The Involvement Of The Akt/fOXO1 Signaling Pathway In The Mechanism Of Cellular Pyroptosis Of Microglia In Alzheimer’s Disease

Objective: Alzheimer’s disease is the most common neurodegenerative disease and the leading cause of mental retardation worldwide.It places a heavy burden on patients and caregivers as there is no effective cure or delay in the progression of the disease.The pathogenesis of Alzheimer’s disease has been extensively studied and has been identified to involve a series of physiopathological processes including the formation of misfolded amyloid beta deposits in neuronal and glial cells resulting in the inability of the cells to perform normal physiological functions,and the clearance of the deposited proteins resulting in an associated cellular immune inflammatory response and subsequent cellular damage and death.Among these processes,neuroinflammation and cell death are closely associated with the development of Alzheimer’s disease.Studies on the relevance of pyroptosis to Alzheimer’s disease have recently increased with the discovery of pyroptosis,a process of programmed cell death associated with the inflammatory response.Microglia play an important role in the clearance of amyloid beta deposits as scavengers of the nervous system.In addition,microglia activation is one of the important manifestations of cellular inflammation and cellular damage,directly responding to the degree of neurological damage.In Alzheimer’s disease,abnormal activation of microglia and overproduction of pro-inflammatory factors are also important contributors to cellular damage and increased disease severity.In particular,microglia are the main site of cellular pyroptosis,and proteins associated with cellular pyroptosis are highly expressed in activated microglia,resulting in the release of pro-inflammatory factors that contribute to the further deterioration of Alzheimer’s disease.Therefore,controlling the pyroptosis activity in microglia is one of the most important approaches to slow down the disease process in AD.Although there is a growing body of research related to the pathogenesis of AD,the mechanisms regulating the pyroptosis activity in microglia are not yet fully understood.FOXO1 is a member of the forkhead box O family of transcription factors,a family of transcription factors that is highly conserved in evolution.It is closely associated with cell proliferation,differentiation and death.Evidence has been found for the involvement of FOXO1 in the regulation of cellular pyroptosis in other diseases including non-alcoholic liver injury and myocardial ischaemic injury.In addition,the PI3K/Akt/FOXO1 signalling pathway was found to directly regulate GSDMD protein in microglia in spinal cord ischaemic injury.The PI3K/Akt signalling pathway is one of the most important regulatory pathways of FOXO1 and plays an important role in the regulation of cell cycle,apoptosis and autophagy,and we screened this pathway by bioinformatic methods to predict that it may also be a signalling pathway related to cell pyroptosis activity.To clarify whether FOXO1 is also involved in the regulation of microglial cell pyroptosis in AD,we designed an experiment to interfere with the expression level of FOXO1 phosphorylation through the action of Akt agonists and to detect microglial cell pyroptosis activity in APP/PS1 transgenic mice to test the experimental speculation.We demonstrated that down-regulation of FOXO1 phosphorylation in AD mice is associated with active cellular pyroptosis.The signalling pathway involved is the PI3K/Akt signalling pathway,and the application of Akt agonist can promote Akt phosphorylation,further phosphorylate FOXO1,reduce its transcriptional activity,decrease the expression of NLRP3,IL-1β and GSDMD activation,reduce the degree of cellular pyroptosis,reduce cellular oxidative damage,increase cell survival and cell viability,and improve the learning and memory ability of AD mice.The results showed that the expression of NLRP3 and IL-1β and the activation of GSDMD reduced the extent of cellular pyroptosis,decreased oxidative damage,increased cell survival and cell viability,and improved the learning and memory abilities of AD mice.We conclude that up-regulation of FOXO1 phosphorylation in microglia of AD mice through PI3K/Akt signaling pathway can improve the impairment of learning and memory ability,improve the symptoms of Alzheimer’s disease and slow down the disease process.Methods:(1)Firstly,the APP/PS1 transgenic mice(AD group)and wild-type mice(WT group)were subjected to Morris water maze behavioural experiments,and immunofluorescence quantification of cellular pyroptosis-related indicators in astrocytes and microglia in the cortical and hippocampal regions of the two groups,and FOXO,GSDMD,IL-1β and IL-18 protein quantification in the brain tissue of each group using western blot.and the quantification of NLRP3,GSDMD,IL-1β and IL-18 proteins.The AD mice were fed with a special diet PLX3397 to inhibit microglia proliferation and then subjected to a water maze experiment and brain tissue was taken to measure the expression of microglia-associated proteins Iba-1,CD68 and pyroptosis-related proteins.(2)The brain tissues of APP/PS1 transgenic mice were quantified for FOXO and focal death-related NLRP3,GSDMD,IL-1β and IL-18 proteins using western blot assay in the WT,AD and ADAS groups(AD mice + FOXO1 inhibitor).To clarify the correlation between FOXO1 and cell pyroptosis.To clarify whether FOXO1 acts on NLRP3 inflammation vesicles,mice were treated with MCC950,an inhibitor of NLRP3,and the MCC group(AD mice + MCC950)and the MCCAS group(AD mice + MCC950 +FOXO1 inhibitor)were added and then subjected to water maze experiments,and brain tissue was taken for protein quantification.(3)To apply the GEO database in NCBI to perform weighted gene co-expression network analysis and gene enrichment analysis of KEGG pathway for FOXO1 differentially expressed genes in Alzheimer’s disease patients by bioinformatic analysis to clarify the related signaling regulatory pathways.The Akt/FOXO1 pathway in the FOXO signaling pathway was selected as the signaling pathway to be studied using protein interactions website prediction.(4)BV2 cells were cultured according to the screened signalling pathways,and the cells were divided into negative control group(group C),positive control group(group L),LPS+Akt agonist group(group LS)and LPS+Akt agonist+FOXO1 inhibitor group(group LSA)using LPS-induced cell pyroptosis,Akt agonist SC79 and FOXO1 inhibitor AS1842856.The morphological changes of BV2 cells in each group were examined by MTT method,and the extent of oxidative damage in the cells was detected by using relevant assay kits and the relationship between the alteration of FOXO1 phosphorylation level and cell pyroptosis activity in BV2 cells was detected by immunofluorescence and protein quantification.(5)APP/PS1 mice given SC79 and AS1842856 were divided into the following four experimental groups: wild group(WT group),Alzheimer’s disease group(AD group),Alzheimer’s disease + Akt agonist group(ADSC group),Alzheimer’s disease + Akt agonist + FOXO1 inhibitor group(ADSCAS group)for Morris water maze Behavioural experiments were performed,and FOXO1 and pyroptosis related indicators were quantified by immunofluorescence and western blot protein assay for each group.Results: Compared to WT mice,AD mice had impaired learning memory,longer latency before finding a plateau in behavioral experiments,slower swimming speed,and fewer mice finding a plateau.The expression of NLRP3+,GSDMD-N+ and IL-1β+ cells was increased in the AD group.Specific inhibition of microglia proliferation improved the learning ability of AD mice and reduced the extent of microglia cell pyroptosis.Inhibition of FOXO1 activity exacerbated the degree of cellular pyroptosis,i.e.increased expression of NLRP3,GSDMD-N and IL-1β,suggesting that FOXO1 is associated with cellular pyroptosis.The expression of other FOXO1-related proteins did not differ in mice after the application of NLRP3 inhibitors,suggesting that NLRP3 is an important target of FOXO1 in regulating cellular scorching activity.The KEGG gene pathway enrichment analysis suggested that the FOXO signaling pathway,cellular senescence and late glycation end products and their receptor signaling pathways are signaling pathways related to the regulation of Alzheimer’s disease by FOXO1.The Akt/FOXO1 signalling pathway was suggested as a possible signalling pathway based on protein interaction site prediction analysis.LPS concentration of 3ug/ml induced cell pyroptosis in BV2 cells,increased expression of cell pyroptosis-related proteins,increased ROS expression,altered SOD enzyme activity and impaired structural integrity of mitochondrial membranes.Up-regulation of FOXO1 activity through the Akt/FOXO1 signaling pathway improved the proliferation viability of BV2 cells and reduced the expression of NLRP3,GSDMD-N and IL-1β.FOXO1 regulated microglia pyroptosis activity in AD mice through the Akt/FOXO1 signaling pathway and improved the learning and memory ability of AD mice.Conclusion: 1.In AD mice with impaired learning and memory abilities,proliferation and activation of astrocytes and microglia and active cellular pyroptosis were observed in brain tissues,manifested by increased expression of NLRP3,GSDMD amino-terminal activation form and IL-1β.Inhibition of whole brain microglia proliferation reduced the extent of cellular pyroptosis activity in the brain and improved impaired intelligence in mice,suggesting that microglia are the main type of cells that undergo cellular pyroptosis.NLRP3 is an important target of FOXO1.FOXO1 was associated with cellular pyroptosis activity in AD mice,and inhibition of FOXO1 activity could exacerbate cellular pyroptosis damage.NLRP3 is an important target of FOXO1.FOXO1 is involved in AD regulation related signaling pathways including FOXO signaling pathway,cellular senescence pathway and AGE and their receptor RAGE signaling pathway.Akt/FOXO1 signaling pathway is the regulatory pathway of FOXO1 in AD cell pyroptosis.LPS induces cell pyroptosis in BV2 cells with oxidative damage.Up-regulation of FOXO1 activity through the Akt/FOXO1 signaling pathway can improve the proliferation viability of BV2 cells and reduce cell pyroptosis activity.FOXO1 regulates microglia pyroptosis in AD mice through the Akt/FOXO1 signaling pathway and improves the learning and memory ability of AD mice.