Protective Effects Of Xingnaojing Injection And Muscone On Synaptic Damage In Alzheimer’s Disease

Background:Alzheimer’s disease（AD）is a neurodegenerative disorder affecting the memory and learning abilities with over 50 million patients worldwide,which brings heavy burdens to the societies and families.One of the characteristics of AD is the deposition of senile plaques composed ofβ-amyloid（Aβ）,and the damage of synaptic structure and function directly or indirectly induced by Aβplays a critical role in learning and memory impairments in early stage of AD,which is closely correlated to N-methyl-d-aspartate（NMDA）receptor and histone acetylation.However,the underlying mechanisms of AD have not been fully known,and there is no cure for AD.Muscone（Mus）is an active component of Xingnaojing injection（XNJ）,which is benefit in treating alcoholic consciousness disturbance.However,little is known of the effects of XNJ or Mus on AD.In this study,we explored whether XNJ or Mus was able to modulate cognitive functions and synaptic plasticity deficits in AD mice.Furthermore,we elucidated the underlying molecular mechanism of the protective effects of XNJ or Mus.Methods:In the study of XNJ,Aβ_1-42 oligomer was injected into the bilateral hippocampus of C57BL/6 mice using a stereotaxic apparatus,and 7 days later injected XNJ intraperitoneally（i.p.）every day.One month later,we assessed the cognitive function by Novel object recognition test（NOR）and Morris water maze（MWM）.The levels of synaptic proteins and NMDA receptors were evaluated as shown by western blotting（WB）,and synaptic structures were measured by Golgi and immunofluorescence staining.The functions of synapse and NMDA receptor were detected by electropsychological experiment,and the activation of AKT/mTOR signaling pathway and the level of NMDA receptors with or without rapamycin treatment were detected by WB.Meanwhile,APP/PS1 mice were used to verify the conclusions by NOR and long-term potentiation（LTP）experiment.In the study of Mus,the wild type（WT）and APP/PS1 mice were injected Mus i.p.daily for one month,and were assessed the cognitive function by NOR and MWM.The levels of Aβwere evaluated by immunofluorescence staining and enzyme linked immunosorbent assay（Elisa）.The level of synaptic protein was evaluated by WB,and the synaptic structure and function were detected by Golgi staining and LTP.In vitro,primary cortical neurons were stimulated by Aβ_1-42,and the cell viability was measured by CCK8.The neuron morphology and synaptophysin expression were detected by immunofluorescence staining.At last,WB was used to detect the acetylation level of H3K9 and the expression of histone deacetylase 2（HDAC2）protein with or without the treatment with cyclohexidine（CHX）.Quantitative PCR（Q-PCR）was used to detect the m RNA level of HDAC2.In addition,MG132 was used to inhibit the function of proteases and Co-Immunoprecipitation（co-IP）was applied to assess the level of ubiquitination of HDAC2.Molecular docking analysis by computer modeling was used to detect whether Mus might directly interact with HDAC2.Results:1)XNJ alleviated the searching time to explore the new object in NOR and the explore ability for platform in MWM.And the levels of synaptic proteins,the density of dendritic spines and the synaptic plasticity in CA1 region of hippocampus of AD mice with XNJ treatment were enhanced.2)NMDA receptor expression and function impairment in the hippocampus of AD mice after XNJ treatment were relieved.AKT/mTOR signaling pathway was activated after XNJ treatment in AD mice.Inhibiting mTOR was able to block the protective effect of XNJ on NMDA receptor and cognitive dysfunction.3)Mus alleviated the searching time to explore the new object in NOR and the explore ability for platform in MWM,and decreased the levels of Aβin brains.Moreover,the level of synaptic protein in hippocampus was increased with Mus treatment.In addition,Mus improved the complexity of neuron dendrites,the density of mushroom dendritic spines and the synaptic plasticity in CA1 region of hippocampus of AD mice.In vitro,Mus alleviated neuronal death and morphological and structural damage in the primary cortical neurons stimulated by Aβ.4)In vivo and vitro,Mus could enhance the expression of NR2B and the acetylation level of H3K9,and downregulate the level of HDAC2 protein.Meanwhile,Mus could significantly increase the level of ubiquitination of HDAC2 compared with Aβstimulated neurons.The docking results implied that Mus and HDAC2 might have direct interactions.Conclusions:1)XNJ attenuated cognitive dysfunction and synaptic damage in the hippocampus of AD mice via activating AKT/mTOR signaling pathway.2)As one of the major effective components of XNJ,Mus attenuated cognitive dysfunction and synaptic damage in the hippocampus of AD mice.Besides,Mus decreased the levels of Aβ,alleviated neuronal death and improved the morphology of neurons and synapse.Mechanistically,Mus increased the ubiquitination of HDAC2,which might lead to the acetylation level of H3K9 to increase the level of NR2B.