BMSC-exos Ameliorate Streptozotin-induced Neuropsychiatric Damage In SAD Mice By Inhibiting Hippocampal Microglia Activation

Alzheimer’s disease（AD）is a neurodegenerative disease characterized by progressive cognitive decline.Memory loss,difficulty interpreting or understanding things and performing past daily chores,and changes in personality and mood are the most common clinical symptoms of AD.Exosomes are a class of extracellular vesicles with a diameter of about 30-200 nm,which carry important genetic information such as m RNA,mi RNA,and proteins,and the transmission of information between cells can be mediated by exosomes.Bone marrow mesenchymal stem cells（BMSC-exos）are extracellular vesicles that can perform the function of bone marrow mesenchymal stem cells（BMSCs）.In recent years,BMSC-exos has shown a certain therapeutic effect in the research of heart,kidney and other diseases.In this study,a mouse model of sporadic Alzheimer’s disease（SAD）was established by injecting streptozotocin（STZ）into the bilateral ventricle.The behavioral tests were conducted to observe the effects of lateral ventricle injection of BMSC-exos on the movement,learning and memory ability of SAD mice.Western blot,quantitative polymerase chain reaction（q PCR）,immunofluorescence staining（IF）and other experiments were performed to detect the expression of related molecules in the hippocampal tissue to explore the potential therapeutic effect and possible mechanism of BMSC-exos on SAD.ObjectiveTo investigate the protective effect of BMSC-exos against the behavioral dysfunction of SAD mice and explore the potential mechanism,especially the activation of glial cells and the expression changes ofβamyloid（Aβ）,microtubule-related proteins and synapse-related proteins in the hippocampus.Method1.Establishment of a SAD mouse model and the changes of neuropsychiatric behavior and neuropathological features.Sixteen male C57BL/6 mice aged 4 weeks were randomly divided into two groups including a control group and a SAD group.The mice in the SAD group were received an injection of STZ drug solution（3 mg/kg）into the bilateral ventricles,and the control mice was given the same amount of artificial cerebrospinal fluid（ACSF）.Two weeks later,the neuropsychiatric behavioral changes were observed via a series of behavioral tasks including open field test（OFT）,novel object recognition test（NOR）and tail suspension test（TST）.Brain tissue samples were collected after the last behavioral study.Brain tissue sections were subjected to immunofluorescence staining for astrocytes,microglia,and synapse-related proteins.Western blot assay detected hippocampal Aβ_1-42protein,amyloid precursor protein cleaving enzyme（BACE）,interleukin 1β（IL-1β）,Interleukin 6（IL-6）,tumor necrosis factorα（TNF-α）,glial fibrillary acidic protein（GFAP）,microtubule-associated protein Tau5,phosphorylated Tau protein（p-Tau）（Ser396）,brain-derived neurotrophic factor（BDNF）,synaptotagmin-1（Syt-1）,synapsin 1（Syn-1）protein expression level.The m RNA expression levels of IL-1β,IL-6 and TNF-αin hippocampus were detected by q PCR experiment.2.The protective effect of BMSC-exos on the neuropsychiatric injury of SAD mice and the potential mechanism.BMSC-exos were extracted from the supernatant of mouse BMSCs isolated and cultured from the femur and tibia of adult C57BL/6 mice,which were purified and sorted by flow cytometry,and cultured in vitro.The morphology of BMSC-exos was observed by transmission electron microscopy,and the typical marker proteins of exosomes,such as cluster of differentiation 63（CD63）,heat shock protein 70（HSP70）and tumor susceptibility gene 101 protein（TSG101）were detected by Western blot assay.Twenty-four male C57BL/6 mice were randomly divided into control group,model group,lateral ventricle group（0.5μg/day）and tail vein group（25μg/day）.Six weeks after the establishment of the SAD model according to the above method,BMSC-exos were administered by lateral intracerebroventricular injection or tail vein injection for five consecutive days,and an equal amount of ACSF was administered to the mice in the control and model groups.The behavioral changes were observed by OFT,elevated plus maze test（EPM）,NOR,Y maze test（Y-maze）and TST.The m RNA expression levels of IL-1β,IL-6 and TNF-αin hippocampus were detected by q PCR.The protein expression levels of Aβ_1-42,BACE,GFAP,IBA1,p-Tau（Ser396）,Tau5,IL-1β,IL-6,TNF-α,BDNF,Syt-1 and Syn-1 in hippocampus were detected by Western blot assay.The expression of GFAP,IBA1,Aβ,p-Tau and DCX in the hippocampus was detected by immunofluorescence staining（IF）,and the expression of Aβin the hippocampus was observed by thioflavin S staining（Th S）.Result1.Compared with the control group,the duration in the center,number of crossing,grooming and rearing in the OFT,and the novel object preference index was significantly reduced the SAD mice injected with STZ in the bilateral ventricles had significantly reduced,but there was no statistical difference in the immobility time in the TST between the two groups;IF results showed that the activation of astrocytes in the hippocampus of SAD mice was significantly enhanced,also the expression of p-Tau was significantly increased,and the expression of Syt-1 was significantly decreased;q PCR and Western blot results showed that the m RNA expression of IL-1β,IL-6 and TNF-α,the protein expression levels of Aβ,BACE,IL-1β,IL-6 and TNF-αof SAD mice was significantly increased.The Tau protein was phosphorylated significantly and the expression levels of synapse-related proteins such as BDNF,Syt-1,and Syn-1 of SAD mice were significantly decreased.2.Compared with SAD mice,the neuropsychiatric behavior of SAD mice can be improved by the lateral ventricle injection of BMSC-exos,the duration,frequency,time and distance in the centre,number of crossing and rearing in the OFT,and the novel object preference index were increased,but there was no significant influence on preference index of the novel arm in the Y-maze,moving distance of closed arm in the EPM and immobility time in the TST;The IF results showed that injection of BMSC-exos into the lateral ventricle could significantly inhibit the activation of glial cells in the hippocampus of SAD mice,promote neurogenesis and lower the expression levels of hippocampal Aβand p-Tau;q PCR and Western blot results showed that the m RNA expression levels of IL-1β,IL-6,and TNF-αand the protein expression levels of Aβ,BACE,p-Tau,IL-1β,IL-6,and TNF-αof were decreased,and the protein expression of BDNF SAD mice was increased by the injection of BMSC-exos into the lateral ventricle.Conclusion1.The SAD mouse model can be successfully induced by bilateral intracerebroventricular injection of STZ,which is manifested in reduced exploratory behavior and cognitive dysfunction.The mechanism of action may be related to the activation of inflammatory response in the brain,the accumulation of amyloid plaques,the abnormal phosphorylation of Tau protein,and the damage of prominent plasticity.2.The cognitive function of SAD mice can be improved by lateral ventricle injection of BMSC-exos,and its mechanism may be related to alleviating neuroinflammation,reducing the expression of Aβand p-Tau,and regulating the expression of prominent plasticity-related protein BDNF.