A total of 10 neonatal Sprague-Dawley (SD) rats (weighing, 5–6 g, uncertain gender, fed by breastfeeding, kept in an environment at 21±2°C) were obtained from the Experimental Animal Center of Southern Medical University, (Guangzhou, China). The institutional review boards of all participating centers approved the protocols used in the study, and all experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Southern Medical University (Guangzhou, China; permit number: 2016213). Neurons were isolated through enzymatic digestion of the brain tissue of the rats, as previously described (20). Briefly, the rats were sacrificed by an overdose of 1% sodium pentobarbital (40 mg/kg per SD rat) administered via intraperitoneal injection, and then the cerebral cortices from neonatal SD rats (0–2 days old) were dissected and trypsinized with 0.125% trypsin for 30 min at 37°C. Samples were passed through a 200-μm mesh sieve and cells were collected by centrifugation (500 × g for 5 min). Cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml streptomycin and 100 U/ml penicillin, then seeded onto poly-D-lysine-coated 25-mm dishes and cultured at 37°C in a 5% CO₂ atmosphere. Culture medium was replenished twice a week with fresh complete DMEM.

At 24 h after plating, neurons were divided into the following five groups: Control, model, NAR-L (low-dose), NAR-M (medium-dose) and NAR-H (high-dose). Neurons in the control group were cultured under normal conditions for 6 days. Neurons in the model group were cultured under normal conditions for 4 days, followed by oxygen deprivation (90% N₂, 5% O₂ and 5% CO₂) for 12 h and oxygen restoration (95% air and 5% CO₂) for 36 h. In the NAR treatment groups, neurons were cultured under normal conditions for 4 days, then co-cultured with different concentrations of NAR (20 μM for NAR-L, 40 μM for NAR-M and 80 μM for NAR-H) for 8 h, followed by oxygen deprivation for 12 h and oxygen restoration for 36 h. In the time-course assay, neurons were co-cultured with 80 μM NAR for 0, 2, 4 and 8 h prior to oxygen deprivation/reoxygenation, and assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

The generation of ROS was measured by chloromethyl-2′,7′dichlorodihydro fluorescein diacetate (CM-H₂DCFDA) staining. For CM-H₂DCFDA staining, cells were washed with ice cold PBS and treated with 10 μM CM-H₂DCFDA (Invitrogen, Thermo Fisher Scientific) in DMEM for 45 min. Cells were then trypsinized and suspended in phosphate-buffered saline (PBS) and fluorescence was measured at 538 nm. Furthermore, the activities of superoxide dismutase 1 (SOD1) and glutathione (GSH), and the content of malondialdehyde (MDA), in the different groups were determined to assess the anti-oxidative effects of NAR using commercial kits (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China).

The authors determined mitochondrial ANT transport activity using an inhibitor termination method. A total of 20 μl ³H-ADP solution (0.3 μmol/l) was added into 50 μl of prepared mitochondrial suspension for 10 sec, then 50 μl of the ANT inhibitor atrac-tyloside (3.2 nmol/l) was added and mixed immediately to terminate ANT transporter function. The mixture was centrifuged for 20 min at 3,000 × g and 4°C and the supernatant was discarded. After resuspension and washing with 1 ml of separation medium, 400 μl H₂O₂ (8.8 mol/l) was added to the precipitate, which was subsequently incubated in a 70°C water bath for 40 min. ANT transport activity was measured using the liquid scintillation counting method (Triathler liquid scintillation counter; Triathler, Turku, Finland). ANT transport activity is positive proportional to the number of scintillation counts (dpm/sec), and the number of scintillation counts can reflect the change in ANT transport activity of different groups.

The authors used JC-1 staining to measure mitochondrial depolarization in neurons of the control, model, NAR-L, NAR-M and NAR-H groups. Following treatment, 200 μl cells were collected and incubated for 20 min with an equal volume of JC-1 staining solution at 37°C, then rinsed twice with PBS. Δψm was determined by measuring the relative amount of dual emissions from mitochondrial JC-1 monomers or aggregates using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Δψm was presented in units of fluorescence intensity.

The levels of adenine nucleotides in the neurons of each group were determined by high-performance liquid chromatography (HPLC), according to the method described by Yang et al (21). The following conditions were used for HPLC: wavelength, 254 nm; sensitivity, 0.01 AUFS; mobile phase, 2 mmol/l PBS (pH 5.5); and speed, 1 ml/min. A YWG-ODS C18 (10 μm) column (4.6×250 nm) was used.

Cells were harvested during the logarithmic growth phase. Small interfering RNAs (siRNAs) against Nrf2 (siNfr2) and negative control siRNAs (siRNA-NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into cells using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific), according to the manufacturer's protocol. The sequences of siNrf2 and siRNA-NC are presented in Table I. Following transfection, western blotting was used to assess the transfection efficiency, and model and NAR treatment groups were established prior to further experiments.

Cell viability was determined using an MTT assay. Briefly, neurons were plated onto 96-well plates at a density of 1×10⁴ cells/well. Following treatment, 20 μl MTT (5 mg/ml) was added to each well and incubated for 4 h. The medium was then removed and the formazan crystals were dissolved with dimethyl sulfoxide (DMSO). Absorbance was read at 570 nm with a microplate reader (Multiskan Spectrum; Thermo Fisher Scientific).

Apoptotic cells were recognized and distinguished from normal cells using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit for flow cytometric analysis, according to the manufacturer's instructions (Invitrogen, Thermo Fisher Scientific). After treatment, neurons were harvested and washed twice with cold PBS, then incubated with 5 μl FITC-Annexin V and 5 μl PI working solution (100 μg/ml) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometric analysis (BD Accuri C6; BD Biosciences) (22).

Neurons were lysed in a radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40] containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Basel, Switzerland) using standard procedures. The total proteins concentration was determined using a Bio-Rad protein assay system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total of 50 μg proteins were subjected to 12% SDS-PAGE, and then transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% (w/v) skimmed milk in 0.05% Tris-buffered saline with Tween-20 at room temperature for 2 h. Immunoblotting was performed with primary antibodies against Nrf2 (ab31163, dilution 1:1,000; Abcam, Cambridge, MA, USA) and its downstream targets heme oxygenase 1 (HO-1, ab1324, dilution 1:250; Abcam) and NAD(P)H quinone dehydrogenase 1 (NQO1, ab28947, dilution 1:1,000; Abcam). β-actin levels were used for normalization. The protein bands were scanned using ECL western blotting detection reagents (Pierce, Rockford, IL, USA) and quantified using a ChemiDoc image analysis system (Bio-Rad Laboratories).

Total RNA was extracted from the cultured neurons of each group using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). A cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to synthesize cDNA according to the manufacturer's instructions. qPCR was performed using an iCycleriQ Detection System (Bio-Rad Laboratories) and interaction dye SYBR-Green. The sequences of the primers used are listed in Table II. GAPDH mRNA levels were used for normalization. DNA was amplified with an initial denaturation at 95°C for 5 min, followed by 39 cycles of 95°C (15 sec) and 60°C (15 sec). RT-qPCR data were analyzed and expressed as relative mRNA levels using CT values (23) and were subsequently converted to fold changes.

All the original experimental data were analyzed using the SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). Comparisons among the groups were performed by one-way analysis of variance (ANOVA), and Fisher's least significant difference test was used for comparisons between two groups. P<0.05 was considered to indicate statistically significant differences.

To assess the antioxidant effect of NAR, ROS levels in the neurons of the different cell groups were analyzed by CM-H₂DCFDA staining. As presented in Fig. 1A, the authors observed an increased accumulation of ROS in the model group when compared with the control group (P<0.01). In turn, the levels of ROS were significantly decreased in the NAR-H group when compared with the model group (P<0.05). Additionally, compared with the model group, the high-dose NAR group exhibited significant increases in the activities of SOD1 and GSH (P<0.05; Fig. 1B and C). Furthermore, the level of MDA was significantly decreased in the NAR-H group when compared with the model group (P<0.05; Fig. 1D). The levels of MDA and ROS, and activities of SOD1 and GSH, did not differ significantly between the NAR-L, NAR-M and model groups (P>0.05).

Mitochondria are considered to be a major producer of ROS in mammalian cells, and overproduction of ROS may be an indicator of mitochondrial dysfunction (24). Thus, the authors initially measured the concentration of adenylate in the different cell groups using a HPLC method. The data indicated that, compared with the control group, the levels of ATP, ADP and AMP decreased significantly in the model group, whereas the NAR-H groups exhibited notable improvements in adenylate levels when compared with the model group (Table III; P<0.05).

ANT transport activity can reflect ATP synthesis and normal cell function. Therefore, the authors subsequently detected mitochondrial ANT transport activity in the neurons of the different cell groups. The data indicated that mitochondrial ANT transport activity was significantly decreased in the model group when compared with the control group (P<0.05), and significantly increased in NAR-H group when compared with the model group (P<0.05). However, ANT transport activity did not differ significantly between the NAR-L, NAR-M and model groups (P>0.05). These results are presented in Fig. 2A.

As in Fig. 2B, Δψm was significantly reduced in the model group when compared with the control group (P<0.05), while Δψm was markedly increased in the NAR-M and NAR-H groups when compared with the model group (P<0.05). Δψm did not differ significantly in the NAR-L group (P>0.05).

It is well established that intracellular oxidative stress and mitochondrial dysfunction cause cell injury and neurotoxicity (25). Therefore, flow cytometry was performed to assess cell apoptosis in the different groups. As shown in Fig. 3A, the number of apoptotic cells was significantly increased in the model group when compared with the control group (P<0.01), while the number of apoptotic cells was significantly decreased in the NAR-H group when compared with the model group (P<0.05). To verify results of the apoptosis assay, western blot analysis was performed to detect markers of apoptosis, namely B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cytosolic cytochrome c and cleaved caspase-3 (Fig. 3B and C). A strong increase was observed in the expression of Bax and reduced expression of Bcl-2 in the model group (P<0.01). Furthermore, the expression levels of cytosolic cytochrome c and cleaved caspase-3 were markedly increased in the model group (P<0.01). However, high-dose NAR could reverse these expression changes (P<0.05). An MTT method was subsequently used to measure cell viability in the different groups. The data indicated that the viability of cells was significantly decreased in the model group and increased in the NAR-H group (P<0.05; Fig. 4A). However, neuronal viability, along with the rate and markers of apoptosis, did not differ significantly in the NAR-L and NAR-M groups (P>0.05).

NAR increases the gene expression of Nrf2 and its target genes. The authors performed RT-qPCR and western blot analysis to measure the expression level of Nrf2 and its target genes in oxidative stressed neurons following treatment with different concentrations of NAR. The experiments demonstrated that the protein and mRNA levels of Nrf2 and its target genes HO-1 and NQO1 were significantly decreased in the model group and increased in the NAR-H group (P<0.05; Fig. 4B–D). In addition, NAR induced the transcription of Nrf2, HO-1 and NQO1 in an apparent dose- and time-dependent manner (Fig. 5).

To further explore the effect of NAR on neurons, Nrf2 was knocked down using RNA interference. Compared with the siRNA-NC cell group, the expression levels of Nrf2 and its target genes HO-1 and NQO1 were markedly reduced in the siNrf2 group, as demonstrated by western blot analysis (P<0.01; Fig. 6). These data indicated that knockdown of Nrf2 using RNA interference was successful. Moreover, in both the siRNA-NC and siNrf2 cell groups, NAR-H treatment could increase the expression levels of Nrf2 when compared with transfected model neurons (P<0.05; Fig. 7).

In addition, the authors assessed the viability and apoptosis of neurons following Nrf2 knockdown. Compared with the siRNA-NC group, the viability of neurons was significantly decreased and the apoptosis of neurons was markedly increased in the siNrf2 cell group (P<0.05). These results indicated that Nrf2 could regulate the viability and apoptosis of neurons. Following NAR treatment, the data indicated that NAR could rescue the viability of neurons in the siRNA-NC and siNrf2 cells groups. Notably, NAR-H treatment significantly rescued the viability of transfected model neurons (P<0.05; Fig. 7C). In addition, the data indicated that NAR treatment could inhibit the apoptosis of neurons in the siRNA-NC and siNrf2 cell groups; NAR-H treatment significantly rescued the apoptosis of transfected model neurons (P<0.05; Fig. 7D).

Taken together, these data suggested that NAR could activate the Nrf2/ARE signaling pathway and regulate the gene expression of Nrf2 and its downstream targets.