Fluoxetine protects against IL-1β-induced neuronal apoptosis via downregulation of p53

Highlights

• Fluoxetine alleviated neuronal injury and p53 increase in a mouse model of tMCAO.

• Fluoxetine attenuated IL-1β-induced apoptosis and p53 elevation in cortical neurons.

• Downregulation of p53 was required for the neuroprotective effects of fluoxetine.

• Fluoxetine down-regulated p53 expression via a p38-dependent pathway.

Abstract

Fluoxetine, a selective serotonin reuptake inhibitor, exerts neuroprotective effects in a variety of neurological diseases including stroke, but the underlying mechanism remains obscure. In the present study, we addressed the molecular events in fluoxetine against ischemia/reperfusion-induced acute neuronal injury and inflammation-induced neuronal apoptosis. We showed that treatment of fluoxetine (40 mg/kg, i.p.) with twice injections at 1 h and 12 h after transient middle cerebral artery occlusion (tMCAO) respectively alleviated neurological deficits and neuronal apoptosis in a mouse ischemic stroke model, accompanied by inhibiting interleukin-1β (IL-1β), Bax and p53 expression and upregulating anti-apoptotic protein Bcl-2 level. We next mimicked neuroinflammation in ischemic stroke with IL-1β in primary cultured cortical neurons and found that pretreatment with fluoxetine (1 μM) prevented IL-1β-induced neuronal apoptosis and upregulation of p53 expression. Furthermore, we demonstrated that p53 overexpression in N2a cell line abolished the anti-apoptotic effect of fluoxetine, indicating that p53 downregulation is required for the protective role of fluoxetine in IL-1β-induced neuronal apoptosis. Fluoxetine downregulating p53 expression could be mimicked by SB203580, a specific inhibitor of p38, but blocked by anisomycin, a p38 activator. Collectively, our findings have revealed that fluoxetine protects against IL-1β-induced neuronal apoptosis via p38-p53 dependent pathway, which give us an insight into the potential of fluoxetine in terms of opening up novel therapeutic avenues for neurological diseases including stroke.

Introduction

Previous studies have demonstrated that neuroinflammation is one of the key factors responsible for the pathogenesis of many neurological disorders, including ischemic stroke (Kim et al., 2015a), neurodegenerative diseases (Tuon et al., 2015), and depression (Alcocer-Gomez et al., 2015). There are accumulative evidences that several inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), contribute directly to diverse forms of neuronal injury (Rothwell, 2003). Inhibiting inflammation has currently being considered as a promising neuroprotective strategy for treatment of stroke (Murray et al., 2015), epilepsy (Tao et al., 2015) and Alzheimer's disease (AD) (Sheng et al., 1996). Therefore, seeking potential therapeutic targets and effective neuroprotectants against inflammation-induced neuronal injury is crucial for the treatment of neurological diseases.

Fluoxetine, a classical selective serotonin reuptake inhibitor (SSRI), plays a crucial role in anti-inflammation, anti-tumor and neuroprotection apart from the effect on serotonin (5-HT) (Ghosh et al., 2015). Fluoxetine is widely used as the treatment of a wide variety of psychiatric disorders, including depression (Sakr et al., 2015), bipolar disorder (Detke et al., 2015), anxiety disorders (Gupta et al., 2015). Recently, fluoxetine has been demonstrated several beneficial effects on ischemic stroke patients (Chollet et al., 2014) as well as several animal models of stroke (Lim et al., 2009, Ng et al., 2015, Schaz et al., 2011, Singh and Chopra, 2014). For example, fluoxetine reduces infarct volume and neurological deficits in a rat acute ischemic stroke (Lim et al., 2009). It also reduces TNF-α, IL-1β, and nuclear factor-κB (NF-κB) levels, and protects the loss of biogenic amines in brain tissue of ischemic rats (Singh and Chopra, 2014). Therefore, the protection of fluoxetine against cerebral ischemic injury may be associated with it's anti-inflammatory role. However, the exact mechanism for fluoxetine inhibiting neuroinflammation-induced neuronal death remains unknown.

p53, as a tumor suppressor protein, is involved in numerous signaling pathways including cell cycle arrest, apoptosis, senescence, and DNA repair (Wang et al., 2014a). Currently, p53 is recognized as an important pro-apoptotic factor in the progress of neurological disorders, such as stroke (Zhai et al., 2014), depression (Ruan et al., 2015), and epilepsy (Engel et al., 2013), neurodegenerative diseases (Singh and Pati, 2015). For example, p53 induces neuronal apoptosis via death-associated protein kinase 1 (DAPK1) pathway in ischemic brain injury (Wang et al., 2014b). Inflammatory cytokines, especially IL-1β, induce NF-κB nuclear translocation, increasing p53-upregulated modulator of apoptosis (PUMA). Activated PUMA liberates p53 from Bcl-X_L, causing Bax activation, and leading to apoptosis eventually (Suzuki et al., 2009). Based on this, we made a hypothesis that p53 may be involved in the protection of fluoxetine against inflammation-induced neuronal apoptosis.

In the present study, we performed serial experiments in vivo and in vitro to prove this proposal. We found that treatment of fluoxetine reduced neuronal apoptosis and suppressed the upregulation of IL-1β and p53 expression in transient middle cerebral artery occlusion (tMCAO) model of mice. Consistently, in vitro studies showed that fluoxetine prevented IL-1β-induced apoptosis and p53 upregulation in primary cortical neurons. Furthermore, by use of Neuroblastoma N2a cell line, we demonstrated that p53 downregulation mediated the neuroprotective effects of fluoxetine and p38 phosphorylation contributed to the p53-dependent neuronal apoptosis. This finding has provided an insight into a novel anti-inflammatory mechanism of fluoxetine that may contribute to the use of fluoxetine therapy for ischemic stroke and other neuronal injured diseases.