese medicine Eucommia ulmoides can prevent calcium overload in neurons of APP/PS1 mice by regulating the expression of glutamate receptor system protein NMDAR1,GluR2 and CaMK Ⅱ in hippocampus,and play a neuroprotective role in AD mice.4.Chinese medicine Eucommia ulmoides can reduce neuron apoptosis by increasing the expression of Bcl-2,and decreasing the expression of Caspase-3 in hippocampal neurons of APP/PS1 mice,and exert neuroprotective effect on AD mice.5.Chinese medicine Eucommia ulmoides can increase the content of Ach and the activity of ChAT,and decrease the activity of AchE in the hippocampus of APP/PS1 mice,and increase the activity of cholinergic neurons,and exert the neuroprotective effect on AD mice.6.The Chinese herbal medicine,Eucommia ulmoides,can increase the expression of ERα,ERβ and estrogen secretion regulatory proteins FSHR and LHR in the cortex and hippocampus of APP/PS 1 mice,and then regulate the expression of related proteins in the downstream hippocampus,and play a neuroprotective role in the AD model mice.7.The extract of Eucommia ulmoides can increase the expression of ERβ,ERα,FSHR and LHR in the hypothalamus of APP/PS 1 mice,and increase the content of GnRH,β-EP and SP in hypothalamus,suggesting that Eucommia ulmoides extract can activate the hypothalamus-pituitary-Gonad axis,exert anti-aging effect.8.Eucommia ulmoides ingredients baicalein may regulate the expression of ERβ,P-JNK/JNK,NMDAR1,GluR2 and CaMK Ⅱ in PC12 cells injured by Aβ,so as to improve the morphology of PC 12 cells,reduce the apoptosis rate of PC cells induced by Abeta,and exert the protective effect on AD model cells. |
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