Fig 1: Fluoxetine inhibited chronic mild stress (CMS)-induced NLRP3 inflammasome activation via suppression of the association RNA-dependent protein kinase (PKR) with NLRP3 in hippocampus of mice. (A) Sucrose preference (n=18/groups; * P<.05, # P<.05 vs CMS). (B) Tail suspension test (n=18/groups). (C) Forced swim test (n=18/groups). Representative immunoblots (D) and quantitative analysis of NLRP3 (E), caspase-1 (F) and IL-1β (G) in hippocampus of CMS mice (n=4/groups). (H) Representative immunoblots and quantitative analysis of phosphorylated and total PKR (n=4/groups). (I) Immunoprecipitation and immunoblot analysis of the PKR-NLRP3 interaction (n=4/groups). Data are represented as means ± SEM. * P<.05, ** P<.01 vs CMS.
Fig 2: Fluoxetine reduced ATP-induced ROS production, RNA-dependent protein kinase (PKR) phosphorylation and the association of PKR with NLRP3 in macrophages. Bone marrow-derived macrophages (BMDMs) were pretreated with fluoxetine (Flx, 10 μM) for 1 hour, then stimulation with LPS+ATP. (A) Cells were stained with H2DCF-DA labeling and images captured with a fluorescence microscope. (B) The quantitative analysis of ROS levels by a Titertek Fluoroskan II microtiter fluorescence plate reader. (C) LPS-primed BMDMs were stimulated with ATP at indicated time points and phosphorylated and total PKR were determined by Western-blot analysis (** P<.01, *** P<.001 vs LPS+ATP 30min). (D-E) BMDMs were pretreated with fluoxetine (Flx, 10 μM) for 1h, followed by stimulation with LPS+ATP for 30 minutes. (D) Representative immunoblots and quantitative analysis of phosphorylated and total PKR. (E) Immunoprecipitation and immunoblot analysis of the PKR-NLRP3 interaction. Data are represented as means ± SEM from 2 replicates in 3 independent experiments. ** P<.01, *** P<.001 vs LPS+ATP. There were no significant differences among -LPS-ATP, -LPS-ATP+Flx, and +LPS+ATP+Flx.
Fig 3: Schematic layout of showing fluoxetine inhibits NLRP3 inflammasome activation via ROS- RNA-dependent protein kinase-NLRP3 signaling pathway in depression. Fluoxetine decreases the level of ROS, which attenuates PKR phosphorylation and the interaction of PKR with NLRP3, and subsequently inhibits the NLRP3 inflammasome activation.
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