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b-CARYOPHYLLENE PROTECTS THE C6 GLIOMA CELLS AGAINST GLUTAMATE-INDUCED EXCITOTOXICITY THROUGH THE NRF2 PATHWAY L. C. ASSIS, a M. R. STRALIOTTO, a D. ENGEL, a M. A. HORT, a R. C. DUTRA b,c*  AND A. F. DE BEM a 

cytoprotective effects of BCP were mediated by the amelioration of cellular antioxidant responses via Nrf2 activation, which is, in part, dependent on cannabinoid receptor type 2 (CB2R) activation. This functional nonpsychoactive CB2R ligand, could represent an important molecule for protection of glial cells against oxidative stress induced by glutamate. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a Departamento de Bioquı´mica, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil b Laborato´rio de Autoimunidade e Imunofarmacologia, Campus Ararangua´, Universidade Federal de Santa Catarina, Ararangua´, SC, Brazil c Departamento de Farmacologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal de Santa Catarina, Ararangua´, SC, Brazil

key words: b-caryophyllene, C6 cell line, glutamate, Nrf2 pathway, CB2R.

Abstract—b-Caryophyllene (BCP), a natural bicyclic sesquiterpene present in several essential oils, displays analgesic and anti-inflammatory properties in vitro and in vivo. Astrocytes are a major class of glial cells that regulate extracellular ion balance, repair and scarring processes in the CNS following neuroinflammatory conditions and traumatic injuries. This study sought to determine the protective effect of BCP against glutamate (Glu)-induced cytotoxicity in the C6 glioma cell line on neurochemical parameters as well as their biochemical mechanism. Glu increases intracellular reactive oxygen species (ROS) production and induces mitochondrial dysfunction as well as decreasing antioxidant defenses such as glutathione (GSH) and glutathione peroxidase activity. BCP prevented C6 cells from Glu-induced cytotoxicity by modulating the cellular antioxidant response, mainly by inhibiting ROS production and reestablishing the mitochondrial membrane potential (Dwm). Moreover, BCP per se induced the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) which was reflected by improvement in the cellular GSH antioxidant system. Taken together, our results suggest that

INTRODUCTION b-caryophyllene (BCP), a natural bicyclic sesquiterpene, is a major volatile plant compound found in large amounts in the essential oils of different species, such as Cinnamomum spp., Piper nigrum (Mockute et al., 2001; Prat et al., 2005), and Cannabis sativa (Sibbald, 2005). A recent study demonstrated that BCP selectively binds to cannabinoid receptor type 2 (CB2R) and acts as a full agonist (Gertsch et al., 2008). In particular, CB2R ligands have been shown to inhibit carrageen-induced mouse paw edema (Iwamura et al., 2001), act on primary afferent neurons to inhibit nociception (Ibrahim et al., 2001), and play a protective role in hepatic ischemia– reperfusion injury (Batkai et al., 2007). In addition, recent studies showed that BCP showed anti-inflammatory (Gertsch et al., 2008), antiviral (Astani et al., 2011), antinociceptive (Ghelardini et al., 2001) and antioxidant effects (Kawakami et al., 2005; Sevcik et al., 2005). We and others have also previously demonstrated that BCP inhibits inflammatory bowel diseases (IBDs) such as ulcerative colitis through CB2R activation and the peroxisome proliferator-activated receptor (PPARc) pathway (Kimball et al., 2006; Bento et al., 2011). Other results suggest that BCP oxide blocks the signal transducer and activator of transcription 3 (STAT3) signaling cascade and thus shows potential for the treatment of cancers (Kim et al., 2013). More recently, accumulated evidence suggests involvement of the cannabinoid system in neuroinflammation (Yokote et al., 2008; Downer, 2011) and neurodegeneration (Palazuelos et al., 2008; Bhaskaran and Smith, 2010). Glutamate (Glu), the most prominent neurotransmitter in the body, is present in over 50% of nervous tissue and plays a key role in neuronal excitation through ionotropic

*Corresponding author. Address: Laborato´rio de Autoimunidade e Imunofarmacologia, Campus Ararangua´, Universidade Federal de Santa Catarina, 88900-000 Ararangua´, SC, Brazil. Tel: +55-483721-6448. E-mail addresses: [email protected], [email protected] (R. C. Dutra).   These authors contributed equally to this work. Abbreviations: Dwm, mitochondrial membrane potential; AD, Alzheimer disease; AMPA, a-amino-3-hyroxy-5-methylisoxazole propionic acid; ARE, antioxidant response element; BCP, b-caryophyllene; CB2R, cannabinoid receptor type 2; CHX, cycloheximide; CNS, central nervous system; DCFH-DA, 2,7dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified eagle medium; GCLC, glutamate cysteine ligase; Glu, glutamate; GSH, glutathione; GPx, glutathione peroxidase; FBS, fetal bovine serum; NMDA, N-methyl-D-aspartate; Nrf2, nuclear factor (erythroidderived 2)-like 2; MS, Multiple Sclerosis; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide; PD, Parkinson’s disease; ROS, reactive oxygen species; SOD, superoxide dismutases. http://dx.doi.org/10.1016/j.neuroscience.2014.08.043 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 220

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(NMDA and AMPA or metabotropic (mGlu1–mGlu8) receptors (Greenamyre and Porter, 1994; Dingledine et al., 1999). However, for the last few decades, overstimulation of Glu receptors, especially the NMDA subtype, has been hypothesized to play a pivotal role in the pathogenesis of neuronal death (Olney, 1990; McElnea et al., 2011; Niciu et al., 2012). Upon their activation, these open their associated ion channels to allow the influx of Ca2+ and Na+ ions, suggesting a strong relationship between excessive Ca2+ influx and Glu-triggered neuronal injury (Johnson et al., 2004). In addition, the metabotropic Glu receptor mGluR1 and mGluR5 can also mobilize Ca2+ from the internal stores from the endoplasmic reticulum (ER) (Berridge et al., 2000; Rae and Irving, 2004; Sohn et al., 2011). Excessive Ca2+ uptake potentially opens the mitochondrial permeability transition pores, which in turn can further stimulate reactive oxygen species (ROS) production, a consequence of activation of Ca2+dependent enzymes such as nitric oxide synthase (Gautier et al., 2012), leading to both caspase-dependent and independent apoptotic-like cell death (Lipton, 2008) and finally to progressive neurodegeneration (Lee et al., 2009). Generation of high levels of ROS and down regulation of anti-oxidant mechanisms result in neuronal cell death during neurodegenerative diseases like traumatic brain injury, Multiple Sclerosis (MS), and Alzheimer (AD) and Parkinson’s (PD) diseases (Farooqui and Farooqui, 2009). In this regard, a recent study showed that cannabinoid agonist WIN55212 prevented early neuronal death via CB1-independent mechanisms during hypoxic–ischemic brain damage by attenuating glutamatergic excitotoxicity (Fernandez-Lopez et al., 2006). The nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ antioxidant response element (ARE) pathway is a critical signaling pathway regulating antioxidants and phase II detoxification enzymes, such as hemeoxygenase 1 (HO-1), NAD(P)H quinoneoxido reductase 1 (NQO1), glutathione peroxidase (GPx), glutamate cysteine ligase (GCLC) and thioredoxin reductase 1 (TrxR1) (Keum et al., 2003; Ichiyama et al., 2008). The importance of the antioxidant activity of Nrf2 has been clearly demonstrated in cell culture and animal models of neurodegenerative diseases, including AD and PD (van Muiswinkel and Kuiperij, 2005; Yang et al., 2008). Moreover, natural products such as quercetin and phenolic acid have been identified for their ability to attenuate oxidative damage by activating Nrf2 (Yeh and Yen, 2006; Weng et al., 2011). Glial cells such as astrocytes and microglia are the most abundant cells in the human brain (Wang et al., 2008; Belanger et al., 2011). Astrocytes provide trophic and metabolic support to neurons, modulate synaptic activity and neuronal plasticity, even as they influence neuronal survival by regulating: (i) free radical scavenging, (ii) water transport, (iii) production of cytokines/NO and, especially, (iv) Glu uptake and release (Barbeito et al., 2004; Rossi et al., 2007), which through glutamine synthetase catalyzes Glu to form glutamine for export to neurons, thus completing the Glu–glutamine cycle (Mates et al., 2002). However, a recent report demonstrated that sustained ERK1/2 activation contributes to

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Glu-induced apoptosis of astrocytes in vitro and in vivo (Szydlowska et al., 2006). Moreover, Glu-induced astrocyte degeneration has been described by others in the context of oxygen–glucose deprivation (Paquet et al., 2013), such as ischemia and Amyotrophic Lateral Sclerosis (Bruijn et al., 1997; Van Damme et al., 2007; Yamanaka et al., 2008). Keeping the above data in mind, the purpose of the present study was to investigate the role of BCP, a functional nonpsychoactive CB2R ligand, on astroglial cell excitotoxicity induced by Glu and the possible involvement of the antioxidant/Nrf2 pathway in this process, for possible identification of novel neuroprotective agents.

EXPERIMENTAL PROCEDURES Cell culture and treatments C6 rat glioma cells (CCL-107) were obtained from the American Type Culture Collection (ATCC), cultured as a monolayer in polystyrene dishes and maintained in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2. Cells were subcultured at 80% confluence and used between the 10th and 16th passage. In order to evaluate Glu-induced cytotoxicity, the cells were plated into a 96-well plate at equal density (3  103 cells/well) in FBS free medium and exposed to different Glu concentrations (0.05–10 mM) for 24 h. To evaluate the potential effects of BCP on Glu-induced excitotoxicity different protocols were used: (i) co-administration: the cells were concomitantly exposed to Glu (1 mM) and BCP (0.5–1 lM) for 1 or 24 h; and (ii) pre-treatment: C6 glioma cell line were pre-treated with BCP (0.5–3 lM) for 24 h before Glu exposure (1 mM). The dose of each drug was chosen based on preliminary studies or previous publications (Bento et al., 2011; Kataria et al., 2012). Cell viability The viability of C6 cells in the presence or absence of Glu or BCP was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) assay, as previously described (Siqueira et al., 2009). After treatment, 200 lL of MTT solution (0.5 mg/mL) was added and incubated for 2 h. Then the MTT was removed and 200 lL of DMSO/ well was added to dissolve the intracellular crystalline formazan product. Finally, the solution was collected and the optical density was measured at 550 nm using a microplate reader. The results were expressed as a percentage of the absorbance of non-treated cells. Measurement of ROS production Intracellular ROS production was detected using the nonfluorescent cell permeating compound 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). C6 cells were plated into 12-well plates (6  104 cells/well) and

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exposed to different treatments as described above. Following this, the cells were incubated with DCFH-DA (10 lM) for 30 min. The intensity of fluorescence was measured using a spectrofluorometer (488 nm excitation and 520 nm emission), and ROS was monitored for 1 h. Measurement of mitochondrial membrane potential (Dwm)

Dwm was assessed using the lipophilic cationic probe fluorochrome JC-1, as previously described (Reers et al., 1991). In the presence of physiological Dwm, JC-1 forms aggregates that fluoresce with an emission peak at 588 nm. Loss of membrane potential favors the monomeric form of JC-1, which has an emission peak at 530 nm. To examine the effect of BCP on modulating the Dwm induced by Glu (1 mM), C6 rat glioma cells were plated into 24-well plates (3  104 cells/well) and pretreated as described previously. Afterward, C6 glioma cell line were incubated with JC-1 (5 lM) for 20 min at 37 °C and JC-1 fluorescence intensity was measured using a fluorimetric microplate reader (Tecan, Gro¨dig/Salzburg, Austria) with excitation at 488 nm and emission at 525 nm and 590 nm. Dwm was inferred from the ratio of red/green fluorescence intensity. The images were acquired from three randomly chosen fields using an inverted epifluorescence microscope (Olympus IX70). Measurement of cellular reduced glutathione (GSH) Intracellular levels of GSH were determined by fluorimetric assay as described previously (Hissin and Hilf, 1976). In brief, cells (9  104 cells/well) were plated into six-well plates and submitted to different treatments as described above. After treatment, the cells were scraped into 0.6 M perchloric acid and recovered into 100 mM PBS containing 5 mM EDTA, pH 8.0 at 4 °C. Samples were centrifuged at 14,000 rpm at 4 °C for 10 min. A volume of 100 lL of supernatant was incubated with 100 lL of ortho-phthaldehyde (0.1% w/v in methanol) and 1.8 mL of 100 mM Na2HPO4 for 15 min at room temperature. Fluorescence intensity was read at an emission wavelength of 420 nm and an excitation wavelength of 350 nm in a spectrofluorometer (Tecan). Cellular GSH content was calculated by using a concurrently run standard curve and expressed as lg of GSH per mg of protein. Measurement of GPx activity GPx activity was measured according to Wendel (1981) using tert-butylhydroperoxide as substrate. Cells (9  104 cells/well) were plated into six-well plates and submitted to different treatments as described above. The enzyme activity was determined by monitoring NADPH disappearance at 340 nm in 50 mM potassium phosphate buffer, pH 7.0, containing 1 mM EDTA, 1 mM GSH, 0.2 U/mL GSH reductase, 0.2 mM tert-butylhydroperoxide, 0.2 mM NADPH and the supernatant containing 0.2–0.3 mg protein/mL. GPx activity was expressed as nmoles of NADPH oxidized/min/mg of protein, using an extinction coefficient of 6.22  103/M/ cm for NADPH.

Western blot analysis Cytosolic and nuclear fractions of C6 glioma cell line were prepared to investigate the translocation of Nrf2 by BCP treatment. Equal amounts of protein for each sample (50 lg) were loaded per lane and electrophoretically separated using 12% denaturing polyacrylamide gel electrophoresis (SDS–PAGE). Afterward, the proteins were transferred to nitrocellulose membranes, saturated by incubation with 10% non fat dry milk solution and then incubated overnight with Nrf2 antibody (1:300 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (1:25,000, Cell Signaling Technology, Danvers, MA, USA). The immunocomplexes were visualized using the ECL chemiluminescence detection system (GE Healthcare, Sa˜o Paulo, SP, Brazil). Densitometric values in the nuclear fraction were normalized using monoclonal mouse laminin antibody (1:2000, Santa Cruz Biotechnology) and in the cytosolic fraction with b-actin (1:3000, Santa Cruz Biotechnology). Protein levels were quantified by optical density using Image-J Software and expressed as their ratio to laminin represented by arbitrary units. RNA extraction, cDNA synthesis, and quantitative real-time PCR Total RNA was extracted from C6 glioma cells using the SV Total RNA Isolation Kit and 0.4 lg of each sample was reverse transcribed to cDNA using TaqMan RT reagents. The reaction mixture was incubated at 25 °C for 10 min, 48 °C for 1 h, and 95 °C for 5 min. Quantitative real-time PCR, using the ABI 7900HT cycler and SYBR-Green Master Mix reagent was performed according to the manufacturer’s protocol (Applied Biosystems). The primers were, for HO-1: forward, 50 CTTTCAGAAGGGTCAGGTGTC-30 ; reverse, 50 -TGCTT GTTTCGCTCTATCTCC-30 ; for GPx-1: forward, 50 -CAG TTCGGACATCAGGAGAA-30 ; reverse, 50 -AGGGCTTCT ATATCGGGTTC-30 ; for GCLC forward, 50 -TTACCGA GGCTACGTGTCAGAC-30 ; reverse, 50 -TGTCGATGGT CAGGTCGATGTC-30 and for GAPDH: forward, 50 CCCTCTGGAAAGCTGTGGCGTG-30 ; reverse, 50 -TCCT CAGTGTAGCCCAGGATGC-30 (Dasgupta et al., 2007; Lin et al., 2011; Fujimura and Usuki, 2014). Primers were used in a final concentration of 0.3 lM. The reaction conditions were 50 °C for 2 min, then 90 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The data were analyzed using Sequence Detection Systems (SDS) version 2.4 Software (Applied Biosystems). A dissociation step was added for SYBR-Green runs. For each sample, gene expression was quantified using a standard curve and normalized against the expression of the GAPDH gene. Drugs and reagents BCP, GSH, GPx, JC-1, NADPH, tert-butylhydroperoxide, cycloheximide (CHX) and DCFH-DA were obtained from Sigma–Aldrich (St. Louis, MO, USA). AM630 was

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purchased from Cayman Chemical (Ann Arbor, Michigan, USA). The BCP was solubilized in a 0.9% NaCl solution. All-vehicle solutions were used for the respective control treatments. DMEM, MTT, FBS, L-glutamine, HEPES, penicillin and streptomycin were obtained from GIBCO (Carlsbad, CA, USA). Polyclonal rabbit Nrf2 and monoclonal mouse laminin antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Secondary antibodies conjugated to horseradish peroxidase were obtained from Cell Signaling Technology (Danvers, MA, USA). The primers and probes for HO-1, GPx-1, GCLC and GAPDH were purchased from Applied Biosystems (Foster City, CA, USA). The other reagents used were of analytical grade and obtained from different commercial sources. Statistical analysis All data are expressed as mean ± SEM from at least three independent experiments. Differences between mean values of multiple groups were analyzed by a one- or two-way analysis of variance (ANOVA) followed by Newman–Keuls or Bonferroni’s post hoc multiple range test, respectively. P-Values less than 0.05 (P < 0.05 or less) were considered significant. The statistical analyses were performed using GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA, USA).

RESULTS Prolonged exposure of C6 cells to Glu triggers a dose-dependent cytotoxic effect First, we determined the toxic effect of Glu in C6 cells cultures in a concentration-dependent assay. The cell viability of C6 glioma cell line was measured by MTT assay. Glu treatment of C6 cells induced a progressive and significant reduction in cell viability in a concentration-dependent manner (Fig. 1A). At a dose of 1 mM Glu the number of viable cells decreased by roughly 50% compared to untreated cells; this dose was then selected for subsequent experiments.

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Exposure of cells to BCP prevents Glu-induced excitotoxicity In order to evaluate the possible cytoprotective effect of BCP per se in C6 cells, we assessed different concentrations of BCP (0.5–3 lM) using the MTT assay. Importantly, BCP per se did not affect astroglial viability in any tested concentrations (Fig. 1B) and for this reason concentrations of 0.5 lM and 1 lM were used in subsequent experiments. Next, we investigated the neuroprotective effect of BCP upon excitotoxicity induced by Glu on C6 cells. As shown in Fig. 2A, Glu treatment (1 mM) of C6 led to a significant reduction in cell viability, whereas cotreatment with BCP (0.5 lM) and Glu partially protects C6 cells from Glu excitotoxicity. (Fig. 2A). Interestingly, pre-treatment of cells with BCP for 24 h before Glu exposure protected C6 cells from death by counteracting the toxic effect of Glu (Fig. 2B). Notably, BCP (0.5 lM and 1 lM) fully protected C6 cells (87.7% and 95.1% survival, P < 0.01, respectively) against the harmful effects of Glu, with cell viability attaining levels equivalent levels to those of the untreated control group (Fig. 2B).

BCP prevents Glu-induced ROS production and mitochondrial dysfunction Increasing evidence has shown that impairment of cellular Ca2+ homeostasis, mitochondrial disturbance, activation of NOS, and generation of ROS provoked by Glu-linked oxidative stress plays a crucial role in cell death (McElnea et al., 2011). In this set of experiments, we investigated whether the glial-protective effects of BCP were also linked to decreased ROS production after Glu stimulation using 20 ,70 -dichlorfluorescein-diacetate (DCFH-DA). As shown in Fig. 3, incubating cells with Glu stimulated an increase in intracellular ROS after 20 min of Glu exposure (Fig. 3A) for up to 24 h (Fig. 3C). The direct effect of BCP (co-treatment) is modest when compared to the pre-treatment, which suggest that BCP could act indirectly by activating gene

Fig. 1. Glutamate-induced excitotoxicity in astroglial cells. (A) Glutamate-dependent toxicity in C6 cells is concentration dependent. C6 cells (80% confluent) were treated with the indicated concentrations of glutamate for 1 h and assessed 24 h later. (B) Effect of BCP (0.5–3 lM) exposure per se for 1 h on C6 cell viability, assessed 24 h later. Cell viability was assessed by reduction of MTT and expressed as a percentage of control (nontreated cells, 100%, dotted line). Each bar represents the mean ± SEM of at least three independent experiments. ⁄P < 0.05, ⁄⁄P < 0.01 indicate significant differences when compared to control (a one-way ANOVA followed by Newman–Keuls test).

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Fig. 2. Neuroprotective effects of BCP against glutamate-induced toxicity. (A) C6 cells were concomitantly exposed to Glu (1 mM) and BCP (0.5– 1 lM) for 1 h. (B) The cells were pre-treated with BCP (0.5–1 lM) for 24 h before Glu (1 mM) exposure. Cell viability was assessed by reduction of MTT and expressed as a percentage of control (non-treated cells, 100%, dotted line). Each bar represents the mean ± SEM of at least three independent experiments. ⁄P < 0.05 indicates the difference when compared to control; #P < 0.05 indicates the difference when compared with Glu-treated groups (a two-way ANOVA followed by Bonferroni test).

Fig. 3. Glutamate-evoked production of ROS is blocked by BCP treatment in C6 cells. (A) The cells were concomitantly exposed to Glu (1 mM) and BCP (0.5–1 lM) for 5 min. (B) C6 cells were pre-treated with BCP (0.5–1 lM) for 24 h before Glu (1 mM) exposure, then ROS was monitored for 1 h. (C) C6 cells were pre-treated with BCP (0.5–1 lM) for 24 h before Glu (1 mM) exposure, and DCFH-DA was measured 24 h later. After the treatment, cells were incubated with DCFH-DA (10 lM) for 30 min and the amount of ROS (fluorescence intensity) was measured by a fluorimetric microplate reader. The results are expressed as fluorescence intensity. Each bar represents the mean ± SEM of at least three independent experiments. ⁄P < 0.05 indicates the difference when compared with control; #P < 0.001 indicates the difference when compared with Glu-treated groups (a two-way ANOVA followed by Bonferroni test).

expression related with the cell’s redox enzyme system, whereas a period of 24 h is required to activate the system. Notably, pre-treatment of C6 cells with BCP for 24 h completely prevented Glu-induced ROS production even after a long period of Glu exposure (Fig. 3B, C), with inhibitions of 35.3% (0.5 lM) and 30.3% (1 lM) (P < 0.05). In fact, mitochondria have emerged as key regulators of oxidative stress and cytotoxicity (Jou, 2008). Mitochondrial dysfunction and the ensuing bioenergetic impairment are likely to promote ion pump failure, loss of mitochondrial membrane integrity and consequent cell death (Reers et al., 1991). In this set of experiments, we investigated whether the same treatment with BCP could modulate Dwm following Gluinduced excitotoxicity. As expected, Glu drastically reduced mitochondrial potential (P < 0.01) after 10 min of exposure (Fig. 4A) for up to 24 h (Fig. 4C, D). Interestingly, pre-treatment with BCP (0.5 lM and 1 lM) 24 h before Glu exposure completely prevented the Dwm dysfunction (Fig. 4B–D). However, the co-treatment (Glu + BCP) displayed only a moderate effect on Dwm induced by Glu (Fig. 4A).

Effect of BCP on GSH antioxidant system The antioxidant system, including both enzymes and specific molecules, acts as scavengers of ROS and their downstream products to minimize the buildup of ROS and lessen the potential inflicted damage. As such, the balance between ROS and the antioxidant system plays a critical role in determining the fate of cells (Sheng et al., 2013). The GSH system is critically involved in protecting cells against oxidative stress (Sheng et al., 2013). To ascertain how BCP confers protection against Glu-induced cell death, we measured the levels of intracellular GSH and GPx activity. Glu-treated C6 cells showed 23% and 24.5% (P < 0.05) reductions in GSH levels (Fig. 5A) and GPx activity (Fig. 5B) compared to untreated cells, respectively. Pre-treatment with BCP 24 h before Glu exposure significantly reversed the effects of the Glu-dependent decrease in GSH levels in the cells by 29% (0.5 lM) and 30.2% (1 lM) (P < 0.05) (Fig. 5A), as well as reducing GPx activity by 37% (0.5 lM) and 34% (1 lM) (P < 0.05) (Fig. 5B). Of note, pre-treatment with BCP was able to upregulate GSH levels and GPx activity when compared to untreated cells (Fig. 5A, B).

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Fig. 4. BCP prevents mitochondrial membrane potential (Dwm) dysfunction. (A) C6 cells were concomitantly exposed to Glu (1 mM) and BCP (0.5 lM – 1 lM) for 5 min. The cells were pre-treated with BCP (0.5 lM – 1 lM) for 24 h before Glu (1 mM) exposure; JC-1 was measured at 1 h (B) and 24 h (C) after. The change in mitochondrial membrane potential (Dwm) was assessed based on the signal intensity from JC-1 monomeric and aggregate fluorescence; JC-1 was monitored for 1 h. (D) JC-1 fluorescence was quantified fluorometrically at a magnification of 400. Each bar represents the mean ± SEM of the ratio (red/green fluorescence) of at least three independent experiments. ⁄P < 0.05 indicates the difference when compared with control; #P < 0.001 indicates the difference when compared with Glu-treated groups (a two-way ANOVA followed by Bonferroni test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. BCP upregulates and completely restores glutamate-induced depletion of GSH and GPx activity in C6 cells. (A) Effect of BCP per se on GSH biosynthesis and cell death induced by Glu (1 mM) in C6 cells. (B) Effect of BCP on GPx activity in C6 cells and cell death induced by Glu (1 mM). Rat C6 glioma cells were pre-incubated with BCP (0.5–1 lM) for 24 h before Glu (1 mM) exposure. Each bar represents the mean ± SEM of at least three independent experiments. ⁄P < 0.05 indicates the difference when compared with control; #P < 0.05 indicates the difference when compared with Glu-treated groups (a two-way ANOVA followed by Bonferroni test).

BCP induces Nrf2 activation To further define some of the signaling systems activated by Glu-induced excitotoxicity that could mediate the glial-protective effect of BCP, we used western blot and

qRT-PCR analysis to assess the activation of transcriptional factor Nrf2. As shown in Fig. 6, BCP per se markedly promoted Nrf2 nuclear translocation after three (Fig. 6A) and 12 h (Fig. 6B) in the C6 glioma cell line.

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Fig. 6. BCP induces Nrf2 nuclear translocation. C6 glioma cells were pre-treated with BCP (0.5–1 lM) for 3 and 12 h. The cells were then submitted to SDS polyacrylamide gel electrophoresis, electroblotted and incubated overnight with a primary polyclonal antibody against Nrf2 antibody. b-Actin bands are also shown (protein loading control) for the cytosolic fraction. Laminin A/C bands are also shown (protein loading control) for the nuclear fraction. Each bar represents the mean ± SEM of at least three independent experiments. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001 indicates the difference when compared with control (a one-way ANOVA followed by Newman–Keuls test).

Since we found activation of Nrf2 with BCP treatment, we than evaluated the mRNA expression of some its target genes: HO-1, GPx-1 and GCLC (Lin et al., 2011). As shown in Fig 7A–C, BCP treatment increased the mRNA levels of Nrf2-target genes after six and 24 h. Moreover, to determine whether the modulation of anti-oxidant mechanisms by BCP is a genomic effect, dependent on Nrf2, we determined the level of GSH in the presence of CHX, a classical protein synthesis inhibitor. As shown in Fig. 7D, co-treatment with 0.3 lM of CHX opposed the BCP-induced upregulation of GSH levels in the C6 glioma cell line. Taken together, these results (Figs. 6 and 7) support our hypothesis that BCP can improve the cellular antioxidant response by inducing the nuclear transcriptional factor of Nrf2. Antioxidant effects of BCP is dependent on CB2R BCP was recently identified as a natural selective agonist of the peripherally expressed CB2R, which represents a dietary phytocannabinoid (Gertsch et al., 2008). In this set of experiment, we investigated whether a CB2Rselective antagonist could prevent the antioxidant activity of BCP in vitro. For this purpose, C6 glioma cell line were pre-treated with the selective CB2R antagonist AM630 (0.3 lM) alone or in combination with BCP. As shown in Fig. 7E, BCP per se induced a significant upregulation of intracellular GSH levels; however, CB2R antagonist AM630 notably abolished the antioxidant effect of BCP in C6 glioma cell (P < 0.05) (Fig. 7E). Relevantly, the CHX inhibitor and CB2R antagonist AM630 significantly blocked the neuroprotective effect of BCP upon excitotoxicity induced by Glu on C6 cells (Fig. 7E). These results support the concept that antioxidant effect of BCP is dependent on CB2R express in the C6 glioma cell.

DISCUSSION Neurodegenerative diseases share a common mechanism of pathophysiology such as oxidative stress, mitochondrial

aberrations, and inflammation that lead to neuron degeneration and death (Fernandez-Checa et al., 2010). Developing therapeutic modulators of these universal mechanisms could have a significant impact in the management of neurodegenerative diseases like AD, PD, MS, traumatic brain and spinal cord injury through delaying disease onset or progression (Onyango and Khan, 2006). The interaction of Glu with specific membrane receptors is responsible for several neurological actions mediated by neuronal and glial cells in the CNS (Kulawiak and Szewczyk, 2012). However, excessive Glu can lead to neuronal and glial cell death in a variety of pathological conditions and is thought to play a crucial role in the pathogenesis of many neuropsychiatric and neurodegenerative disorders (Farber et al., 1998). So there is a great need to search for new and better neuronal and glial-protective drugs. The results reported herein demonstrate the effectiveness of the natural sesquiterpene BCP, a functional nonpsychoactive CB2R ligand, in blocking Glu-induced excitotoxicity in C6 cells. Our data also show that mechanisms responsible for BCP’s C6 cells’ protective actions are primarily associated with its ability to inhibit ROS production and mitochondrial dysfunction, as well as upregulate the GSH antioxidant system through the CB2R/Nrf2 pathway (see scheme proposed in Fig. 8). The relevant effects of BCP occur when C6 cells are pre-exposed for 24 h to BCP, therefore, an indirect effect seems to be involved in the protective effect of BCP against Gluinduced cytotoxicity. Moreover, the increase of cellular GSH activity was accompanied by an increase in GPx activity when cells were exposed to BCP. These results suggest that BCP could act indirectly by activating gene expression related with the cell’s redox enzyme system, however a period of 24 h is required to activate the system. Our results suggest that induction of such enzymes is governed by the transcriptional factor Nrf2, which represents a key step in endogenous cellular protection and it is becoming a promising therapeutic target for neuroprotection. There is an increasing evidence that induction of the

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Fig. 7. Effect of BCP on Nrf2-targets gene expression, as well as result of protein synthesis inhibitor and CB2R-selective antagonist on BCP antioxidant effect. (A–C) C6 glioma cells were pre-treated with BCP (1 lM) for 3, 6 and 24 h and qRT-PCR was carried. Results were normalized with the housekeeping gene GAPDH. The treated groups were compared with control cells (considered as 100%). (D) C6 cells were concomitantly exposed to cycloheximide (CHX) (0.3 lM) plus BCP (1 lM) or AM630 (0.3 lM) plus BCP (1 lM) for 30 min; (E) Glu (1 mM) alone, BCP (1 lM) alone, cycloheximide (CHX) (0.3 lM) alone, AM630 (0.3 lM) alone, cycloheximide (CHX) (0.3 lM, 30 min before) plus BCP (1 lM) or AM630 (0.3 lM, 30 min before) plus BCP (1 lM) for 24 h before Glu exposure. Cell viability was assessed by reduction of MTT and expressed as a percentage of control (non-treated cells, 100%, dotted line). Each bar represents the mean ± SEM of at least three independent experiments. (A– C) ⁄P < 0.05; ⁄⁄P < 0.001 indicates the difference when compared with control. (D, E) DDP < 0.001 versus control group; #P < 0.05 indicates the difference when compared with control (A) or glutamate-treated group (B); ⁄P < 0.05; ⁄⁄P < 0.001 indicates the difference when compared with BCP alone treated group (a one-way ANOVA followed by Newman–Keuls test).

Nrf2/ARE signaling pathway confers protection in the CNS (Jing et al., 2013). Excessive ROS production can lead to lipid, protein and DNA oxidation, and these events are associated with age-related diseases (Arundine and Tymianski, 2004). Herein, BCP decreased DCFH oxidation, emerging as an important molecule providing protection against cellular toxicity. In agreement with our data, it was recently found that the essential oil of Salvia fruticosa demonstrated remarkable protective activity in a model of oxidative stress in vitro in primary astrocyte cultures. Noteworthy, this protective effect of Salvia fruticosa was attributed to a-humulene and a-pinene (Elmann et al., 2009). Moreover, another group has demonstrated that resveratrol, a polyphenolic compound found in grapes and red wine, decreased DCFH oxidation and prevented H2O2-oxidative insult (Quincozes-Santos et al., 2013) as well as preventing oxidative damage in C6 cells (Quincozes-Santos et al., 2009). Extending this idea, a recent report demonstrated that the CB1/CB2R agonist, CP55, 940, and the CB2R agonist, JWH015 significantly protected human retinal pigment epithelial cells (RPE)

from oxidative damage. In addition, CP55, 940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway (Weiss et al., 2009). Dwm has been implicated as a factor in impaired mitochondrial function (Bai et al., 2001). This organelle has been shown to be impaired in various models of cell injury (Moncada and Bolanos, 2006). A decrease in Dwm following an intense burst of ROS production induces mitochondrial disruption, inhibition of the mitochondrial respiratory chain, reduction in ATP synthesis, and cell death (Choi et al., 2000). Previous studies and our data showed that Glu treatment induced a significant decrease in Dwm (Sun et al., 2012). However, our results demonstrated that pre-treatment of BCP prevented the reduction in Dwm induced by Glu in C6 cells, suggesting that the neuroprotective effect of BCP may be related to the mitochondrial death pathway. In line with this, it has recently been demonstrated that paeoniflorin, a monoterpene glycoside, inhibits the reduction in Dwm induced by Glu in PC12 cells (Sun et al., 2012). Furthermore, other

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Fig. 8. Schematic representation of the mechanism by which BCP regulates glutamate-induced excitotoxicity in C6 glioma cell line. Glutamate is the most prominent neurotransmitter in the body, present in over 50% of nervous tissue and playing a key role in neuronal excitation. However, overstimulation of glutamate receptors plays a pivotal role in the pathogenesis of neuronal and astroglial death. Glutamine is converted to glutamate by glutaminase (1) in presynaptic neuronal cells. On the other hand, in astrocytes, glutamate is converted to glutamine by glutamine synthetase (GS) and exported extracellularly to be taken up again by neurons (2). Glu receptors are present on presynaptic and postsynaptic neurons as well as on glial cells. These include both ionotropic (NMDA and AMPA/KA) and metabotropic receptors (mGluRs) (3). The activation of NMDA receptors on astrocytes induces Na+ and Ca2+ influx (4). Moreover, cytoplasmic Ca2+ overload in astroglial cells leads to free radical production, including ROS (5), oxidative stress, and mitochondrial membrane potential (Dwm) dysfunction, which will lead to energy failure (6). BCP, a natural bicyclic sesquiterpene, rescued C6 glioma cell line from glutamate-induced cytotoxicity by modulating oxidative responses, mainly by inhibiting ROS production (7) and reestablishing mitochondrial membrane potential (Dwm) (8), maintaining the cellular redox environment through the Nrf2 signaling pathway and consequently upregulating levels of GSH and GPx activity and downregulating ROS production (9). Abbreviations used in the figure: BCP, b-caryophyllene; GSH, glutathione; GPx, glutathione peroxidase; Nrf2, nuclear factor (erythroid-derived 2)-like 2; ROS, reactive oxygen species; GS, glutamine synthetase; Dwm, mitochondrial membrane potential; ARE, antioxidant responsive element ( ), inhibition; ( ), stimulation.

results have shown that activation of the CB2R by JWH133 prevented apoptosis during ischemia/reperfusion through inhibiting the intrinsic mitochondria-mediated apoptotic pathway and involved the PI3K/Akt signal pathway (Li et al., 2013). More importantly, a very recent study showed that WIN 55, 212–2, a potent cannabinoid receptor agonist, reduced apoptotic cell death in different brain regions through maintaining mitochondrial integrity and functionality (Alonso-Alconada et al., 2012). Taken together, this series of experiments suggests that the glial-protective effects of BCP upon glutamatergic excitotoxicity depend, at least in part, on its abilities to prevent the synthesis and/or release of ROS and prevent mitochondrial dysfunction. The levels of reactive species can be determined by the balance between their rate of production and clearance by different antioxidant compounds/enzymes, such as superoxide dismutases (SOD), catalase (CAT), GSH and GPx (Akerboom and Sies, 1981). To guard against cell damage, the formation and elimination of ROS must be balanced. Cells are normally equipped with antioxidant scavengers and enzymes to prevent high levels of ROS mediated damage, but neurons express them only at very low concentrations/activity levels (Heales and Bolanos, 2002). It is therefore imperative that these cells co-operate with neighboring astrocytes to achieve a complete (and complex) cycle of ROS detoxification and

neuroprotection (Fernandez-Fernandez et al., 2012). Elevated levels of extracellular Glu inhibit cysteine uptake by inhibiting the anti-porter system and the precursor of GSH, induces a marked decrease in intracellular GSH levels, thereby inducing oxidative stress in the cell (Conrad and Sato, 2012). Herein, our data showed that Glu-induced a significant downregulation of intracellular GSH levels and GPx activity, thereby inducing oxidative stress in C6 cells. Also of relevance, treatment with BCP per se or after Glu-stimulation markedly improved the cellular levels of GSH, as well as increased GPx activity in C6 glioma cells. A very interesting study conducted by (Chen et al., 2005) showed that D9-tetrahydrocannabinol (THC), a psychoactive cannabinoid receptor ligand, significantly decreased NMDA-induced ROS generation in AF5 cells. Furthermore, a recent report demonstrated that COR167, a CB2R agonist, potently protected rat brain cortical slices against oxygen glucose deprivation (OGD) and reperfusion injury, partly through CB2R (Contartese et al., 2012). These results suggest that BCP may prevent Glu-induced excitotoxicity through increasing GSH levels and GPx activity in glial cells. It has been demonstrated that depletion of GSH in glial cells induces neurotoxicity through activation of the p38 MAPkinase, jun-N-terminal kinase, and NF-jB inflammatory pathways (Lee et al., 2010). Based on these results, it

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is tempting to speculate that BCP could represent a valuable tool for the control/treatment of neurodegenerative conditions associated with oxidative imbalance, such as MS, AD and PD. Major oxygen and redox-sensitive transcriptional factors (TFs) include nuclear factor-(erythroid-derived 2) like 2 (Nrf2), hypoxia-inducible factor 1 (HIF1), and nuclear factor kappa-B (NF-jB). Expression of antioxidant response element (ARE)-driven genes and enzymes is directed by Nrf2 cap’n’collar bZIP transcription factors, which include enzymes involved in GSH synthesis, GPx, glutathione-S-transferase (GST), heme oxygenase (HO), SOD, and ferritin (Hybertson et al., 2011). Conversely, NF-jB activates genes, particularly those involved in the inflammatory response. Recent studies have demonstrated that flavonoids extracted from Ixeris sonchifolia, in particular luteolin, contribute to its neuroprotective effects against ischemia-induced cellular injury through direct antioxidant effects associated with upregulation of Nrf2 (Zhang et al., 2013). In addition, sesquiterpene lactones isolated from Tanacetum parthenium activated Nrf2 in mouse primary cortical cultures (Fischedick et al., 2012). Our results are in accordance with a recent report stating that Cordyceps sinensis, an edible mushroom growing in Himalayan regions, increases hypoxia tolerance by inducing HO-1 and metallothionein via Nrf2 activation in human lung epithelial cells (Singh et al., 2013). Supporting and extending the above data, pre-treatment with BCP, a functional nonpsychoactive CB2R ligand, activated Nrf2 resulting in increases in both intracellular GSH levels and GPx activity. Moreover, the co-treatment with a classical protein synthesis inhibitor or with a CB2R-selective antagonist could prevent the antioxidant and neuroprotective activities of BCP in vitro. Taken together, our results suggest that cytoprotective effects of BCP were mediated by amelioration of cellular antioxidant responses via Nrf2 activation, which is dependent on CB2R activation.

CONCLUSION In summary, the data presented herein extend our previous results (Bento et al., 2011) and demonstrate, for the first time, that BCP, a natural bicyclic sesquiterpene, prevented C6 glioma cell line from Glu-induced cytotoxicity by modulating antioxidant responses, mainly by inhibiting ROS production and reestablishing Dwm, maintaining the cellular redox environment through the CB2R/Nrf2 signaling pathway. Altogether, our findings provide evidence that BCP might constitute a novel and attractive molecule and open-up new perspective for the development of new drugs to treat neurodegenerative diseases.

CONFLICTS OF INTEREST There are no conflicts of interest. Acknowledgments—This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), and the Fundac¸a˜o de Apoio a Pesquisa

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do Estado de Santa Catarina (FAPESC)/PRONEX, all in Brazil. LCA and MRS are PhD students in neuroscience and biochemistry, respectively, receiving grants from CAPES. AFB is supported by a research fellowship from CNPq.

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(Accepted 26 August 2014) (Available online 4 September 2014)