Ethanol directly induced HMGB1 release through NOX2/NLRP1 inflammasome in neuronal cells

Highlights

• Ethanol directly induced HMGB1 release from neuronal cells.

• Ethanol-induced HMGB1 release was oxidative stress dependent.

• NOX2 played an important on in ethanol-induced HMGB1 release.

• Ethanol induced activation of NLRP1 inflammasomes in neuronal cells.

• Prevention activation of caspase 1 alleviated HMGB1 release

Abstract

It has been reported that ethanol contributes to neuronal damage. However, the mechanisms mediating the actions of ethanol on neurons remain elusive. The present study was designed to test whether ethanol directly induced HMGB1 release and to explore the cellular and molecular mechanism mediating its action. It was found that ethanol increased significant HMGB1 release from SH-SY5Y cells and from cultured primary cortical neurons as detected by ELISA assay. Meanwhile, ethanol induced the expression of NOX2 subunits such as gp91 and p47^phox and increased the activation of NLRP1 inflammasome. However, when cells were pretreated with NADPH oxidase inhibitor, apocynin, HMGB1 release was significantly decreased. Moreover, apocynin also prevented the activation of NLRP1 inflammasome as detected the levels of cleaved caspase-1. In addition, z-YVAD-fmk, an inhibitor of caspase-1, decreased the ethanol-induced HMGB1 release. It is concluded that ethanol-induced HMGB1 release is associated with NOX2/NLRP1 inflammasome signaling, which represents a novel mechanism of ethanol-associated neuron injury.

Introduction

Long-term alcohol consumption can cause disturbances to the organs of the body. It has been shown that ethanol can cross through the blood-brain barrier and directly induce neuronal injury (Imam, 2010). Therefore, alcohol consumption will result in significant alterations of brain structure and function (Kennedy et al., 1990). At last, cognition will be impaired and neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s diseases, will develop (Luo, 2014). However, the mechanisms underlying alcohol neurotoxicity remain largely elusive.

It has been shown that proinflammatory cytokines, such as TNF-α, IL-1, and IL-6, play key roles in the chronic ethanol-induced damage in liver, gastric mucosal and bowel (Gonzalez-Reimers et al., 2014). In recent years, many studies have reported that inflammatory mediators and cytokines are also increased in human post-mortem alcoholic brain, as well as following ethanol treatment of animals (Vetreno et al., 2013, Zou and Crews, 2012). Neuroimmune activation contributes to brain damage and behavioral changes associated with alcohol consumption (Crews et al., 2011). Moreover, studies have shown that cognitive deficit is associated with increased neuroinflammation (oxidative–nitrosative stress, TNF-α, IL-1β, and TGF-β1) in both cerebral cortex and hippocampus of ethanol-exposed rats (Tiwari and Chopra, 2012). As cell types of inflammation responding to injury in the brain, it has been long thought that microglial cells are the major CNS inflammatory cells, which are activated by injurious and infectious stimuli to produce inflammatory mediators that initiate and reinforce the inflammatory response. Moreover, studies have demonstrated that microglial cells play an important role in ethanol-induced neuroimmune activation, neurodegeneration and behavioral pathology (Fernandez-Lizarbe et al., 2013, Zhang et al., 2014b). However, recent studies have demonstrated that neurons also play an important role in mediating inflammatory stimulus (de Rivero Vaccari et al., 2014, Denes et al., 2012).

Recent studies have shown that high-mobility group box 1 protein (HMGB1) can be released from neurons after injury and may contribute to the initial stages of inflammatory response (Shin et al., 2014, Sun et al., 2014). HMGB1 protein is a ubiquitous widespread nuclear protein present in most cell types (Keyel, 2014). It is typically localized in the nucleus and functions as a nuclear cofactor in regulation of transcription. However, HMGB1 can also be present in the cytoplasm and be released into the extracellular matrix, where it has crucial roles in carcinogenesis and inflammation. Once secreted, HMGB1 is thought to drive inflammatory response (Andersson et al., 2014, Fang et al., 2012). Then HMGB1 induces signal transduction through the receptor RAGE (receptor for advanced glycation end products), and perhaps the toll-like receptors (TLR2 and TLR4) (Nadatani et al., 2013, Weber et al., 2015). It has been suggested that brain HMGB1 is highly expressed in neurons and is released from neurons (Sun et al., 2014). These findings are consistent with brain releasing HMGB1 that impacts neuronal signaling (Zhang et al., 2014a). Further studies support the hypothesis that HMGB1 protein is linked to ethanol-induced increase in expression of brain neuroimmune genes (Zou and Crews, 2014).

However, whether and how ethanol directly induces the release of HMGB1 from neuronal cells need to be further studied. An abnormal generation of reactive oxygen species (ROS) is thought to contribute to ethanol-induced inflammation (Jung and Metzger, 2010). ROS dramatically increase the ability of extracellular HMGB1 to activate the downstream transducing signals (Maugeri et al., 2014, Xie et al., 2014). It has also been shown that HMGB1 release is regulated and promoted by ROS production. NOX2 appears to be a major source of pathological oxidative stress in the central nervous system (CNS) (Fu et al., 2014, Nayernia et al., 2014). However, whether NOX2 regulates HMGB1 release remains unclear in ethanol-treated neuronal cells. This hypothesis was tested in this study.