| ObjectiveAlzheimer's disease(AD)is a kind of neurodegenerative disease of central nervous system with complicated etiology.Its characteristic is occult onset,the course of AD is a continuous and progressive decline of intelligence,and memory impairment is the first and the most important symptom.In AD patients,early memory loss,progressive language ambiguity(difficulty in finding words,naming disorders,etc.)gradual disappearance of concepts of familiar things and people,impaired executive ability,visual exploration in a certain space,The ability of thinking,especially abstract thinking,degenerates,and some patients are characterized by moodiness and abnormal behavior,which bring great mental and physical pain to patients and their families.AD patients account for about 50%-60% of the total dementia population.As to the pathogenesis of AD,?-amyloid deposition and tau protein theory have been widely accepted.With further study,it has been found that the core of pathogenesis of AD in early stage is synaptic dysfunction.Synaptic dysfunction in AD is associated with synaptic proteins,including presynaptic and postsynaptic membrane proteins.Abnormal expression of synaptophysin can impair normal synaptic function of the brain and lead to synaptic plasticity change,which is closely related to cognitive dysfunction and affects the development of AD throughout the later stages.Therefore,the changes of synaptophysin in AD were systematically studied,and the changes of synaptophysin and behavioral changes were further observed.The changes of protein expression and the improvement of overall cognitive function have become the focus of our research.The pathogenesis of AD is very complex,and there is no effective treatment drug.The research and development of traditional Chinese medicine and its multi-component and multi-target features provide the possibility for the treatment of AD.Our group took APP/PS1 double transgenic mice as the model,and used Shenzhiling Oral liquid,which was composed of Codonopsis pilosula,Cassia twig,Poria cocos,Radix Polygala,Acorus calamus,as an intervention drug.It has the function of reinforcing heart qi,connecting heart yang and expelling phlegm to enlighten the orifices.To observe the improvement of learning and memory in model mice by Shenzhiling,and to detect the synaptic related proteins by Western-blot and immunohistochemistry,and to observe the expression and distribution of synaptic proteins.To explore the protective effect of Shenzhiling on cognition and synapse in AD model mice,and to provide methods and approaches for the study of how to control the pathological changes and clinical symptoms of AD in the later stage.Materials and Methods 1 75 APP/PS1 double transgenic male mice were randomly divided into 5 groups,15 in each group: model control group(equal volume of distilled water)and donepezil group(0.92 mg/kg,dissolved in equal volume of distilled water.)The Shenzhiling large,medium,and low dose groups(50 g/kg,25 g/kg,12.5g/kg,dissolved in an equal volume of distilled water).Fifteen wild-type C57 mice of the same age and same background were selected as the normal control group(equal volume of distilled water).The rats were given intragastric administration once a day for 3 months.2 To use the step-down experiment to observe the effect of Shenzhiling Oral Liquid on learning and memory ability and memory retention ability of 3 months old APP/PS1 double transgenic mice,and evaluate the improvement of cognitive function after three months of continuous intervention.3 Immunohistochemistry and Western-blot methods were used to observe the distribution and expression of the synaptic-associated proteins mGluR5,?-synuclein and ?7-nAChRs in the central nervous system of 3 months old APP/PS1 double transgenic mice after three months of continuous intervention.To evaluate the regulatory effect of “Shenzhiling Oral Liquid” on synaptic plasticity.4 Transmission electron microscopy was used to observe the effect of Shinzhiling Oral Liquid on the synaptic ultrastructure of 3-month-old APP/PS1 double transgenic mouse hippocampus after three months of continuous intervention.Results 1 Shenzhiling can improve the memory retention ability of APP/PS1 double transgenic mice.The step-down experiment results can reflect the memory retention.In the step-down test,the number of errors of the model group was significantly increased compared with the normal group(P<0.01),compared with the model group,the number of errors of the donepezil group and medium dose group was significantly decreased(P<0.01).The time of the mice in the model group was significantly shorter than that in the normal group(P<0.01);compared with the model group,the incubation period of the mice in the donepezil group was increased(P<0.01);The latency time of the mice in the medium and low dose group was increased(P<0.05).2 Immunohistochemical staining showed that the positive cells of mGluR5 in the hippocampus of mice in the model group were significantly higher than those in the normal control group(P<0.01).Compared with the mice in the model group,the mGluR5 positive cells in the hippocampus of the mice in each group were decreased(P<0.01).The positive cells of ?7-nAChRs in the hippocampus of mice in the model group were significantly lower than those in the normal control group(P<0.01).Compared with the mice in the model group,the donepezil group,the middle group,and the low group of the expression of ?7-nAChRs positive cells in hippocampus increased(P<0.01).The positive cells of ?-Syn in the hippocampus of mice in the model group were significantly higher than those in the normal control group(P<0.01).Compared with the mice in the model group,the ?-Syn positive cells in the hippocampus of the donepezil group and the low-dose group were significantly decreased(P<0.01).Compared with the mice in the model group,the expression of ?-Syn positive cells in the hippocampus of the mice in the middle-dose group was decreased(P<0.05).3 Western-blot results showed that the expression of mGluR5 protein in the model group was significantly higher than that in the normal group(P<0.01).Compared with the model group,the expression of mGluR5 protein in the donepezil group was significantly reduced(P<0.01);Compared with the mice in the model group,the protein expression of mGluR5 protein was decreased in the middle and low dose group(P<0.05).The expression of ?7-nAChRs protein in the model group was significantly lower than that in the normal group(P<0.01).Compared with the model group,the expression of ?7-nAChRs protein in the donepezil group and the middle-dose group was increased(P<0.05).Compared with the normal group,the ?-Syn protein expression in the model group was significantly increased(P<0.01);compared with the model group,the donepezil group,the middle and the low dose group was significantly reduced(P<0.01);compared with the model group,the large dose group indicated that the protein expression was reduced(P<0.05).4 The results of electron microscopy showed that compared with the synapse structure in the hippocampus of the normal mice,the synaptic structure of the mouse hippocampus in the model group had abnormal changes,the synaptic cleft was wide,the synaptic mitochondria were swollen,and the anterior and posterior membrane structures were unclear.These phenomena indicate that there was a synaptic structure change in AD model mice;compared with the model group,the synaptic structure of the hippocampal CA1 region was basically normal in the donepezil group,the large,middle,and low dose groups.These results suggest that Shenzhiling oral liquid has improved synaptic structure in hippocampal CA1 region of APP/PS1 double transgenic mice.ConclusionImproving the memory retention ability of APP/PS1 double transgenic mice,Shenzhiling oral liquid may be mediated by regulating the key proteins mGluR5,?7-nAChRs and ?-synuclein in hippocampal synapses of APP/PS1 mice.Shenzhiling oral liquid could reduce the increased mGluR5 and ?-synuclein,and increase the reduced ?7-nAChRs,thus improving the memory retention ability of AD model mice. |
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