The Protective Role Of Fluoxetine Against BV2 Microglia Pyroptosis Induced By Aβ_25-35 And The Mechanisms

Objective:Pyroptosis is a new programmed inflammatory cell death that is implicated in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease（AD）,which mainly depends on the activation of NLRP3 inflammasome.Clinical studies have confirmed that fluoxetine could delay the process of AD and exert anti-inflammatory,anti-oxidative and neuroprotective effects in depression therapy.However,whether fluoxetine benefit AD development via inhibiting pyroptosis is still unknown.In this study,we used amyloid-βpeptide(Aβ_25-35)-stimulated BV2 microglia to simulate an inflammatory environment of AD and investigate the protective role of fluoxetine on AD neuroinflammation and its underlying mechanism,especially the role of NLRP3inflammasome activation and microglia pyroptosis.Methods:1.BV2 cells were treated with different concentrations of fluoxetine for 12h,24h,36h and 48h,cell viability was detected by CCK-8 assay to determine the optimal intervention concentration（20μM）and time（24h）of Aβ_25-35.2.BV2 cells were divided into a spectrum of groups:controls,Aβ_25-35（20μM）,Aβ_25-35+low-dose（5μM）fluoxetine,Aβ_25-35+medium-dose（10μM）fluoxetine,Aβ_25-35+high-d ose（10μM）fluoxetine,Aβ_25-35+10μM disulfiram（the GSDMD inhibitor）.Pyroptosis was assessed by LDH assay,Annexin V/FITC/PI staining assay and transmission electronmicroscopy.Western blot was used to analyze the expression of inflammasome and pyroptosis-associated proteins（NLRP3,ASC,caspase-1,cleaved-caspase-1,GSDMD）;ELISA assay and DCFH-DA fluorescence staining were used to detect cytokines expressions and ROS generation respectively.Results:1.Fluoxetine reversed the decline of cell viability induced by Aβ_25-35:compared with the untreated controls,the cell viability of Aβ_25-35group was significantly decreased（P<0.01）,and pretreatment with fluoxetine（10,20μM）for 2h increased the BV2 cell viability（P<0.05）.2.Fluoxetine relieved Aβ_25-35-induced pyroptosis in BV2 cell:compared with the Aβ_25-35group,pretreatment with high-dose（20μM）fluoxetine reduced the release of lactate dehydrogenase（LDH）（P<0.05）.Fluoxetine（20μM）alleviated the morphological change and cell pyroptosis（P<0.01）induced by Aβ_25-35in BV2 cells.3.Fluoxetine alleviated Aβ_25-35-induced NLRP3 inflammasome activation:compared with the Aβ_25-35group,high dose of fluoxetine（20μM）downregulated the expression of inflammasome-associated proteins（NLRP3,ASC,caspase-1,activated caspase-1,GSDMD）（P<0.01）,and pretreatment with fluoxetine（5,10,20μM）decreased the release of IL-1βand IL-18 induced by Aβ_25-35（P<0.05）.4.Fluoxetine inhibited the production of ROS in Aβ_25-35-induced BV2 cells:compared with the control group,the production of ROS significantly increased in the Aβ_25-35group（P<0.05）,which could be effectively reversed by fluoxetine in a dose-dependent way（P<0.05）.Conclusion:The present study demonstrated that fluoxetine ameliorated Aβ_25-35-induced ROS production,NLRP3 inflammasome activation and pyroptosis in BV2 cells.Our findings indicate that fluoxetine might play a neuroprotective role in AD via inhibiting neuroinflammatory reactions,which might be a promising therapeutic target for AD.