Melatonin Protects Methamphetamine-Induced Neuroinflammation Through NF-κB and Nrf2 Pathways in Glioma Cell Line

Abstract

Methamphetamine (METH) is known as a toxin for neuronal and glial cells. Previous studies have found that METH-induced glial cell death and inflammation is mediated by oxidative stress. However, the exact mechanisms of the inflammatory response remain unclear. Therefore, we hypothesized that the activation of nuclear factor-κB (NF-κB) signaling, a key mediator of inflammation, and the inhibition of nuclear factor erythroid 2-related factor-2 (Nrf2) signaling, a regulator of the antioxidant response, would be significant events occurring in response to METH-induced inflammation in a rat glioma cell line (C6 cells). Our results show that METH increased the production of nitric oxide (NO) and up-regulated the expression of its main regulatory protein, inducible nitric oxide synthase (iNOS). METH also induced NF-κB activation by increasing inhibitory κBα (IκBα) degradation and translocation of the NF-κB (p65) subunit into the nucleus. Additionally, METH inhibited the activation of the Nrf2 pathway by decreasing the translocation of Nrf2 into the nucleus and also by suppressing the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO-1), and glutamate-cysteine ligase catalytic subunit (γ-GCLC), resulting in the suppression of superoxide dismutase (SOD) activity. Pretreatment with melatonin effectively promoted Nrf2 activation and reversed the METH-induced NF-κB response. Melatonin increased the expression of HO-1, NQO-1, and γ-GCLC, resulting in increased SOD activity. In addition, melatonin also decreased IκBα degradation, translocation of the p65 subunit, and expression of iNOS, resulting in decreased NO production. Taken together, our results indicate that melatonin diminishes the proinflammatory mediator in METH-stimulated C6 cells by inhibiting NF-κB activation and inducing Nrf2-mediated HO-1, NQO-1, and γ-GCLC expression.

Melatonin suppresses methamphetamine-triggered endoplasmic reticulum stress in C6 cells glioma cell lines

“…Our study demonstrated that melatonin protects glial cells against ER stress-induced cell death. Recent studies have demonstrated that METH is a neurotoxic drug that can induce neuronal (Ajjimaporn et al, 2005;Zhu et al, 2006;Deng et al, 2007) and glial cell death by several pathways, including oxidative stress (Riddle et al, 2006;Tocharus et al, 2008), mitochondrial dysfunction (Brown et al, 2005;Jumnongprakhon et al, 2014), excitotoxicity (Staszewski and Yamamoto, 2006;Ernst and Chang, 2008), inflammatory responses (Jumnongprakhon et al, 2015), and ER stress (Shah and Kumar, 2016). We have previously reported that exposure to METH causes an overproduction of ROS (Tocharus et al, 2010) that leads to oxidative stress.…”

“…Cells were pretreated with melatonin (1, 10, and 100 nM) for 2 hr in the presence or absence of METH (1.6 mM) for 9 hr as described in previous studies (Jumnongprakhon et al, 2014(Jumnongprakhon et al, , 2015. Cells were harvested, and the total RNA was isolated using TRIZOL reagent according to the manufacturer's protocol.…”

“…The cells were pretreated with melatonin at concentrations of 10, 100, and 500 nM for 2 hr prior to treatment with 1.6 mM METH (Jumnongprakhon et al, 2014(Jumnongprakhon et al, , 2015 for 9 hr or 12 hr and lysed in lysis buffer containing 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 40 mM β-glycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate and 1X protease inhibitors at 4°C for 15 min. The protein concentration was determined by using the Bradford protein assay (Bio-Rad Laboratories), and equal amounts of protein were electrophoresed in a 10-15% sodium dodecyl sulfate (SDS) polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) at 400 mA for 30 min.…”

“…Recent studies have suggested that METH is a neurotoxic drug that can cause pathological changes and death in neuronal and glial cells (Thompson et al, 2004;Kitamura et al, 2007Kitamura et al, , 2010Takeichi et al, 2012). METH induces glial cell hyperactivity and death via many pathways, including apoptosis, oxidative stress, inflammation, interruption of mitochondrial function, and endoplasmic reticulum (ER)-stress responses (Tocharus et al, 2010;Jumnongprakhon et al, 2014Jumnongprakhon et al, , 2015Shah and Kumar, 2016). METH also induces neuronal apoptosis in the striatal neurons of mice by disturbing the function of the ER, leading to ER stress (Riddle et al, 2006;Deng et al, 2007;Jayanthi et al, 2014;Wongpayoon and Govitrapong, 2016).…”

“…Our previous studies suggested that melatonin is a neuroprotective agent that can down-regulate the expression of not only reactive oxygen species (ROS) and nitric oxide (NO) but also pro-inflammatory cytokines (Tocharus et al, 2008(Tocharus et al, , 2010. Moreover, melatonin also attenuates METH-induced glial cell death by decreasing the inflammatory responses and the mitochondrial death pathway (Jumnongprakhon et al, 2014(Jumnongprakhon et al, , 2015. Therefore, this study will investigate the effect of METH-mediated glial cell death through the ER stress pathway in glioma cell lines and further evaluate whether inhibition of the ER stress/UPR is an underlying mechanism of the anti-apoptotic effects of melatonin.…”