Anandamide Protects HT22 Cells Exposed to Hydrogen Peroxide by Inhibiting CB1 Receptor-Mediated Type 2 NADPH Oxidase

Background. Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA. Methods. The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression. Results. HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA. Conclusion. Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.


Introduction
Oxidative stress is implicated in the pathology of many central nervous system (CNS) disorders, including Alzheimer's disease, Parkinson's disease, and ischemic stroke [1][2][3]. Hydrogen peroxide (H 2 O 2 ) is produced at nearly every stage of the oxidative cycle and widely applied to induce oxidative stress in vitro [4]. H 2 O 2 -induced oxidative stress can cause lipid peroxidation, mitochondria injury, and DNA damage [5,6].
NADPH oxidase (Nox) is a membrane-associated enzyme complex consisting of several subunits including NADPH oxidase 2 (Nox2). Activation of neuronal Nox2 contributes to oxidative damage of the CNS [7], and inhibition of Nox2 can attenuate cerebral oxidative stress injury [8].
We have previously demonstrated that inhibition of Nox2 reduced the damage induced by oxygen glucose-deprivation to a mouse hippocampal neuron cell line, HT22 [9]. Endogenous cannabinoid anandamide (N-arachidonoylethanolamine, AEA) mimics the bioactivity of Δ 9 -tetrahydrocannabinol (THC), the principal psychoactive component of marijuana [10]. There are two main cannabinoid receptors, CB1 and CB2 [11]. In the CNS, CB1 is mainly expressed in neurons, and CB2 in glial cells, such as microglia and astrocytes [11]. It was recently demonstrated in rodent models that AEA conferred neuroprotection by activating cannabinoid receptors. AEA could protect the newborn brain against excitotoxicity by activating CB1 [12] and attenuated cytotoxic edema caused by administration of Na + /K + -ATPase inhibitor [10]. We have previously reported that electroacupuncture pretreatment induces neuroprotection by stimulating release of AEA through a protein kinase C epsilon-mediated pathway [13]. However, the precise mechanism by which AEA mediated protection in the CNS remains undefined. The aim of this study was to determine whether AEA could protect HT22 cells against H 2 O 2 -induced 2 Oxidative Medicine and Cellular Longevity injury and whether Nox2 was involved in the AEA-induced protection from oxidative stress via activation of CB1.

Cell
Culture. HT22 cells were cultured in DMEM with 10% FBS (v/v), 100 U/mL penicillin, and 100 g/mL streptomycin at 37 ∘ C in a humidified atmosphere containing 5% CO 2 and 95% air. The medium was replaced every 2 days. When cell density reached roughly 70-80%, cells were exposed to the indicated drugs for 3 h. Then some evaluations were performed.

Experimental Protocols.
To find a suitable H 2 O 2 concentration, the HT22 cells were assigned into seven groups (Figure 1(a)). Except for control group, the other six groups were exposed to different concentrations of H 2 O 2 for 3 h, ranging from 50 M to 1000 M. Then MTT assay was taken to determine the injury degree of the cells. To find a suitable AEA concentration, the cells were divided into six groups. Except for control and H 2 O 2 only groups, the other four groups were exposed to 200 M H 2 O 2 plus different concentrations of AEA (Figure 1(b)). Then MTT assay was taken to evaluate the injury degree of the cells.
Then the cells were assigned into six groups, including control, AEA, H 2 O 2 , AEA + H 2 O 2 , AM251 + AEA + H 2 O 2 , and AM251 + H 2 O 2 groups. After an incubation of 3 h, MTT assay, LDH release, and western blotting were taken to determine the roles of CB1 and Nox2 in AEA-induced protection (Figure 1(c)). To further investigate the role of CB1 in AEA-induced protection against H 2 O 2 in HT22 cells, the HT22 cells were divided into three groups, including control, CB1-siRNA, and SC-siRNA groups; after an incubation of 5 h, western blotting was used to evaluate the silencing rate of CB1 protein expression (Figure 1(d)). Then, the cells were divided into five groups, including control, H 2 O 2 , AEA + H 2 O 2 , CB1-siRNA + AEA + H 2 O 2 , and SC-siRNA + AEA + H 2 O 2 . Then cells injury was evaluated by MTT and LDH release at 3 h after incubation, and ROS generation was evaluated by measuring fluorescence intensity (Figure 1(e)). Apocynin, a specific Nox inhibitor [14], was used to further investigate the role of Nox2 in AEA-induced protection against H 2 O 2 . The cells were divided into five groups, including control, H 2 O 2 , AEA + H 2 O 2 , apocynin + AEA + H 2 O 2 , and apocynin + AEA + H 2 O 2 ; western blotting, MTT assay, and LDH release were used to measure Nox2 expression and cell injury (Figure 1(f)).

Cell
Viability. HT22 cells were plated at a density of 1 × 10 4 cells/well in 96-well plates. After treatment with different drugs for 3 h, cell injury was evaluated by MTT assay. 20 L of 5 mg/mL MTT solution was added to each well, and after 4 h incubation at 37 ∘ C the supernatant of each well was carefully removed. Then 150 L DMSO was added per well to solubilize the formazan product. The plate was then shaken for 10 min to ensure the formazan had completely dissolved. Absorbance at 490 nm was evaluated using a spectrophotometer (TECAN, CH).

LDH
Release. HT22 cells were plated at a density of 2 × 10 4 cells/well into a 24-well plate. After the treatments, the supernatants of each well were removed for assessment of LDH release, which was measured according to the manufacturer's instructions for the LDH assay kit. In brief, 100 L of cell-free supernatant, 250 L of buffer, and 50 L of coenzyme were mixed homogeneously and the supernatant was incubated with this reaction mixture for 15 min at 37 ∘ C. Next, 250 L of 2,4-dinitrophenylhydrazine was added into the mixture and incubated for an additional 15 min at 37 ∘ C in the dark. Finally, 2.5 mL of 400 mM of NaOH was added into the mixture to stop the reaction. After 3 min, the absorbance of the mixture was determined at 440 nm by spectrophotometry. The absorbance of the sample blank, standard, and standard blank was measured at the same time. LDH activity was calculated according to the following formula: 2.6. Apoptotic Rate. HT22 cells were seeded into a 6-well plate at a density of 1 × 10 5 cells/well. After treatment with H 2 O 2 , AEA, and/or AM251, cells were centrifuged at 1000 rpm for 5 min. After two washes with ice-cold phosphate buffered saline (PBS), cells were resuspended in binding buffer at a density of 1 × 10 6 cells/mL. And 5 L of fluorescein 5-isothiocyanate [2-(3,6-dihydroxy-9H-xanthen-9-yl)-5-isothiocyanatobenzoic acid] FITC-conjugated antiannexin-V staining antibody and 2 L of propidium iodide (PI) solution were added to 100 L of the binding buffer. After thorough mixing, and 15 min incubation at room temperature in the dark, the apoptotic rate was assessed by flow cytometry (BD, USA).  washed three times with PBS, before incubation with Cy3labeled secondary antibody (1 : 200)  2.9. Intracellular SOD, GSH, and GSSG. HT22 cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well. After treatment with H 2 O 2 , AEA, and/or AM251 (Figure 1(c)), cells were harvested and homogenized in 0.5 mL of 0.1 M phosphate buffer (pH 7.4). The mixture was centrifuged at 3000 rpm for 10 min at 4 ∘ C, and supernatants were used for SOD, GSH, and GSSG activity assessments with the corresponding reagent kits by spectrophotometry.

Short
Interfering RNA. The sequence of mouse-CB1 short interfering RNA (CB1-siRNA, sc-39911) and scrambled short interfering RNA (SC-siRNA, sc-37007) were purchased from Santa Cruz Biotechnology. HT22 cells were seeded in 6-well plates at a density of 2 × 10 5 cells/well in 2 mL DMEM without FBS. When the cell density reached 60-80% confluence, cells were washed with 2 mL siRNA Transfection Medium (sc-36868). CB1-siRNA and SC-siRNA were added to the cells and incubated for 5 h at 37 ∘ C before 1 mL of normal growth medium containing 20% FBS was added into each well without removing the transfection mixture. After an additional incubation of 24 h, cells were harvested.
2.11. Immunoblot Analysis. HT22 cells were lysed with modified RIPA-buffer containing a protease inhibitor-cocktail and 100 M phenylmethanesulfonyl fluoride on ice for 30 min. The total protein content was qualified by a bicinchoninic acid kit. Total protein lysates were subjected to 12% sodium dodecyl SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were incubated with rabbit anti-mouse primary antibody (CB1, 1 : 1000; Nox2/gp91phox, 1 : 1000; Abcam, UK; cleaved caspase-3, 1 : 500; Santa Cruz, USA) in PBS with 0.1% Tween-20 overnight at 4 ∘ C and then incubated for 1 h at room temperature with anti-rabbit IgG. -Actin, tubulin, and GADPH served as the control, respectively. Expression was visualized by enhanced chemiluminescence. The signal was quantified by densitometry by an immunoblotting detection system (Alpha Innotech, USA).

Real-Time PCR.
Total RNA was isolated from HT22 cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Total RNA (5 g) was used for first strand cDNA synthesis using the cDNA synthesis kit (TaKaRa, Japan). The PCR conditions for Nox2 were as follows: after initial denaturation at 95 ∘ C for 5 min, 40 cycles of 94 ∘ C for 30 s, 58 ∘ C for 30 s, and 72 ∘ C for 1 min were performed, followed by a 10 min extension at 72 ∘ C. The Nox2 RNA primers were as follows     determined with the relative comparative threshold method [15].

Statistical
Analysis. SPSS 11.0 (SPSS Inc, Chicago, IL) was used to conduct statistical analysis. Values were expressed as means ± standard deviation (SD). Results were compared by one-way ANOVA, followed by Tukey's Multiple Comparison Test. < 0.05 was considered statistically significant.

AEA Protected HT22 Cells Exposed to H 2 O 2 in a Dose-Dependent
Manner. HT22 cells were exposed to H 2 O 2 for 3 h, which decreased the cell metabolic activity in a dosedependent manner. Exposure to 200 M H 2 O 2 decreased the cell metabolic activity by roughly 50% (Figure 2(a)), and we used this condition for the subsequent experiments.
HT22 cells were exposed to 1 to 20 M of AEA in the presence of 200 M H 2 O 2 . 10 M and 20 M AEA significantly ameliorated the cytotoxic effect of H 2 O 2 (Figure 2(b)). We used 10 M of AEA for subsequent experiments.

AEA Upregulated CB1 Expression in HT22 Cells.
We used immunofluorescence and western blotting to assess whether AEA could up-regulate CB1 expression in HT22 cells. We observed CB1 staining in the cell membrane and cytoplasm of HT22 cells, consistent with a previous study [11]. Treatment with 10 M AEA induced a significant upregulation of CB1 expression ( < 0.05), and the selective CB1 antagonist AM251 reversed the AEA-induced up-regulation of CB1 expression (Figure 3).

Protection of AEA against Oxidative Stress in HT22
Cells Involved CB1. In the absence of AEA, AM251 did not affect the cytotoxic impact of H 2 O 2 (Figure 4(a)); however AM251 abolished the AEA-induced protection of HT22 cells, reducing the cell metabolic activity from 66.9 ± 2.4% to 49.5 ± 7.1% ( < 0.05). AM251 also reversed the influence of AEA on LDH release, increasing the LDH release from 29.1 ± 7.6 U/L to 51.2 ± 7.9 U/L ( < 0.05) (Figure 4(b)). We also evaluated cleaved caspase-3 expression and apoptotic rate by western blotting (Figure 4(c)) and flow cytometry (Figures 4(d)-4(i)), respectively, to assess the apoptosis of HT22 cells. AEA significantly decreased the expression of cleaved caspase-3 and the apoptotic rate of HT22 cells in response to H 2 O 2 ( < 0.05). And AM251 abolished these effects caused by AEA. In addition, AEA ameliorated the changes in cellular morphology elicited by H 2 O 2 and maintained the integrity of HT22 cells, and AM251 reversed this effect ( Figure 5). These results indicated AEA protected HT22 cells from the damage caused by H 2 O 2 , and AM251 reversed this protection, suggesting that the protective effects of AEA may be mediated via CB1.

AEA Decreased Intracellular ROS and Maintained Intracellular Redox Status via CB1.
Exposure of HT22 cells to H 2 O 2 led to accumulation of intracellular ROS, and simultaneous treatment with AEA markedly reduced the generation of ROS ( Figure 6). SOD plays a vital role in protecting cells against oxidative injury. H 2 O 2 treatment sharply decreased SOD activity in HT22 cells to 32.2 ± 5.0% (Figure 7(a)), and AEA restored SOD activity to 70.3 ± 4.0% of baseline ( < 0.05). GSH is also an important cellular antioxidant. H 2 O 2 treatment sharply decreased GSH activity in HT22 cells from 18.8 ± 2.7 M to 6.1 ± 1.0 M (Figure 7(b)). Simultaneous application of AEA partially restored GSH levels to 12.1 ± ( Figure 7(c)), and this effect was almost entirely abolished by AEA, which reduced GSSH levels to 1.6 ± 0.4 M ( < 0.05). The GSH/GSSG ratio was reduced from 12.3 ± 1.1 to 2.2 ± 0.3 by H 2 O 2 treatment, and AEA partially restored this balance, increasing the ratio to 7.3 ± 0.7 (Figure 7(d)). The influences of AEA on intracellular ROS, SOD, GSH, GSSG, and GSH/GSSG ratio were abolished by the CB1 antagonist AM251, indicating that the antioxidative effects of AEA may be mediated via CB1 of HT22 cells.

CB1 Knockdown Reversed the Beneficial Effects of AEA on HT22 Cells Exposed to H
To further determine whether AEA-induced antioxidative ability was mediated by CB1 in HT22 cells, we used CB1-siRNA to knock down the expression of CB1. CB1-siRNA was effective in reducing the expression of CB1 (Figure 8(a)) and reversed AEA-induced cytoprotection, leading to a significant reduction of cell metabolic activity (Figure 8(b)), an increase of LDH release (Figure 8(c)), and a rise of intracellular ROS level ( Figures  8(d)-8(i)).  Results are expressed as means ± SD ( = 6). * < 0.05 versus the control (no H 2 O 2 , no AEA, and no AM251), # < 0.05 versus the cells exposed to H 2 O 2 alone, and ∧ < 0.05 versus the cells exposed to AEA plus H 2 O 2 . (Figure 9(a)). AEA decreased the Nox2 protein expression (Figure 9(b)) and mRNA transcription (Figure 9(c)). However, CB1 antagonist AM251 or CB1-siRNA (Figure 9(d)) abolished AEA-induced influence on Nox2 protein expression and mRNA transcription, suggesting that the Nox2 may be involved in AEA-induced cytoprotection against H 2 O 2 via CB1.

Nox2 Was
To further investigate the role of Nox2 in AEA-induced protection in HT22 cells exposed to H 2 O 2 , we used apocynin, a specific Nox inhibitor. We found that the presence of 50 M apocynin decreased the expression of Nox2 protein significantly (Figure 10 Results are expressed as means ± SD ( = 6). * < 0.05 versus control (no H 2 O 2 , no AEA, and no AM251), # < 0.05 versus the cells exposed to H 2 O 2 alone, and ∧ < 0.05 versus the cells exposed to AEA plus H 2 O 2 . and apocynin did not cause a more significant reduction of Nox2 expression than either AEA or apocynin used alone ( > 0.05). Similarly, we noticed a combination of AEA and apocynin did not induce a more significant increase of cell metabolic activity (Figure 10(b)) and reduction of LDH release (Figure 10(c)) than either AEA or apocynin alone ( > 0.05), indicating that Nox2 inhibition may be involved in AEA-induced cytoprotection against H 2 O 2 .

Discussion
In this study, we found that 10 M AEA treatment of a murine hippocampal neuron cell line, HT22, significantly improved cell injury, decreased apoptosis, and ameliorated the morphological changes induced by oxidative stress in the form of 200 M H 2 O 2 . Treatment with AEA reduced intracellular ROS and Nox2 expression in HT22 cells exposed to H 2 O 2 , and these effects were reversed by application of CB1 antagonist AM251 or CB1-siRNA. In addition, Nox inhibitor apocynin plus AEA did not induce a more significant downregulation of Nox2 or neuroprotection than apocynin or AEA used alone. These findings indicate that Nox2 inhibition is involved in AEA-induced neuroprotection against H 2 O 2 through CB1 activation in HT22 cells.
Oxidative stress is involved in the pathophysiology of many CNS diseases [1][2][3]. Overaccumulation of intracellular ROS causes oxidative stress, which can damage cellular membranes, injure the mitochondria, and induce cell death. Thus enhancement of the cellular processes that suppress ROS generation or remove excess ROS may be effective in treating oxidative stress-induced diseases. Recently Nox proteins have been demonstrated to be major producers of ROS in CNS cells such as neurons, astrocytes, and microglia under pathophysiological conditions [16,17]. Thus, inhibition of Nox proteins may present an effective mechanism to limit oxidative stress in the CNS. The Nox family includes Nox 1, 2, 3, 4, and 5, dual oxidase (DUAX) 1, and DUAX2 [18]. Nox2 appears to be the most important Nox in cerebral injury [19]. The infarct volume of Nox2 deficient mice is smaller than that of the wild-type and Nox2 deficient mice experience less blood-brain barrier injury than wild-type mice in a stroke model [20,21]. Cannabinoids, such as AEA, appear to protect neurons against excitotoxicity, oxidative stress, and hypoxia through the activation of CB1 [12,22]. Moldzio et al. reported that the cannabinoid THC protected dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP + ) induced oxidative injury [23]. Chung et al. reported that cannabinoids WIN55, 212-2, and HU210 decreased lipopolysaccharideinduced activation of Nox in microglia [24]. We, therefore, hypothesize that inhibition of Nox may be involved in cannabinoid-induced protection from oxidative stress. In one of our previous studies, we observed that electroacupuncture induced neuroprotection against cerebral ischemia by increasing cerebral levels of the endogenous cannabinoid AEA [25]. However, AEA-mediated neuroprotection which is mediated via inhibition of Nox remained unknown. Thus, we investigated the protective effects of AEA against H 2 O 2induced neuronal injury.
The cannabinoid receptor CB1 is a G protein-coupled receptor present in both the cellular membrane and cytoplasm of neurons [26]. Nox2 is also mostly localized on the cellular membrane of neurons [19]. We found that antagonism of CB1 or silencing of CB1 expression reversed the AEA-induced inhibition of Nox2 protein expression and Nox2 mRNA transcription. In addition, Nox inhibitor apocynin alone or plus AEA did not induce a significant downregulation of Nox2 expression compared with AEA alone in the HT22 cells exposed to H 2 O 2 . Therefore, we inferred that AEA may activate CB1, which, in turn, inhibits Nox2 expression resulting in a reduction of cellular ROS. Intracellular redox balance reflects the level of oxidative stress levels in cells and is crucial to cell function and survival. Accumulation of oxidants or overconsumption of antioxidants will damage cellular metabolism and even result in cell death [27]. In this study, we found that when in conditions of oxidative stress AEA raised intracellular SOD and GSH, reduced GSSG, and increased the GSH/GSSG ratio, and these effects were reversed by CB1 antagonist AM251, indicating that AEA could restore the balance of intracellular antioxidative and oxidative substances via CB1 receptor.
The ability of the endocannabinoid AEA to protect HT22 cells against H 2 O 2 -induced injury recommends AEA as a candidate therapy for oxidative stress-related neurological disorders. Endogenous agents have specific inactivation systems and therefore may run less risk of interfering with ongoing developmental profiles than artificial ligands. This is vital, as low levels of AEA can affect embryonic implantation, neural development, and suckling [28,29]. Furthermore, cannabis use has been associated with the onset of schizophrenia [30,31]. Exposure to synthetic Results are expressed as means ± SD ( = 4). * < 0.05 versus the control (no H 2 O 2 , no AEA, and no AM251 or siRNA), # < 0.05 versus the cells exposed to H 2 O 2 alone, and ∧ < 0.05 versus the cells exposed to AEA plus H 2 O 2 .
cannabinoids WIN55, 212-2 caused disruption of learning and decreased emotional reactivity [32]. Thus interventions targeting the cannabinoid system need to be minimal during development [33] and endogenous agonists may be less deleterious.
However, further work must be done to determine the relevance of our findings in vivo. We have not fully elucidated the precise antioxidative mechanisms induced by AEA. In this study we investigated only the role of Nox2 in AEA efficacy, and whether other components of Nox are associated with AEA activities is not yet known.

Conclusions
This study provides evidence that the endocannabinoid AEA protects the mouse hippocampal neuron cell line HT22 against H 2 O 2 -induced oxidative injury through CB1mediated inhibition of Nox2.