Fluoxetine Inhibits NLRP3Inflammasome Activation

Fluoxetine, a selective serotonin reuptake inhibitors (SSRIs) antidepressant, iscommonly prescribed for treating major depression due to its tolerability and safety.It is well known that the therapeutic action of fluoxetine is ascribed to selectiveinhibition of presynaptic serotonin reuptake. Fluoxetine is also used for treatment ofother mental diseases such as anorexia nervosa, obsessive-compulsive disorders andpremenstrual anxiety disorders. However, the molecular and cellular mechanisms offluoxetine still remain unclear. Recent studies have found that fluoxetine has a widerange of pharmacological effects, such as neuroprotective, anti-tumor andanti-inflammatory. Several studies have shown that fluoxetine has anti-inflammatoryeffects in animal models of peripheral and central inflammation by inhibitingpro-inflammatory cytokines and activation of microglia.Major depression is a severe psychiatric disorder with lifetime prevalence inexcess of15%and is the fourth leading cause of disability worldwide. Several linesof evidence have demonstrated that the pro-inflammatory cytokine interleukin-1β(IL-1β) plays an important role in the etiology and pathophysiology of majordepression. Levels of IL-1β were elevated in patients with major depression orminor depression, which are correlated with the severity of depression. Exogenousadministration of IL-1β, either peripherally or directly into the brain, producesdepressive-like symptoms. In contrast, blockade of the IL-1receptor, inhibits thesestress-like effects. These findings suggest that IL-1β contributes importantly to theinduction of depressive symptomatology. Thus, controlling IL-1β production mayprove to be a therapeutic benefit in the treatment of depression. NLRP3inflammasome plays a key role in IL-1β secretion and inflammatoryresponse. Active, mature IL-1β is produced by cleavage of the inactive pro-IL-1βprecursor by caspase-1, which is activated in a large multi-protein complex called theinflammasome. The NLRP3inflammasome, composed of the Nod-like receptorprotein NLRP3(also called cyopyrin or NALP3), the adaptor protein ASC andcaspase-1, is vital for the production of mature IL-1β in response to a variety ofsignals. NLRP3can detect endogenous stress-associated danger signals, such asATP, monosodium urate crystals (MSU) or β-amyloid. Although reactive oxygenspecies (ROS), together with low cytoplasmic concentrations of potassium (K+), areknown to be prerequisites for activation of the NLRP3inflammasome, the signalingpathways that lead to NLRP3activation are still poorly understood. Therefore, inthis study, we sought to examine the pharmacological effect and molecularmechanism of fluoxetine on NLRP3inflammasome.AIM: To investigate the effect of fluoxetine on NLRP3inflammasome and illustrateits mechanism.METHODS: C57BL/6J mice were used to divide into three groups: control, CMSgroup, CMS+Flx (fluoxetine,10mg/kg per day, i.p) group, which NLRP3inflammasome components were detected by western blotting. Bone marrow-derivedmacrophages (BMDMs) were prepared from the femurs and tibias of C57BL/6miceof last three groups, and NLRP3inflammasome components were detected bywestern blot while IL-1β was detected by ELASA. RAW264.7macrophages or BV2cells were stimulated with LPS (100ng/ml) and then pulsed with5mM ATP for30min or250μg/ml MSU for6h. In the experiments, cells were pretreated with theindicated concentrations of fluoxetine1h before LPS stimulation. Cell extracts andprecipitated supernatants were analysed by immunoblot and ELISA. IntracellularROS was detected by molecular probe DCFH-DA; western blot was used to detectMAPK/p65/PKR phosphorylation. RESULTS:1. Fluoxetine inhibited CMS-induced NLRP3inflammasome activation inhippocampus and in BMDMsWe exploited the relationship between NLRP3inflammasome activation anddepression in vivo using animal model of depression and showed that fluoxetineeffectively suppressed CMS-induced NLRP3inflammasome activation in primarycultured BMDMs and in hippocampus, and attenuated CMS-induced depressiveresponses.2. Fluoxetine suppressed LPS+ATP/MSU-induced NLRP3inflammasomeactivationFluoxetine significantly reduced NLRP3inflammasome-mediated caspase-1activation and subsequent production of IL-1β in both peripheral RAW264.7macrophages and central BV2microglia cells after stimulation with ATP or MSU.3. Fluoxetine suppressed NLRP3inflammasome activation by inhibition ofROS production and PKR/MAPK phosphorylation in RAW264.7macrophageFluoxetine significantly reduced NLRP3inflammasome-mediated caspase-1activation and subsequent production of IL-1β in peripheral RAW264.7macrophages,which result from the inhibition of ROS production, PKR/MAPK phosphorylationand the interaction of PKR with NLRP3.CONCLUSIONS:1. NLRP3inflammasome are involved in the pathogenesis of depression, which maybe developed to potential therapeutic targets for depression.2. Our results indicate that the therapeutic effects of fluoxetine are partially mediatedby negatively modulating NLRP3inflammasome activation in central andperipheral systems.