Study On The Mechanism Of PPT Regulating FOXO3 To Increase Newborn Neurons And Improve Cognitive Function In Alzheimer’s Disease

Alzheimer’s disease（AD）is a progressive neurodegenerative disorder that is the most common cause of dementia in older adults,affecting more than 45 million people worldwide.The pathologic features of AD are extracellular plaques resulting from Aβaggregation and fibrous tangles resulting from hyperphosphorylation of intracellular tau protein.Patients eventually show extensive loss of neurons that cannot be compensated by endogenous regeneration.Neural stem cells（NSCs）pool exists in the subgranular region of the dentate gyrus of hippocampus,which can replenish neurons through adult neurogenesis and is closely related to the fate determination mechanism of NSCs.Recent studies have shown that adult hippocampal neurogenesis（AHN）,the process by which NSCs form neurons,occurs throughout human life.Therefore,regulating the fate of endogenous NSCs to differentiate into neurons is a promising therapeutic strategy.Impaired adult neurogenesis of AD has been demonstrated in human AD patients and in rodent models of AD.With the progression of AD,the proportion of quiescent NSCs increases and neurogenesis is impaired,but the regulatory mechanism is still unclear.Therefore,maintaining the balance of NSCs quiescence and activation and promoting the change of NSCs fate to neurogenesis is the key to improve cognitive dysfunction in AD.Studies have shown that FOXO3 inhibits the cell cycle by regulating genes that remain quiescent and prevent premature differentiation,regulates the quiescence and survival of NSCs by regulating gene programs that control oxygen metabolism,inhibits normal NSCs neurogenesis,and contributes to the maintenance of NSCs reserves throughout the life cycle.Knocking out FOXO3 can significantly reduce the dormancy of NSCs and promote their proliferation.Although there is evidence that the expression and activity of FOXO3 in hippocampal neurons are up-regulated during AD,whether it leads to the increase in the proportion of resting NSCs and the key molecular mechanism of neurogenesis obstruction remains unclear.FOXO3 is mainly regulated by post-translational modifications such as phosphorylation,which is regulated by upstream PI3K-AKT kinase phosphorylation and regulates FOXO3 activity through nucleolar shuttling.FOXO3 is a transcription factor whose DNA binding region recognizes the DNA structure and promotes the transcription of downstream genes.Therefore,the discovery of drugs that can inhibit FOXO3 activity may have the effect of driving NSCs out of quiescence to promote adult neurogenesis in AD and improve cognition.Panax ginseng Meyer,a medicinal plant with the same origin as medicine and food,has a history of thousands of years in our country as a tonic of"calming the spirit and educating the brain".Studies have shown that ginseng can improve symptoms and inhibit AD progression in patients with AD by reducing Aβdeposition and tau hyperphosphorylation through the PI3K-AKT signaling pathway.However,ginseng has many active components against AD,and whether it could promote adult neurogenesis through PI3K-AKT-FOXO3 needs to be further clarified.In this study,we first explored the active ingredients of ginseng with anti-AD function through systematic review and meta-analysis,and found that ginsenotriol type has better anti-AD effect.In view of the advantages of 20（S）-Protopanaxatriol（PPT）as a ginsenoside type metabolite,such as low dosage,strong effect of passing the blood-brain barrier and improving cognitive function of AD,the therapeutic effect of PPT on AD was further evaluated on the animals and cellular models.To systematically investigate the mobilization of endogenous NSCs to differentiate into neurons to improve cognitive dysfunction in AD,and to clarify the mechanism of PPT regulating FOXO3 to drive AD NSCs to exit from the quiescent state,so as to provide a theoretical basis for the discovery of new targets in AD.The research content of this paper is divided into five parts:（1）Systematic review and meta-analysis of active ingredients of ginseng in the treatment of cognitive function in AD.Methods:In this chapter,Pub Med,Web of Science,Scopus,CNKI database and Wanfang database were searched to find relevant studies on active ingredients of ginseng in the treatment of cognitive function in AD,and the included studies were systematically reviewed and meta-analyzed according to PRISMA guidelines.Morris water maze（MWM）was the main outcome marker,and Aβdeposition,inflammatory response and oxidative stress were the secondary outcome markers.Results:（1）A total of 493 relevant articles were retrieved,and 37 studies were included according to inclusion and exclusion criteria.（2）The active ingredients of ginseng decreased the MWM escape latency of AD animals,increased the number of platform crossing and the time spent in the destination quadrant;（3）The active components of ginseng decreased the Aβdeposition of AD animals;（4）The active components of ginseng decreased the oxidative stress level of AD animals,increased the expression of SOD and GSH,decreased the expression of MDA;（5）The active ingredients of ginseng decreased the inflammatory level,IL-1βexpression and TNF-αexpression in AD animals;（6）The source of bias was investigated by subgroup analysis and funnel plot analysis.Among them,PPT,as a ginsenoside triol type metabolite,has a small dose,can pass the blood-brain barrier and improve AD cognitive function.（2）Study on the effect of PPT on cognitive dysfunction in AD animals.Methods:Several AD animal models were established,including Al Cl₃-induced AD zebrafish model,Aβ1-42 brain stereotaxic injection AD mouse model and 5×FAD mouse model,and PPT treatment intervention was given.The changes of cognitive function were observed by detecting the Y maze and T maze of zebrafish,and the Y maze,new object recognition,DNMP experiment and MWM of mice.The changes of the structure and number of neurons were observed by HE and Nissl staining.Results:（1）After PPT administration,AD zebrafish spent more time on the new arm of Y maze,decreased the incubation period of T maze,and significantly extended the cumulative time in EC region.The number of Nissl bodies in the telencephalon was increased with close arrangement of neurons.The results showed that PPT could improve cognitive function and number of neurons in AD zebrafish.（2）After PPT administration,Aβ1-42 induced mice and5×FAD mice had increased preference in new arm of Y maze,significantly increased NOR new object discrimination index,significantly increased DNMP selection stage accuracy,improved MWM localization cruise and space exploration ability.The hippocampus cells were densely arranged,the number of cells increased significantly,the cell structure was relatively complete,and the number of Nissl bodies gradually increased.The results showed that PPT could improve the cognitive function and number of neurons in Aβ1-42 induced mice and 5×FAD mice brain.（3）Effect of PPT on cognitive function in AD through adult neurogenesis.Methods:（1）We established AD zebrafish model induced by Al Cl₃,Aβ1-42 brain stereotaxic injection AD mouse model and 5×FAD mouse model,and gave PPT treatment intervention.In order to clarify the changes of PPT on adult neurogenesis in vivo,we detected the changes of neonatal neurons（HUC）in zebrafish,NSCs（Sox2）,neural progenitor cells（Tbr2）,immature neurons（DCX）,mature neurons（MAP2）,presynaptic membrane proteins SYN and PSD-95.（2）To clarify the changes of NSCs differentiation and function induced by PPT in vitro.we established N2a/APPswe and NE-4C NSCs cell line co-culture and Aβ1-42 oligomer intervention in primary NSCs to simulate the pathological state of AD and give PPT treatment.The expression changes of neuron（MAP2）,astrocyte（GFAP）,and synaptic membrane proteins SYN and PSD-95 were detected.（3）The mechanism by which PPT exerts anti-AD effect mediated by newborn neurons was further clarified.A mouse model of neurogenetic elimination of AD was established and given PPT treatment.Behavioral indicators such as Y maze,new object recognition,DNMP test and MWM were detected to observe the changes in cognitive function.HE,Nissl staining,mature neuron MAP2,pre-and post-synaptic membrane protein SYN,and PSD-95 were detected to observe the changes in the structure and number of neurons.Results:（1）After PPT administration,the telencephalic newborn neuron marker HUC and the transcription levels of huc and neurod1 were significantly increased in AD zebrafish.After PPT administration,the expression of Sox2,Tbr2,DCX,MAP2,SYN and PSD-95 were increased in Aβ1-42 induced AD mice and 5×FAD mice.The results showed that PPT could improve the neurogenesis of AD in vivo.（2）N2a/APPswe cells and NE-4C co-culture model:After PPT treatment,NE-4C cell viability was significantly improved,and the cells showed differentiation morphology of cell body shrinkage and elongated axonal dendrite growth,and DCX and MAP2 expression were increased.Aβ1-42 oligomer intervention of primary NSCs:After PPT administration,NSCs MAP2 expression was increased,GFAP expression was decreased,SYN and PSD-95 expression were also increased.The results showed that PPT can induce NSCs differentiation into neurons and increase synaptic function in AD pathological state in vitro.（3）After PPT administration of AD mice with neurogenetic elimination,compared with PPT administration of the model group,the preference of new arm in Y-maze was decreased,the new object discrimination index of NOR experiment was decreased,the accuracy of DNMP experiment selection stage was increased,the ability of water maze localization cruise and space exploration was decreased,the number of Nissl bodies was decreased,and the expression of MAP2 was decreased.The expression of SYN and PSD95decreased.The results showed that PPT improved the cognitive function of AD animals by promoting neurogenesis.（4）Effect of PPT on neurogenesis progression of neural stem cells.Methods:（1）The Aβ1-42 brain stereotaxic injection AD mouse model and 5×FAD mouse model were established,and PPT treatment was given.In order to clarify that PPT protects NSCs from survival and drives NSCs to enter the cell cycle for neurogenesis NSCs apoptosis protein（Sox2⁺Cleaved-Caspase3⁺）,quiescent NSCs（Sox2⁺BMP4⁺）,proliferating NSCs（Sox2⁺Ki67⁺）,NSCs differentiated NPCs（Sox2⁺Tbr2⁺）,newborn granular neuron（Neu N⁺Brd U⁺）were detected.The changes of Cld U and Id U before and after PPT administration were detected to clarify the role of PPT in the recruitment of quiescent NSCs.（2）By establishing N2a/APPswe and NE-4C co-culture to simulate the pathological state of AD and giving PPT treatment.In order to investigate the protective effect of PPT on NSCs and the effect of cell cycle,the expression of apoptotic protein Cleaved-Caspase3,Bim,Bcl2/Bax,and the changes of cell cycle and cell cycle-related proteins p53,p21,p27^Kip1,Cyclin E1,Cdk2,EGFR,and quiescence BMP4 were detected.Results:（1）We found that apoptotic NSCs（Sox2⁺Cleaved-Caspase3⁺）were decreased,quiescent NSCs（Sox2⁺BMP4⁺）were decreased,and proliferative NSCs（Sox2⁺Ki67⁺）were increased in AD mice after PPT administration,.The proportion of Sox2⁺Cld U⁺Id U⁺cells and Sox2⁺Cld U^-Id U⁺cells were also increased.The number of NSCs differentiated NPCs（Sox2⁺Tbr2⁺）was increased,and the number of newborn mature neurons（Neu N⁺Brd U⁺）was increased.It was suggested that PPT protected NSCs survival and drove NSCs to recruit quiescent NSCs for neurogenesis.（2）After PPT administration,expression levels of Cleaved-Caspase3 and Bim were decreased in NE-4C,Bcl2/Bax ratio was increased.The cell cycle arrest was significantly improved,G1/G0 ratio was decreased significantly,BMP4 was decreased,EGFR was increased.The expressions of cell cycle inhibitors p53,p21 and p27^Kip1were decreased,while the expressions of Cyclin E1 and Cdk2 were increased.It was shown that PPT protected NSCs horizontally and entered the cell cycle in vitro.（5）Molecular mechanisms by which PPT promotes adult neurogenesis.Methods:（1）After knocking down Foxo3,the expression of cell cycle,related proteins and apoptotic proteins was detected.（2）We use network pharmacology and molecular docking techniques to explore the molecular mechanism of PPT therapy for AD.We established the Aβ1-42 brain stereotaxic injection AD mouse model and 5×FAD mouse model and co-culture of N2a/APPswe and NE-4C,PPT was administered.In order to clarify the mechanism of PPT indirectly regulating FOXO3 through PI3K-AKT signaling pathway,PI3K p110,PI3K p85,phosphorylation AKT,phosphorylation Foxo3and karyoplasmic distribution of Foxo3 were detected.（3）Through PPT and FOXO3-DBD molecular docking,molecular dynamics simulation,DNA pull down and Bim promoter luciferase reporter gene test,Foxo3-WT-FLAG and Foxo3-Mut（H212R）-FLAG were overexpressed in FOXO3 knockout cells,and the key amino acid sites of PPT regulating Foxo3 binding to DNA were identified.Results:（1）After FOXO3 knockdown in the model group and PPT treatment in the model group,Bcl2/Bax ratio increased,Bim expression level decreased,EGFR increased,BMP4decreased,and the proportion of G0/G1 stage decreased.The proportion of S phase decreased,the expressions of Cyclin E1 and Cdk2 increased,and the expressions of p53 and p21 decreased.（2）The intersection of AD disease targets and PPT drug targets was used to obtain a total of154 PPT potential targets for AD treatment.The results of network pharmacology show that the anti-AD effect of PPT may be through the core target of PIK3CA and the PI3K-AKT signaling pathway.The docking energy of PPT and PIK3CA is-36.82855,indicating that they have a high possibility of interaction.The expression levels of PI3K p110,p-PI3K p85 and p-AKT in NE-4C were increased after PPT administration.Compared with PPT treatment group,PI3K inhibitor LY294002 reversed the protective effect of PPT on cell viability and PPT-induced AKT phosphorylation.PPT administration can increase the p Foxo3/Foxo3 ratio of the downstream AKT target,and the Foxo3 content in the nucleus of NE-4C decreases,while the Foxo3 content in the cytoplasm increases.After PPT administration,the Foxo3 content in NSCs cytoplasm of AD mice was significantly increased and the nuclear content was decreased.The results show that PPT indirectly regulates Foxo3 through the PI3K-AKT pathway.（3）The molecular docking shows that PPT could bind to the Helix surface of FOXO3-DBD,and PPT have hydrogen bonding with N208,R211 and H212 on FOXO3-DBD protein,and the binding affinity score is-5.1.It showed that PPT and FOXO3-DBD protein had the possibility of binding.Molecular dynamics simulation shows that FOXO3-DBD＿PPT can move stably and interact closely in the middle and late stages of simulation.After PPT administration,the Foxo3 pulled down by DNA probe decreased,and the luciferase activity of Bim promoter decreased.In FOXO3 gene knockout HEK293T cells,after Foxo3-WT-FLAG overexpression was given PPT intervention,the DNA probe pull-down FLAG was significantly decreased.However,after Foxo3-Mut（H212R）-FLAG overexpression,there was no statistical difference in DNA probe pull-down FLAG after PPT treatment.After Foxo3-WT-FLAG overexpression was administered with PPT,the Bim promoter luciferase activity decreased significantly with the increase of PPT dose,but there was no significant change in the Bim promoter luciferase activity after Foxo3-Mut（H212R）-FLAG overexpression was administered with PPT.The results showed that PPT can directly target FOXO3 to inhibit DNA binding.In summary,this paper draws the following conclusions:1.PPT could improve the cognitive dysfunction of AD animal models by driving NSCs to exit the quiescent state and increasing new neurons,which provides a theoretical basis for PPT to improve the cognitive function of AD.2.On the one hand,PPT could indirectly increase FOXO3 phosphorylation through the PI3K-AKT signaling pathway to increase the nuclear output of FOXO3;on the other hand,PPT could directly block the interaction between FOXO3-DBD and DNA,reduce FOXO3 activity to induce NSCs to exit the quiescence state,and provide a new target for AD treatment.