Chronic Glucocorticoids Promotes Aβ Deposition Via Activating NLRP1 Inflammasome In Primary Hippocampal Neurons Of APP/PS1 Mice

ObjectiveTo study the effects and mechanisms of chronic glucocorticoids（GCs）on Aβdeposition in primary hippocampal neurons of APP/PS1 mice.And to study the effects of inflammasome and intracellular calcium on Aβdeposition in chronic GCs-induced primary hippocampal neurons of APP/PS1 mice.Methods1.The primary hippocampal neurons of postnatal（24 h）APP/PS1 mice or WT mice were identified by PCR.After 7 days of cell culture,the neurons were divided into four groups:WT group,WT+1μM DEX group,APP group,and APP+1μM DEX group.Digital holography time-lapse imaging was used to detect cell morphology;The release of LDH was detected by LDH kit;The apoptosis was detected by Annexin V-FITC/PI assay;The intracellular calcium([Ca²⁺]_i)level was detected by calcium imaging;DHE and H₂DCFDA staining was used to detect ROS production;Thioflavin S staining was used to detect Aβdeposition;PSD95 and Aβ1-42 expression were indicated by immunofluorescence staining;The expressions of APP,BACE1,NCSTN,NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1 and IL-1βm RNAs were detected by Real-time PCR;The expressions of NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1,IL-1β,APP,BACE1,NCSTN,TAU,P-TAU,PLC,P-PLC,CN,NFAT1 and IL-6 proteins were detected by western blot.2.The primary hippocampal neurons of postnatal（24 h）APP/PS1 mice were identified by PCR.After 7 days of cell culture,the neurons were divided into six groups:APP group,APP+1μM DEX group,APP+LV-NLRP1-siRNA group,APP+LV-NLRP1-siRNA+1μM DEX group,APP+LV-control group,APP+LV-control+1μM DEX group.The release of LDH was detected by LDH kit;ROS production was explored by H₂DCFDA staining;Thioflavin S staining was used to detect Aβdeposition;PSD95 and Aβ1-42 expression were indicated by immunofluorescence staining;The expressions of NLRP1,ASC,Caspase-1,IL-1β,APP,BACE1,NCSTN,TAU,P-TAU and IL-6 proteins were detected by western blot.3.The primary hippocampal neurons of postnatal（24 h）APP/PS1 mice were identified by PCR.After 7 days of cell culture,the neurons were divided into six groups:APP group,APP+1μM DEX group,APP+2-APB（50μM）group,APP+2-APB（50μM）+1μM DEX group,APP+SKF-96365（50μM）group,APP+SKF-96365（50μM）+1μM DEX group.The release of LDH was detected by LDH kit;The[Ca²⁺]_ilevel was detected by calcium imaging;H₂DCFDA staining was used to detect ROS production;Thioflavin S staining was used to detect Aβdeposition;PSD95 and Aβ1-42 expression were indicated by immunofluorescence staining;The expressions of NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1,IL-1β,APP,BACE1,NCSTN,TAU,P-TAU,PLC,P-PLC,CN,NFAT1 and IL-6 proteins were detected by western blot.Results1.For DEX experiment,the results showed that compared with WT group,the primary hippocampal neurons in WT+1μM DEX group only had slightly damage.Compared with WT group,the primary hippocampal neurons in APP group were significantly damaged,APP group had increased LDH release and apoptosis,decreased PSD95expression,increased[Ca²⁺]_ilevel,increased ROS production,increased IL-6 and IL-1βrelease,and increased Aβdeposition.The expressions of APP,BACE1,NCSTN,NOX2,P22phox,P47phox,NLRP1,ASC,caspase-1 and IL-1βm RNAs were significantly increased.The expressions of APP,BACE1,NCSTN,TAU,P-TAU,PLC,P-PLC,CN,NFAT1,NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1,IL-1βand IL-6 proteins were significantly increased.Compared with APP group,the primary hippocampal neurons were significantly damaged after chronic DEX treatment,including increased LDH release and apoptosis,decreased PSD95 expression,increased intracellular calcium level,increased ROS production,increased IL-6 and IL-1βrelease,and increased Aβdeposition.The expressions of APP,BACE1,NCSTN,NOX2,P22phox,P47phox,NLRP1,ASC,Caspase-1 and IL-1βm RNAs were significantly increased.The expressions of APP,BACE1,NCSTN,TAU,P-TAU,PLC,P-PLC,CN,NFAT1,NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1,IL-1βand IL-6 proteins were significantly increased.2.For LV-NLRP1-siRNA assay,the results showed that compared with APP group,the primary hippocampal neurons were significantly damaged after chronic DEX treatment,APP+1μM DEX group had increased LDH release and apoptosis,decreased PSD95expression,increased IL-6 and IL-1βrelease,increased Aβdeposition.The expressions of APP,BACE1,NCSTN,TAU,P-TAU,NLRP1,ASC,Caspase-1,IL-1βand IL-6proteins were significantly increased.Compared with APP+1μM DEX group,LV-NLRP1-siRNA treatment significantly reduced chronic DEX-induced primary hippocampal neuronal damage,including reduced LDH release and apoptosis,increased PSD95 expression,down-regulated IL-6 and IL-1βrelease,and reduced Aβdeposition.The expressions of APP,BACE1,NCSTN,TAU,P-TAU,NLRP1,ASC,Caspase-1,IL-1βand IL-6 proteins were significantly decreased.3.For 2-APB and SKF-96365 experiments,the results showed that compared with APP group,the primary hippocampal neurons were significantly damaged after chronic DEX treatment,APP+1μM DEX group had increased LDH release and apoptosis,decreased PSD95 expression,increased IL-6 and IL-1βrelease,increased Aβdeposition.The expressions of APP,BACE1,NCSTN,TAU,P-TAU,PLC,P-PLC,CN,NFAT1,NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1,IL-1β and IL-6 proteins were significantly increased.Compared with APP+1μM DEX group,2-APB and SKF-96365treatment significantly reduced chronic DEX-induced primary hippocampal neuronal damage,including reduced LDH release and apoptosis,increased PSD95 expression,down-regulated IL-6 and IL-1βrelease,and reduced Aβdeposition.The expressions of APP,BACE1,NCSTN,TAU,P-TAU,PLC,P-PLC,CN,NFAT1,NOX₂,P22phox,P47phox,NLRP1,ASC,Caspase-1,IL-1βand IL-6 proteins were significantly decreased.ConclusionChronic GCs exposure might accelerate Aβaccumulation via activating Ca²⁺-mediated NFAT1-NOX2-NLRP1 signaling in primary hippocampal neurons of APP/PS1 mice,eventually promoting progression of AD.Down-regulation of NLRP1 prevents Aβaccumulation.More importantly,inhibiting[Ca²⁺]_iprotects against chronic DEX-induced hippocampal neuronal injury in APP/PS1 mice,which may be a potential therapeutic target for preventing chronic DEX-induced hippocampal neuronal injury.