Melatonin, minocycline and ascorbic acid reduce oxidative stress and viral titers and increase survival rate in experimental Venezuelan equine encephalitis

Highlights

• VEE virus replicates and induces oxidative stress in mouse brain.

• VEE virus replicates and induces oxidative stress in neuroblastoma cell cultures.

• Melatonin, minocycline and ascorbic acid reduce those parameters.

• Those compounds have potential therapeutic use in VEE infection.

Abstract

Venezuelan equine encephalitis (VEE) virus causes an acute central nervous system infection in human and animals. Melatonin (MLT), minocycline (MIN) and ascorbic acid (AA) have been shown to have antiviral activities in experimental infections; however, the mechanisms involved are poorly studied. Therefore, the aim of this study was to determine the effects of those compounds on the viral titers, NO production and lipid peroxidation in the brain of mice and neuroblastoma cultures infected by VEE virus. Infected mouse (10 LD₅₀) were treated with MLT (500 μg/kg bw), MIN (50 mg/kg bw) or AA (50 mg/kg bw). Infected neuroblastoma cultures (MOI: 1); MLT: 0.5, 1, 5 mM, MIN: 0.1, 0.2, 2 μM or AA: 25, 50, 75 μM. Brains were obtained at days 1, 3 and 5. In addition, survival rate of infected treated mice was also analyzed. Viral replication was determined by the plaque formation technique. NO and lipid peroxidation were measured by Griess׳ reaction and thiobarbituric acid assay respectively. Increased viral replication, NO production and lipid peroxidation were observed in both, infected brain and neuroblastoma cell cultures compared with uninfected controls. Those effects were diminished by the studied treatments. In addition, increased survival rate (50%) in treated infected animals compared with untreated infected mice (0%) was found. MLT, MIN and AA have an antiviral effect involving their anti-oxidant properties, and suggesting a potential use of these compounds for human VEE virus infection.

Introduction

Venezuelan equine encephalitis (VEE) virus produces an acute infection in human and murine central nervous system (CNS) with neuronal damage (Weaver, 1998, Schoneboom et al., 2000a, Schoneboom et al., 2000b). During an anti-viral immune response a balance between two opposing pathways is observed: the production of pro-inflammatory cytokines and cytotoxic cells to limit the viral infection and regulators to control the excessive immune response and avoid tissue damage. Nitric oxide (NO) can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection (Burrack and Morrison, 2014). In this regard, NO has been implicated in the experimental spread of Venezuelan equine encephalitis (VEE) virus and other viruses (Bonilla et al., 2004, Perrone et al., 2013, Mannick, 1995). The increased presence of NO in VEE infection is probably due to the activation of induced nitric oxide synthase (iNOS) (Valero et al., 2006, Schoneboom et al., 2000a, Schoneboom et al., 2000b). Oxidative stress and/or altered immune response could be involved in VEE viral infection. In this regard, pro-oxidant and immune modulator properties of NO have been reported (Burrack and Morrison, 2014, Mannick, 1995); therefore, approaches to inhibit the effect of NO on viral replication and oxidative stress need to be used appropriated compounds. Melatonin (MTL), a molecule with antioxidant properties, has shown to have protective effects on experimental murine VEE virus infection (Bonilla et al., 2003, Bonilla et al., 2004, Valero et al., 2007, Valero et al., 2009). In addition to anti-oxidant properties, MLT has the capability to regulate leukocyte function and contributes to the control of inflammation in tissues acting as both an activator or inhibitor of the inflammatory and immune responses (Silvestre and Rossi, 2013). MLT also decreases the toll like receptor mediated downstream gene expression and the subsequent NFkB-dependent gene expression, such as those encoding for TNF-α and iNOS produced during viral replication, preventing injury through the inhibition of the oxidative stress and of the production of pro-inflammatory cytokines (Huang et al., 2008). Minocycline (MIN), a tetracycline derivative, and ascorbic acid (AA) have shown antiviral activities in cell cultures and animal model of infections by Japanese encephalitis, human immunodeficiency and measles viruses (Dutta et al., 2010a, Dutta et al., 2010b, Bhaskar et al., 2012, Mishra et al., 2009, Zink et al., 2005). Beside the capacity to inhibit iNOS (Garrido-Mesa et al., 2013), minocycline combines both immunomodulatory and anti-microbial properties that can be neuroprotector. It has also been shown that MIN reduces virus infection and immune responses in several experimental models. This anti-viral effect seems to be linked to its ability to reduce the activation of monocytes and their permissiveness to viral infection. Anti viral mechanisms of MIN include, decreased p38 activation and viral replication in lymphocytes (Zink et al., 2005), reduced production of MCP-1 altering chemotaxis (Zink et al., 2005), blunting changes in activation/proliferation markers and cytokine secretion necessary for the activation pathway that regulate viral replication, decreasing viral production by inhibition of DNA integration or transcription (Si et al., 2004, Zink et al., 2005, Follstaedt et al., 2008, Ratai et al., 2010, Szeto et al., 2010). Several mechanisms of action have been proposed for the potential beneficial effect on viral infections of AA. The anti-oxidative ability of ascorbate, reducing oxidative stress could be important in detoxification and neutralization of reactive oxygen species associated with infection (Wintergerst et al., 2006). AA is also necessary for neutrophil function (Wintergerst et al., 2006) and can stimulate the production of interferon and other anti-viral cytokines, down regulate inflammation, and reduce the ability of several virus to infect cultured cells (Bissell et al., 1980, White et al., 1986, Gerber, 1975, Mandl et al., 2009, Mikirova et al., 2012, Furuya et al., 2008). These data suggest a potential beneficial effect of these compounds in VEE virus infection. Therefore, the aim of the present study was to analyze the effect of MLT, MIN and AA on the viral replication, NO production and lipid peroxidation in the brain of VEE virus infected mice and in infected neuroblastoma cell cultures.