Melatonin promotes blood-brain barrier integrity in methamphetamine-induced inflammation in primary rat brain microvascular endothelial cells

Brain Research 1646 (2016) 182–192

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Melatonin promotes blood-brain barrier integrity in methamphetamine-induced inflammation in primary rat brain microvascular endothelial cells Pichaya Jumnongprakhon a, Piyarat Govitrapong b,c, Chainarong Tocharus a, Jiraporn Tocharus d,n a

Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Bangkok, Thailand Center for Neuroscience and Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand d Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b c

art ic l e i nf o

a b s t r a c t

Article history: Received 6 April 2016 Received in revised form 24 May 2016 Accepted 25 May 2016 Available online 4 June 2016

Melatonin is a neurohormone and has high potent of antioxidant that is widely reported to be active against methamphetamine (METH)-induced toxicity to neuron, glial cells, and brain endothelial cells. However, the role of melatonin on the inflammatory responses which are mostly caused by blood–brain barrier (BBB) impairment by METH administration has not been investigated. This study used the primary rat brain microvascular endothelial cells (BMVECs) to determine the protective mechanism of melatonin on METH-induced inflammatory responses in the BBB via nuclear factor-ĸB (NF-κB) and nuclear factor erythroid 2-related factor-2 (Nrf2) signaling. Herein, we demonstrated that melatonin reduced the level of the inflammatory mediators, including intercellular adhesion molecules (ICAM)-1, vascular cell adhesion molecules (VCAM)-1, matrix metallopeptidase (MMP)-9, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) caused by METH. These responses were related to the decrease of the expression and translocation of the NF-κB p65 subunit and the activity of NADPH oxidase (NOX)-2. In addition, melatonin promoted the antioxidant processes, modulated the expression and translocation of Nrf2, and also increased the level of heme oxygenase (HO)-1, NAD (P) H: quinone oxidoreductase (NQO)1, γ-glutamylcysteine synthase (γ-GCLC), and the activity of superoxide dismutase (SOD) through NOX2 mechanism. In addition, we found that the protective role of melatonin in METH-induced inflammatory responses in the BBB was mediated through melatonin receptors (MT1/2). We concluded that the interaction of melatonin with its receptor prevented METH-induced inflammatory responses by suppressing the NF-κB signaling and promoting the Nrf2 signaling before BBB impairment. & 2016 Elsevier B.V. All rights reserved.

Keywords: Blood–brain barrier Melatonin Methamphetamine Nf-κb Nrf2

1. Introduction Methamphetamine (METH) is the highly potent neurotoxin that is known worldwide to cause neurodegeneration by inducing dysfunction of neurons, glial cells, and the blood–brain barrier (BBB) (Taylor et al., 2013; Loftis and Janowsky, 2014; Cheng et al., 2015; Jumnongprakhon et al., 2014, 2015; Parameyong et al., 2015; Northrop and Yamamoto, 2015; Sun et al., 2015). The primary event of neurodegeneration that is caused by METH has been direct impairment of the BBB prior to mediation of the central nervous system (CNS) damage (Banks and Ericson, 2010). BBB impairment by METH has been reviewed in several mechanisms, n

Corresponding author. E-mail address: [email protected] (J. Tocharus).

http://dx.doi.org/10.1016/j.brainres.2016.05.049 0006-8993/& 2016 Elsevier B.V. All rights reserved.

including the hyperactivity of NADPH oxidase (NOX)-2 which generates excessive amounts of free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Ramirez et al., 2009; Park et al., 2012); the dysfunction of cytoskeleton and the trans-membrane protein of tight junction, which is the controlling of paracellular permeability (Park et al., 2013; Fernandes et al., 2015); the dysfunction of the uptake and the efflux activities (ElAli et al., 2012); and the activation of caspase cascade in cell death response or apoptosis (Abdul-Muneer et al., 2011; Ma et al., 2014; Fisehr et al., 2015). Moreover, overexpression of inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), interleukin (IL)-1, and tumor necrosis factor (TNF), which is an important factor in inflammatory response, has also been reported (Fernandes et al., 2014; CoelhoSantos et al., 2015; Parikh et al., 2015; Zhang et al., 2015; Skaper et al., 2014; Husain et al., 2015; Kothur et al., 2015).

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Previous studies have demonstrated that the release of cytokines causes progressive BBB impairment by the upregulation of leukocyte-endothelial cell adhesion molecules such as intercellular adhesion molecules (ICAM)-1, vascular cell adhesion molecules (VCAM)-1, and matrix metallopeptidase (MMP)-9 leads to tight junction impairment (Urrutia et al., 2013; Fernandes et al., 2014; Wang et al., 2014a, 2014b; Parikh et al., 2015). The impairment of tight junction of BBB reduces transendothelial electric resistance (TEER) values and the expression of tight junction proteins such as ZO-1, occludin, and claudin-5, leading to increased paracellular permeability (Mahajan et al., 2008; Rosas-Hernandez et al., 2013). The nuclear factor-ĸB (NF-κB) signaling is the major pathway that regulates the inflammatory cytokine expression and has been widely studied in inflammatory responses (Kawai and Akira, 2007). Previous studies have reported several stimuli such as lipopolysaccharide (LPS) and METH-induced inflammation in both neuronal cells and glial cells by the overactivation of NF-κB signaling (Flora et al., 2003; Shah et al., 2012; Wires et al., 2012; Perpoonpattana et al., 2013; Coelho-Santos et al., 2015; Jumnongprakhon et al., 2015). Therefore, the inhibition of these negative effects may be beneficial in protecting BBB impairment. Upon inflammation, the cells maintain homeostasis by regulating the defense system. The inflammatory defending system mostly mediates the phase II antioxidant enzymes, including heme oxygenase (HO)-1, NAD (P) H: quinone oxidoreductase (NQO)-1, and γ-glutamylcysteine synthase (γ-GCLC), superoxide dismutase (SOD), catalase (CAT) which is regulated by the nuclear factor erythroid 2-related factor-2 (Nrf2) mechanism (Huang et al., 2015). The activation of Nrf2 signaling triggers the Nrf2 protein to translocate into the nucleus and bind to the antioxidant response element (ARE) (Chen et al., 2015). Several reports have reviewed that the Nrf2 mechanism is the important target of toxin, including METH, prior to mediating the cellular stress, inflammation, dysfunction, and death (Pacchioni et al., 2007; Granado et al., 2011; Jumnongprakhon et al., 2015; Ramkissoon and Wells, 2013, 2015). Moreover, the Nrf2 could reduce the activity of NF-κB signaling (Li et al., 2008). Thus, the activation or protection of this system may increase BBB integrity. Melatonin (N-acetyl-5-methoxytryptamine) is mainly produced from the pineal gland that plays an important role of strong antioxidant, anti-inflammation, and anti-apoptosis, and it is widely accepted that it has high potential as regards protecting the CNS from toxic exposure via melatonin receptors (MT1/2), including exposure to METH. Moreover, melatonin has demonstrated that it could protect BBB by ischemic and hemorrhagic stroke, and sepsis or inflammatory responses to infection (Chen et al., 2006; Reiter et al., 2010; Tocharus et al., 2010; Chern et al., 2012; Acuna-Castroviejo et al., 2014; Garcia et al., 2014; Wang et al., 2014a, 2014b; Lacoste et al., 2015; Nguyen et al., 2015; Manchester et al., 2015; Zhao et al., 2015). According to its exerting effects, melatonin might be beneficial for protecting BBB in METH administration. In the present study, we hypothesized that melatonin might be able to attenuate METH-induced inflammatory responses in the BBB by suppressing the NF-κB signaling and promoting the Nrf2 signaling through NOX2 mechanism.

2. Results 2.1. Melatonin protect against METH-induced NF-κB mechanism via both NF-κB and NOX2 in BMVECs Our present study demonstrated that METH-induced the excessive expression of ICAM-1, VCAM-1, MMP-9, iNOS, and NO, and that it was also closely related with the activation of inflammation through NF-κB mechanism through NOX2 activity which is possible

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cause of BBB impairment. Pretreatment with melatonin could promote the BBB integrity that was impaired by METH. To elucidate the protective effect of melatonin on METH-induced BBB impairment as to whether it is directly against the NF-κB mechanism or the NOX2 activity, 10 mM of JSH23 (the selective inhibitor of NF-κB translocation) and 100 mM of apocynin (NOX2 inhibitor) were used in this experiment. The treatment with METH alone significantly increased the expression of ICAM-1, VCAM-1, and MMP-9 (Fig. 1(A)); iNOS (Fig. 1(B)); and NO (Fig. 1(C)), and these effects were significantly reversed by melatonin treatment (po0.001). Especially, JSH23 completely abolished the expression of these proteins, nearly to the basal level. Herein, it is clearly indicated that melatonin protects against METH-mediated expression of ICAM1, VCAM1, MMP-9, iNOS, and NO via directly suppressing the NF-κB mechanism and partially protecting via the NOX2 mechanism that might be involved with the increasing of BBB integrity. Next, we investigated the role of melatonin on the NF-κB expression and activity via directly inhibiting NF-κB or NOX2. The results showed that melatonin significantly reduced the translocation and expression of the p65 subunit in BMVECs, as shown in Figs. 1(D)–(F) (po0.001). The cotreatment of JSH23 with melatonin showed results similar to those of the control group. In the presence of the NOX2 inhibitor and by co-treatment with melatonin, the translocation of p65 significantly decreased, in comparison to METH treatment; however, the level was still higher than those in JSH23 treated group. These data suggested that NF-κB mechanism is the major pathway of melatonin to protect from METH-induced inflammation in BMVECs and that it is partly mediated by NOX2 activity. 2.2. Melatonin attenuates METH-suppressed Nrf2 mechanism via NOX2 in BMVECs One of the major factors that caused progressive inflammation in BBB by METH is the decreasing activity of the cellular defending system, especially the reduction of antioxidant enzymes such as glutathione peroxidase (GPx), SOD, HO-1, and NQO-1. Next, we determined the protective effect of melatonin on METH-reduced antioxidant enzymes regarding whether it is closely related to NFκB mechanism or NOX2 mechanism. METH treatment alone significantly decreased the expression of HO-1, NQO-1, GCLC, and SOD activity, respectively, as shown in Fig. 2(A)–(D), and these effects significantly increased in melatonin treatment (po0.001). As regards the inhibition of NF-κB, the METH treatment showed results similar to those of METH treated alone. In contrast, the apocynin treatment significantly increased the level of these proteins, similar to the control group (po0.001). Herein, these results indicate that melatonin protects against METH-reduced expression of HO-1, NQO-1, GCLC, and SOD activity via directly suppressing NOX2. Then, we investigated the role of melatonin whether it promoted Nrf2 expression and activity via directly inhibiting NF-κB or NOX2. The results showed that melatonin significantly increased the translocation and expression of Nrf2 in BMVECs compared to METH treated group, as shown in Figs. 2(E)–(G) (po0.001). The cotreatment of JSH23 with melatonin did not alter this effect when compared to METH treatment alone. On the other hand, co-treatment with apocynin and melatonin significantly increased this effect when compared to METH-treated cells. These data suggest that melatonin protects against METH-suppressed Nrf2 signaling in BMVECs mainly through NOX2 mechanism. 2.3. Melatonin diminishes nitrative stress, apoptosis, and cell integrity impairment by METH treatment via NF-κB but not oxidative stress in BMVECs As shown in Fig. 3, we next determined the important role of NF-κB mechanism in inducing cell death and impairment of tight

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Fig. 1. Melatonin protects against METH-induced inflammation via NF-κB signaling in BMVECs. The cells were prior treated with (10 mM) JSH23 or (100 mM) apocynin for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h prior to treatment in the presence or absence of METH (100 mM) for 24 h. Equal amounts of 50 mg protein were separated using electrophoresis and analyzed by western blotting for the expression of ICAM-1, VCAM-1, MMP-9 (A), iNOS (B), nuclear p65 subunit (D), cytosolic p65 (E), and total p65 subunit (F) and actin to normalize for the cytosolic fraction and lamin B1 to normalize for the nuclear fraction. The level of nitrite production was determined by Griess reaction (C). The values present the mean 7 SEM from three independent experiments. ***Po 0.001, in comparison with the control group; ### Po 0.001, in comparison with the METH group; $$$ Po 0.001, in comparison with the METH þ melatonin group.

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Fig. 2. Melatonin attenuates METH- suppressed anti-oxidant defense mechanism of Nrf2 signaling in BMVECs. The cells were prior treated with (10 mM) JSH23 or (100 mM) apocynin for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h prior to treatment in the presence or absence of METH (100 mM) for 24 h. Equal amounts of 50 mg protein were separated using electrophoresis and analyzed by western blotting for the expression of HO-1 (A), NQO-1 (B), GCLC (C), nuclear Nrf2 (E), cytosolic Nrf2 (F), and total Nrf2 (G) and actin to normalize for the cytosolic fraction and lamin B1 to normalize for the nuclear fraction. The SOD activity was determined using the SOD assay kit (C). The values present the mean 7SEM from three independent experiments. ***P o 0.001, in comparison with the control group; ###P o0.001, in comparison with the METH group.

junction by promoting oxidative and nitrative stress caused by METH in BMVECs. The results showed that METH-mediated the ROS level (Fig. 3(A)), RNS level (Fig. 3(B)), paracellular permeability (Fig. 3(D)), caspase-3 level (Fig. 3(E)), and percentage of apoptotic cells (Fig. 3(F)), and also in the attenuation of the TEER values (Fig. 3(C)). When pretreatment with melatonin prior treated with METH, it could prevent these negative responses. To investigate the protective role of melatonin on METH-induced BBB impairment whether involved with NF-κB mechanism. The co-treatment of JSH23 and METH resulted in the reduction of the RNS level

(Fig. 3(B)), paracellular permeability (Fig. 3(D)), caspase-3 level (Fig. 3(E)), and percentage of apoptotic cells (Fig. 3(F)), and also in the promotion of the TEER values (Fig. 3(C)) similar to those of the control group (p o0.001). However, the ROS level did not change in this treatment when compared to the treatment with METH alone. Thus, the impairment of the BBB caused by METH markedly activated the NF-κB mechanism. Moreover, pretreatment with JSH23 and melatonin or JSH23 alone prior to treatment with METH significantly reversed the negative effects when compared to METH treatment alone. This result suggests that melatonin

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Fig. 3. Melatonin protects against METH-induced BBB impairment via NF-κB signaling in BMVECs. The cells were prior treated with (10 mM) JSH23 for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h prior to treatment in the presence or absence of METH (100 mM) for 24 h. The ROS level was investigated by the CM-H2DCDA (A). The RNS level was investigated by the DAF-2DA (B). The formation of the tight junction was investigated using the TEER value by EVOM2 (C). The paracellular permeability was investigated using the FITC-Dextran intensity by permeability assay (D). The apoptosis determined the activity of the caspase-3 level (E) and the apoptotic cells (F) by flow cytometry. The values present the mean 7 SEM from three independent experiments. ***Po 0.001, in comparison with the control group; ### Po 0.001, in comparison with the METH group.

protects against METH-induced BBB impairment by directly suppressing the NF-κB mechanism. 2.4. Melatonin directly prevents METH-induced inflammation by interacted with its receptors (MT1/2) Finally, we determined whether the protective role of melatonin on METH-induced inflammation is mediated via the MT1/2 receptor. The results showed that pretreatment with luzindole, the antagonist of MT1/2 receptors, prior to treated with melatonin, completely ameliorated the effect of melatonin on NF-κB mechanism by significantly increasing ICAM-1, VCAM-1, MMP-9, iNOS, NO, nuclear p65, and total p65, as shown in Fig. 4 (p o0.001). Moreover, the activation of Nrf2 significantly decreased the expression of HO-1, NQO-1, GCLC, SOD activity, nuclear Nrf2, and total Nrf2 (Fig. 5, p o0.001). These findings suggest that melatonin protects against METH-induced inflammation in BMVECs is mediated via MT1/2 receptors and resulted in suppressing NF-κB and activating. 3. Discussion The co-culture of purified primary rat BMVECs and primary mixed glial cells was used as the BBB model in this study for

investigating the protective role of melatonin on METH-induced inflammation. This finding demonstrated that melatonin protected against METH-induced inflammation by directly inhibiting the NFκB signaling and promoting the Nrf2 via NOX2 mechanism. Importantly, the protective role of melatonin in METH-induced inflammation is via interaction with its receptor-mediated signaling. Short-term and long-term METH exposure leading to neurodegenerative diseases has been widely reported (Panenka et al., 2013; Radfar and Rawson, 2014; Yu et al., 2015). The toxicity of METH is manifested in the CNS by impairing the BBB which acts as a strong shield of the CNS through its mediation of cell stress, dysfunction, inflammation, and death (O'Shea et al., 2014; Northrop and Yamamoto, 2015). It is well known that NOX2 is the key regulator of several mechanisms in BBB impairment (Cahill-Smith and Li, 2014), but its role in inflammation is still unclear. Our results demonstrated that apocynin, the NOX2 inhibitor, partly down-regulated the expression of inflammatory mediators via NFκB signaling caused by METH. Based on this finding, we suggested that NOX2 is one of the regulators of NF-κB in METH-induced BBB impairment. NF-κB signaling is the cellular regulation of inflammation by generating several cytokines such as TNF-α, iNOS, and NO, and also the mediation of the expression of monocytes adhesion molecules which interrupt the function of the tight junction in brain endothelium such as ICAM1 and VCAM1.

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Fig. 4. Melatonin interacts with MT1/2 receptors against METH-induced NF-κB signaling activation. The cells were prior treated with luzindole (1 mM) for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h prior to treatment in the presence or absence of METH (100 mM) for 24 h. The western blotting analysis was used for determining the expression of ICAM-1, VCAM-1, MMP-9 (A), iNOS (B), nuclear p65 subunit (D), cytosolic p65 (E), and total p65 subunit (F) and actin to normalize for the cytosolic fraction and lamin B1 to normalize for the nuclear fraction. The level of nitrite production was determined by Griess reaction (C). The values present the mean 7 SEM from three independent experiments. ***Po 0.001, in comparison with the control group; ###Po 0.001, in comparison with the METH group. $$$P o 0.001, in comparison with the melatonin and METH treatment group.

Moreover, this signaling could promote extracellular matrix enzymes such as MMP-2 and MMP-9 for degrading the laminin and collagen type IV in basal lamina (Csiszar et al., 2008; Zhang et al., 2009; Saito et al., 2013; Yuan et al., 2015). The blockage of this signaling could promote BBB integrity by preserving the tight junction. Our study showed that inhibition of the NF-κB or the NOX2 activity, METH did not activate the expressions of ICAM-1, VCAM-1, MMP-9, iNOS, and NO. Similar results were observed in melatonin-treated cells. However, the decreasing of these responses in NOX2 inhibition was not completely inhibited as compared to the NF-κB inhibition group. Thus, these results suggest that melatonin protects from METH-induced inflammation by directly inhibiting the NF-κB pathway and partly inhibiting the NOX2 mechanism. As previous studies have reviewed, the inflammation which triggers the NF-κB signaling was mediated via several mechanisms including NOX2, toll-like receptors (TLRs)-4, and α7-nicotinic acetylcholine receptors (α7-nAChR) (Gloire et al., 2006; Maloney et al., 2009; Gray and Jandeleit-Dahm, 2015; Zhang et al., 2015). From the result, the partial inhibition of NOX2 on NFκB signaling might be co-activated with other mechanisms, especially the α7-nAChR which has been previously reported to have a role in inflammation due to BBB impairment that is caused

by METH administration (Zhang et al., 2015). Inhibition of the NF-

κB activity, METH failed to induce BBB impairment by a decrease

in the oxidative damage and the apoptosis, thereby increasing the BBB integrity, as observed in the increasing of the TEER value and the decreasing of the paracellular permeability. Based on these data, we suggest that NF-κB signaling is the major target of melatonin to modulate BBB function caused by METH. However, the inhibition of NF-κB did not completely decrease the ROS level, which was mostly generated by NOX2 (Park et al., 2012; GarridoUrbani et al., 2014; Xiao et al., 2015). Thus, the inhibition of NF-κB was not the only critical factor in the protection of the BBB. The advantage of melatonin, as this study demonstrated, is the protective effect both on NOX2 mechanism and NF-κB prior to reducing METH toxicity; as a result, it was useful for the protection of the BBB. We further evaluated the protective role of melatonin on METH-induced inflammation via NF-κB regarding whether it required its receptors. The results showed that with luzindole (a non-selective antagonist MT1/2 receptor) pretreatment, melatonin failed to inhibit the translocation of the p65 subunit of NF-κB and the expression of ICAM-1, VCAM-1, MMP-9, iNOS, and NO. Based on this finding, we suggested that melatonin required its receptor for abolishing the inflammation via NF-κB signaling.

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Fig. 5. Melatonin interacts using MT1/2 receptor-mediated Nrf2 signaling in METH treatment. The cells were prior treated with (1 mM) luzindole for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h prior to treatment in the presence or absence of METH (100 mM) for 24 h. The western blotting analysis was used for determining the expression of HO-1 (A), NQO-1 (B), GCLC (C), nuclear Nrf2 (E), cytosolic Nrf2 (F), and total Nrf2 (G) and actin to normalize for the cytosolic fraction and lamin B1 to normalize for the nuclear fraction. The SOD assay kit was utilized to determine the SOD activity level (C). The values present the mean 7 SEM from three independent experiments. ***Po 0.001, in comparison with the control group; ###Po 0.001, in comparison with the METH group. $$$Po 0.001, in comparison with the melatonin and METH treatment group.

As regards inflammatory responses, the cell has its defending mechanism of mediating antioxidant enzymes for protecting against cell stress, dysfunction, inflammation, and death that caused by exposure to toxins (Huang et al., 2015). Previous studies have established that METH caused brain damage by interrupting or reducing antioxidant enzymes, including SOD, catalase (CAT), GPx, HO-1, NQO-1, GCLC, etc. Moreover, the depletion of these enzymes through reduced production induced progressive stress and inflammation of cells prior to dysfunction and death (Kensler et al., 2007; Osburn and Kensler, 2008; Sykiotis et al., 2011; Miyazaki et al., 2011; Perpoonpattana et al., 2013; Huang et al., 2015; Jumnongprakhon et al., 2015). In this study, we found that melatonin could promote the activity of SOD and the expression of HO-1, NQO-1, and GCLC in BMVECs. Next, we proceeded to

investigate the involvement of NF-κB or NOX2 in the protective effect of melatonin in METH-induced inflammation in the BBB. Inhibition of NF-κB signaling pathway, METH did not promote the activity of SOD and the expression of HO-1, NQO-1, and GCLC; so, we suggested that the reduction of the antioxidant enzyme by METH did not involve NF-κB signaling. On the other hand, inhibition of NOX2 activity, METH caused promotion of the SOD activity and expression of HO-1, NQO-1, and GCLC were up-regulated; therefore, we suggest that METH suppressed antioxidant activity via NOX2 mechanism. Subsequently, we investigated the protective role of melatonin in the promotion of antioxidant enzymes and investigated whether it involved with NOX2. Next, the cells were subjected to co-treatment of apocynin with melatonin and METH, which caused the promotion of the SOD activity and

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the expression of HO-1, NQO-1, and GCLC, which is similar to the co-treatment of apocynin with METH. Based on this finding, we suggest that the protective role of melatonin which promotes the antioxidant enzyme activity and expression is closely related to NOX2 mechanism. As demonstrated in a previous study, it has been established that antioxidant enzymes are mostly regulated by Nrf2 signaling, and they reported that a decrease in Nrf2 activation causes depletion of antioxidant enzymes (Perpoonpattana et al., 2013; Jumnongprakhon et al., 2015). In agreement with those reviews, it was observed that METH exposure in BMVECs decreased the expression and translocation of Nrf2 in relation with the low expression of HO-1, NQO-1, and GCLC and SOD activity. Moreover, we found that a decrease in Nrf2 activity did not implicate NF-κB signaling but it was closely involved with NOX2. Importantly, melatonin could promote the expression and Nrf2 activity, and this promotion was closely involved with NOX2 devoid of NF-κB signaling. In contrast, a few studies have demonstrated that the inhibition of NF-κB signaling could promote Nrf2 activity (Wijayanti et al., 2004; Pinkaew et al., 2015). Thus, we suggest that the activation of Nrf2 in the brain endothelial cells may be closely related with NOX2 signaling rather than NF-κB signaling because the NF-κB signaling may be a parallel cascade during inflammation. We also found that in the presence of luzindole, melatonin failed to promote the Nrf2 expression and activity including the expression of HO-1, NQO-1, GCLC, and SOD activity caused by METH. Thus, these data suggest that the protective role of melatonin in METH-induced suppressing of Nrf2 signaling requires the interaction of melatonin and its receptor. 3.1. Conclusions We concluded that the interaction of melatonin with its receptor (MT1/2) protected against BBB impairment caused by METH by directly inhibiting the NF-κB signaling and modulating the Nrf2 signaling via NOX2 mechanism (Fig. 6). Thus, melatonin might be beneficial in protecting the BBB from inflammation caused by METH or other pathogens.

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anti-p65 subunit, anti-lamin B1, Muse Annexin V & Dead Cell Kit, s and Muse Caspase-3/7 Kit were purchased from Merck Millipore (Millipore, MA, USA). The following antibodies were used for the western blot analysis: anti-Nrf2, anti-HO-1, anti-NQO-1, anti-GCLC, anti-ICAM-1, anti-VCAM-1, and anti-MMP-9 (Abcam, Cambridge, UK); and anti-mouse IgG peroxidase-conjugated secondary antibody and anti-rabbit IgG peroxidase-conjugated secondary antibody (Millipore, MA, USA). The superoxide dismutase assay kit was purchased from Cayman Chemical Company (Cayman, MI, USA). 4.2. Primary rat brain microvascular endothelial cells (BMVECs) isolation and culture The isolation of the BMVECs was performed according to the protocol from Liu et al. (2013). Briefly, three whole brains of 10day-old neonatal rats without meninges and large blood vessel were removed, and then were minced into small pieces in a prechilled phosphate buffer saline (PBS) and centrifuged at 1000 rpm at 4 °C for 5 min. Then, the pellets were homogenized in 25% bovine serum albumin (BSA) and centrifuged at 3000 rpm at 4 °C for 10 min. The microvessel pellets were digested with 0.1% collagenase type II (Sigma, St. Louis, MO, USA) for 30 min at 37 °C. Then, the digested microvessels were resuspended in DMEM/F12 and centrifuged at 1000 rpm for 5 min. The precipitate layer was collected and resuspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 3 mg/ml glucose, 0.5 mM glutamine, 3 mM puromycin, and penicillin/streptomycin. The cell suspensions were cultured at 37 °C in a humidified atmosphere of 5% CO2 for 3 days. After the growth of the BMVECs for 90–100% confluence, the cells were passaged with 0.25% trypsin:EDTA and resuspended in DMEM/F12 supplemented with 10% FBS, 3 mg/ml glucose, 0.5 mM glutamine, and penicillin/streptomycin in 37 °C in a humidified atmosphere of 5% CO2. The anti-Von Willebrand factor VIII and the anti-α actin antibody were stained on the cells for the confirming of brain endothelial cells and observed under a fluorescent microscope (Olympus, Tokyo, Japan) (Data not shown). Protocols were performed in compliance with the Guide for the Care and Use of Laboratory Animal (Chiang Mai University) and were approved by Animal Care and Use committee.

4. Experimental procedure 4.3. Primary mixed glial cells isolation and culture 4.1. Reagents and chemicals Melatonin, luzindole, JSH23, apocynin, 4 kDa FITC-dextran, and myeloperoxidase (MPO) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Anti-β-actin, anti-iNOS,

Primary mixed glial cells were prepared following a protocol based on the Mccarthy and De Vellis (1980). Briefly, whole brains of three neonatal rats that were 0–2 days old were removed. After that, the brains were homogenized and centrifuged at 3000 rpm for 10 min. The pellets were resuspended in glial cell maintenance media (DMEM/F12 supplemented with 10% FBS, 0.5 mM glutamine, and penicillin/streptomycin) and cultured at 37 °C in a humidified atmosphere of 5% CO2. The anti-GFAP was stained on the cells for the confirmation of glial cells, and observed under a fluorescent microscope (Data not shown). 4.4. Cultivation of BBB model in vitro

Fig. 6. The interaction of melatonin with its receptor-mediated signaling prevents METH-induced inflammation by suppressing NF-κB and modulating Nrf2 signaling via NOX2 mechanism.

The BBB model was cultivated by co-culturing of the purified BMVECs with primary mixed glial cells. Briefly, the primary mixed glial cells were firstly maintained in 24-well plates at 37 °C in a humidified atmosphere, at 5% CO2, for 7 days. The purified BMVECs were then cultured on collagen/fibronectin (Sigma, St. Louis, MO, USA) coated-inserted 0.4 mm trans-wells and maintained at 37 °C in a humidified atmosphere, at 5% CO2, until 100% confluence was reached. The suitable representation of the BBB model in vitro also confirmed the transendothelial electric resistance (TEER) values by the EVOM2 voltometer with STX-2 electrodes, TEER 4 200 Ω cm2 (Millipore, Inc., MA, USA). Prior to

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investigate in all the experiments, the primary mixed glial cells were discarded and switched the culturing media to media free serum. 4.5. Western blot analysis To perform the western blot analysis, the cultured BMVECs were maintained in collagen/fibronectin coated-inserted transwells of 24-well plates at a density of 1  105 cells/ml at 37 °C until TEER 4200 Ω cm2. The cells were prior treated with apocynin (100 mM) or JSH23 (10 mM) for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h, and then incubated in the presence or absence of METH (100 mM, the cytotoxicity dose which confirming by MTT assay) (Data not shown) for 24 h. The cells were harvested and lysed for the subcellular fraction of cytosolic and nuclear proteins, as described previously (Jumnongprakhon et al., 2015). Briefly, the cytosolic protein was prepared by a hypotonic lysis buffer containing 10 mM HEPES (pH 7.9), 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and a cocktail of protease inhibitors. The nuclear protein was prepared by lysing the pellets after cytosolic preparation with the hypertonic extraction buffer containing 10 mM HEPES (pH 7.9), 0.42 M sodium chloride, 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and a cocktail of protease inhibitors. The Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine the protein concentration. Equal amounts of 50 mg proteins were separated with the 10–15% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA, USA). The membranes were then probed overnight with anti-iNOS, anti-p65, anti-ICAM-1, antiVCAM-1, anti-MMP-9, anti-Nrf2, anti-HO-1, anti-NQO-1, and antiGCLC (1:2000). Then, the blotted cells were then incubated with anti-mouse and anti-rabbit IgG peroxidase-conjugated secondary antibodies (1:4000) (Millipore, MA, USA). Finally, the Immobilon Western HRP substrate (Millipore, MA, USA) was used to incubate the blots prior to exposure to an X-ray film. The densitometry was s analyzed by using the Image-J software. The β-actin was used to normalize the cytosolic fraction and lamin B1 was used to normalize the nuclear fraction.

4.8. ROS and RNS assay The cultured BMVECs were maintained in 96-well microplates at a density of 1  105 cells/ml at 37 °C for 24 h. The cells were prior treated with JSH23 (10 mM) for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h, and then treated in the presence or absence of METH (100 mM) for 24 h. After the cells were exposed to the condition, the incubation solutions of the 20 mM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and the 10 mM 4,5-diaminofluorescein diacetate (DAF-2DA) in PBS for 2 h at 37 °C in the dark were used to determine the ROS and the RNS levels, respectively. The fluorescence values were then measured at excitation/emission wavelengths of 485/535 nm by using a Synergy H4 microplate reader (Biotek, VT, USA). 4.9. TEER measurement and paracellular permeability assay The cultured BMVECs were maintained in collagen/fibronectin coated-inserted trans-wells of 24-well plates at a density of 1  105 cells/ml at 37 °C until TEER 4 200 Ω cm2. The cells were prior treated with JSH23 (10 mM) for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h and then treated in the presence or absence of METH (100 mM) for 24 h. The TEER values were determined by the EVOM2 voltometer with STX2 electrodes for triplication in each group. The paracellular permeability was determined as the fluorescence intensity of FITCdextran. Briefly, 5 mg/ml 4 kDa FITC-dextran in a reaction buffer containing 122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.2 mM MgSO4, 0.4 mM K2HPO4, 1.4 mM CaCl2, 10 mM HEPES, and 10 mM glucose was added to the upper chamber at 37 °C in the dark for 2 h, and the lower chamber media was then used to determine the fluorescence intensity at an excitation/emission wavelength of 488/525 nm by using a Synergy H4 microplate reader. 4.10. Apoptosis and caspase-3 detection assay by flow cytometry

After the cells were exposed to the condition with the phenol red free media, the culture media were collected and the nitrite production determined by Griess reaction assay. An equal volume of the supernatant reacted with an equal volume of a mixture of 1% sulfanilamide in 5% phosphoric acid for 5 min and 0.1% N-(1napthyl) ethylenediamine hydrochloride for 5 min in the dark at room temperature. The absorbance was measured using a microplate reader at 540 nm. The known concentrations of sodium nitrite (NaNO2) were used as the standard curve.

The cultured BMVECs were maintained in collagen/fibronectin coated-inserted trans-wells of 24-well plates at a density of 1  105 cells/ml at 37 °C until TEER 4 200 Ω cm2. The cells were prior treated with JSH23 (10 mM) for 1 h. Then, the cells were pretreated with melatonin (1, 10, and 100 nM) for 2 h, and then treated in the presence or absence of METH (100 mM) for 24 h. Then, the cells were trypsinized and resuspended in culture media at a concentration of 1  104 cells/100 ml. For the determination of the apoptotic cells, the cells were incubated with the reaction assay of Muse™Annexin-V and the dead cell assay kit (EMD Millipore Biosciences) for 20 min in the dark at room temperature. For the determination of the caspase-3 levels, the cells were incubated with caspase-3/7 working solutions (EMD Millipore Biosciences) for 10 min in the dark at room temperature. Finally, the percentage of the apoptotic cells and the caspase-3 level were analyzed by using Muse Cell Analyzer (Merck Millipore, MA, USA), according to the manufacturer's instruction.

4.7. Superoxide dismutase activity assay

4.11. Statistical analysis

After the cells were exposed to the condition, the cells were harvested and lysed with ice-cold lysis buffer containing1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 40 mM βglycerophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate, and a cocktail of protease inhibitors. The SOD activity was determined using an SOD assay kit (Cayman Chemical Company, MI, USA), according to the manufacturer's protocol, which utilized a tetrazolium salt for the detection of the superoxide radicals generated by xanthine oxidase and hypoxanthine.

The data are expressed as mean 7SEM of three independent experiments. The statistical difference was analyzed using oneway analysis of variance (ANOVA) followed by Post Hoc Dunnett's test for comparing the significance between the individual groups (p o0.05).

4.6. Griess reaction assay

Disclosure of interest The authors declare no conflict of interest.

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Acknowledgments This work was supported by the CMU Mid-Career Research Fellowship Program, Chiang Mai University (Grant no. 2558), Thailand, as well as by a research Grant from TRF (DPG 5780001) and Mahidol University (Grant no. IRG5780009) to PG.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres.2016.05. 049.

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