Role Of A? Fiber-induced Astrocyte Activation And Proliferation In Inflammatory Mechanism Of AD

BackgroundAmyloid-?(A?)exist in various aggregation forms such as monomers,oligomers,profibrils and fibers.Plaque deposition of A?,as an important pathological marker of the onset of AD,is mainly formed with A?fibers.A large number of literatures have demonstrated the definite toxic effects of A? oligomers on neurons and astrocytes,while the proinflammatory effects of A? fibers on astrocytes remains unclear.In the brain of AD mouse model,the significant changes in the morphology and gene expression of astrocytes resulted in the disruption of synaptic connections and imbalance of neurotransmitter homeostasis.The astrocytes near the A? plaque become active and undergo reactive proliferation,and then producing more toxic molecules degraded from polyamines.The misfolded and aggregated A? peptides can bind to the pattern recognition receptor on astrocytes,triggering an innate immune response characterized by the release of inflammatory mediators,leading to innate immune response.A?-induced inflammatory response can activate various signaling pathways through various cytokines and chemokines secreted by astrocytes and microglia,leading to neurodegeneration.Therefore,the study on the inflammatory response of astrocytes and its effects on neurons are important targets for the study of the pathogenesis of AD.ObjectiveThe purpose of this study was to investigate the inflammatory response induced by A? fiber to the activation and proliferation of astrocytes,including the expression of iNOS,TNF-?,IL-6,IL-1? inflammatory mediators and the inflammatory pathways related to MAPKs/NF-?B,so as to explore the effects on neurons,which may indicates the role of A? fiber-induced activation and proliferation of astrocytes in the inflammatory mechanism of AD.Methods1.After stimulating astrocytes with A? fiber solution of different concentrations(direct0 ?mol/L,direct1 ?mol/L,direct5 ?mol/L,direct10 ?mol/L,direct15 ?mol/L,direct30 ?mol/L)for 72 hours,the cell activity of astrocytes was detected by CCK-8 method,and the appropriate drug concentration was screened.The supernatant after culture was centrifuged and divided into groups(indirect0 ?mol/L,indirectl ?mol/L,indirect5 ?mol/L,indirect10 ?mol/L,indirect15 ?mol/L,indirect30 ?mol/L),acted on another group of astrocytes for 72 hours,and the activity of astrocytes was detected by CCK-8 method.2.Different concentrations(direct0 ?mol/L,direct15 ?mol/L,direct30 ?mol/L)of A? fiber solution were used to stimulate astrocytes directly for 72 hours.The morphology of astrocytes was observed by immunofluorescence staining with GFAP.The expression of GFAP protein in astrocytes under the action of A? fiber concentration was detected by western blot.3.The protein expression of iNOS,TNF-?,IL-6 and IL-1? in astrocytes was detected by western blot method after A? fiber at different concentrations(direct0?mol/L,direct15 ?mol/L,direct30 ?mol/L,indirect15 ?mol/L,indirect30 ?mol/L)stimulated astrocytes for 72 hours.Total protein levels and phosphorylated protein levels of p38 MAPK,ERK1/2 and p65 were detected by western blot.4.Different concentrations(indirect0 ?mol/L,indirect15 ?mol/L,indirect30?mol/L)of A? fiber were applied to cultured neurons for 24 hours,and the activity of neurons was detected by CCK-8 method.5.After 72 hours of direct stimulation of Ap fiber(direct0 ?mol/L,direct15?mol/L,direct30 ?mol/L),astrocytes were co-cultured with neurons for 24 hours.The neurons were thus grouped into groups of astrocyte-neuron0 ?mol/L,astrocyte-neuronl5 ?mol/L,and astrocyte-neuron30 ?mol/L.Annexin V-FITC/PI double-stained apoptosis detection method was used to detect the apoptosis of neurons.6.The expression levels of RIPK3,MLKL,bcl-2,Bax,caspase-3 proteins in different groups of neurons(astrocyte-neuron0 ?mol/L,astrocyte-neuron15 ?mol/L,and astrocyte-neuron30 ?mol/L)in the co-culture system were detected by western blot.Results1.A?fiber directly promoted the activation of astrocytes in A?concentration-dependent manner within the concentration range of 5-30 ?mol/L.The supernatant promoted the activation of astrocytes in A? concentration range of 10-30?mol/L.2.At concentrations of 15 ?mol/L and 30 ?mol/L,A? fiber increased the cell density,cell body,morphological evolution,GFAP fluorescence intensity and other cell proliferation and morphological changes.The expression of GFAP protein was proportional to the action concentration.3.The expression of inflammatory mediators in astrocytes increased after A?fiber induction.The total and phosphorylation levels of p38,ERK1/2 and p65 pathway proteins were increased.4.A? fiber-induced astrocyte-mediated neuron activity decreased.5.Annexin V-FITC/PI method was used to detect increased apoptosis and necrosis of neurons in the co-culture system.6.The expression of apoptosis related Bax and caspase-3 proteins increased,and the levels of necrotic related MLKL and RIPK3 proteins increased.Conclusions1.At certain concentrations,A? fiber promoted the activation and proliferation of astrocytes.2.A? fiber promotes the expression of TNF-?,IL-1?,IL-6,iNOS and other inflammatory mediators in astrocytes through activating the MAPKs/NF-?B pathway.3.Astrocytes mediated neuronal apoptosis and necrosis were induced by A?fibers.