Myr (CAS no. 607 91 0) was obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). The cell culture materials and reagents were obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). DRG neuron culture was based on previous studies, including our own (20–22). Sprague-Dawley (SD) rats (n=24; weight, 25–30 g; male) were purchased from the Animal Laboratory of Wannan Medical College (Wuhu, China). Rats were kept on a 12 h/12 h light-dark cycle and freely given food and water in a pathogen-free area. All procedures that involved animals were approved by the Institutional Animal Care and Use Committee of Yijishan Hospital (Wuhu, China). DRG neurons were harvested from rats at day 15. The DRG neurons were digested with 0.25% trypsin in Hank's Balanced Salt solution (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 20 min, and added to 10% fetal bovine serum to prevent digestion. Cells were passed through a 74-µm filter and centrifuged in 1,500 × g at 4°C for 5 min. The pellet was resuspended in a neurobasal medium of 2% B-27 supplement, 10 ng/ml nerve growth factor, 1 mmol/l L-glutamine and 1000 U/ml penicillin/streptomycin/neomycin solution. Dissociated DRG neurons were cultured in poly-D-lysine-precoated 6-well culture clusters at 2×10⁶ cells/well in 2 ml. DRG neurons were cultured in media at 37°C with 5% CO₂ and maintained in media containing 20 µmol/l floxuridine for another 24 h to inhibit the growth of non-neuronal cells. The purity of neuronal cells was confirmed by fluorescent labeling of neurofilament protein 200 (cat. no. orb18247; dilution, 1:600; Biorbyt Ltd., Cambridge, UK) and NeuN (cat no. 104225; dilution, 1:500, Abcam, Cambridge, UK) (data not shown). All experimental procedures were performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Committee of Wannan Medical College (Wuhu, China).

Cell viability was measured via a quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich; Merck Millipore). Briefly, DRG neurons were seeded into 96-well plates at 5×10⁴ cells per well. A portion of cells were treated with 1% O₂ (hypoxia) for 24 h, and incubated with 5 mg/ml MTT solution for a further 4 h. A total of 100 µl dimethyl sulfoxide was added to each well and the absorbance was measured at a wavelength of 540 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell viability was expressed as the ratio of clustered DRG neurons to control group The control group were cultured in neurobasal medium with 2% B-27 supplement, 10 ng/ml nerve growth factor, 1 mmol/l L-glutamine and 1,000 U/ml penicillin/streptomycin/neomycin solution, which were all purchased from were purchased from Sigma Aldrich; Merck Millipore.

Dulbecco's Modified Eagle's medium/F12, FBS and neurobasal cell medium were purchased from Gibco; Thermo Fisher Scientific, Inc. The Apoptosis Detection System kit (Roche Diagnostics GmbH, Mannheim, Germany) was used for the TUNEL assay, according to the manufacturer's protocol. DRG neurons were seeded into 96-well plates at a density of 1×10⁵ cells/well. After fixing with 4% paraformaldehyde for 1 h, DRG neurons in each groups were washed with PBS and treated with 1% Triton X-100 in PBS for 30 min on ice. And then incubated for 60 min at 37°C with 50 µl of TUNEL reaction mixture. Cells were subsequently incubated with DAPI (dilution, 1:800; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at room temperature. After washing with PBS, the cells were analyzed by confocal microscopy (magnification, ×40; Zeiss 510; Zeiss AG, Oberkochen, Germany). DRG neurons untreated with myristicin and/or hypoxia were used as a control group. To avoid counting the same cell in more than one region, the authors counted every fifth section (50 µm apart). For each plate, six fields of view were examined.

DRG neurons were washed twice with cold PBS and lysed in radioimmunoprecipitation assay buffer consisting of 50 mM TRIS, 150 mM NaCl, 2% sodium dodecyl sulfate (SDS) and a protease inhibitor mixture (Roche Diagnostics GmbH). Equal amounts of protein (2.0 mg/ml, 10 µl in each lane) were separated by 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk and incubated with primary antibodies at 4°C overnight. The following primary antibodies were used: B cell lymphoma 2 (cat. no. SAB4300339; dilution, 1:500; anti-rabbit; Sigma-Aldrich; Merck Millipore), Bcl-2 associated X protein (cat. no. SAB4502549; dilution, 1:800; anti-rabbit; Sigma-Aldrich; Merck Millipore), cleaved caspase-3 (cat. no. sc-22171; dilution, 1:500; anti-rabbit; Santa Cruz Biotechnology, Inc.), CHOP (cat. no. 2895P; dilution, 1:500; anti-rabbit; Cell Signaling Technology, Inc.), GRP78 (cat. no. G9043; dilution, 1:800; anti-rabbit; Sigma-Aldrich; Merck Millipore), cleaved caspase-12 (cat. no. sc-70227; dilution, 1:500; anti-rabbit; Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. sc-47778; dilution, 1:1,000; anti-rabbit; Santa Cruz Biotechnology, Inc.). Membranes were subsequently incubated with a horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (cat. no. sc-2357; dilution, 1:5,000, Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Following each incubation, membranes were extensively washed in TBS containing Tween-20 and the immunoreactive bands were detected using an enhanced chemiluminescence kit (cat. no. orb90503; Biorbyt, Ltd.). The quantification of Western blotting was conducted using a computerized image-analysis system (Image Pro Plus; version, 6.0; Media Cybernetics, Inc., Rockville, MD, USA) in duplicate.

The DRG neurons were randomly divided into four groups: Control group, control plus Myr group, hypoxia group, and hypoxia plus Myr group. DRG neurons were seeded at 5×10⁴ per well in 6-well plates, cells were harvested and washed twice with cold PBS, before being lysed in radioimmunoprecipitation assay buffer consisting of 50 mM TRIS, 150 mM NaCl, 2% SDS and a protease inhibitor mixture (Roche Diagnostics GmbH). DRG neurons were quantified using the bicinchoninic acid kit for protein determination (cat. no. BCA1-1KT; Sigma-Aldrich; Merck Millipore). SOD (cat. no. 20080829), GSH-PX (cat. no. 20080801) and MDA (cat. no. 20080801) levels in the supernatant were measured using commercially available kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All assays were conducted according to the manufacturer's protocol.

LDH activity was evaluated using a colorimetric LDH assay. DRG neurons were seeded onto a 96-well plate under a 1% O₂ environment for 24 h. Briefly, 100 µl supernatant was transferred from each well to a new 96-well plate and 100 µl fresh reaction mixture was added to each well. After 30 min of incubation at room temperature in the dark, the optical density values were detected at a wavelength of 490 nm using a microplate reader (Thermo Labsystems, Santa Rosa, CA, USA). The quantity of LDH was calculated as a percentage compared with the total amount of LDH present in cells treated with 1% Triton-X 100 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). LDH levels were measured using a commercially available kit (cat. no. 20071129) purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Total RNA was extracted using TRIzol^® solution (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The quantity and quality of isolated RNA were evaluated using absorbance at wavelengths of 260 and 280 nm. Following this, RT was performed in a 20 µl reaction mixture using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). Primer sequences were as follows: Forward, 5′-GGACCTGACCTGCCGTCTAG-3′ and reverse, 5′-GTAGCCCAGGATGCCCTTGA-3′ for β-actin; forward, 5′-GGUAUGAGGACCUGCAAGA-3′ and reverse, 5′-CACCAAGCAUGAACAAUUG-3′ for CHOP; forward 5′-CUACCCAAACAUCGGGAAA-3′ and reverse, 5′-CUCCAGAGAUGCUGAGCGA-3′ for GRP78. qPCR with SYBR^® Green I (Shanghai Hi-Tech Enterprise Bio Co., Shanghai, China) (http://china.53trade.com/web0/disp_company_info.asp?userid=166543) was performed using a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with thermocycling conditions as follows: An initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 60 sec and extension at 72°C for 30 sec. All samples were processed in triplicate. The mRNA expression levels of each gene were calculated using the relative quantitative method (23).

DRG neurons were fixed with 4% PFA for 1 h, washed with PBS containing 0.1% Triton X-100 (PBST), and blocked for 30 min in PBST supplemented with 10% FBS. DRG neurons were subsequently incubated with cleaved antibodies against caspase-12 (cat. no. sc-70227; dilution, 1:800; anti-rabbit; Santa Cruz Biotechnology, Inc.) in the same solution overnight at 4°C, washed and incubated with a mouse anti-rabbit IgG-HRP conjugated secondary antibody (cat. no. sc-2357; dilution, 1:5,000, Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Nuclei were stained with DAPI (1:800; Santa Cruz Biotechnology, Inc.). The cells were examined under a Leica fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).

All data are presented as the mean ± standard deviation of at least three independent experiments. All statistical analyses were performed using SPSS software version 11.0 (SPSS, Inc., Chicago, IL, USA). Statistical comparisons between the different treatments were performed using one-way analysis of variance with Tukey's multiple comparison post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

DRG neurons were exposed to 10–200 µM Myr, the chemical structure of which is presented in Fig. 1, for 24 h. Concentrations of 10–50 µM did not affect cell viability; however, viability was reduced with concentrations >50 µM (Fig. 2A). Therefore, 10–50 µM Myr was selected to treat the 1% O₂ (hypoxia)-exposed DRG neurons for 24 h. Myr inhibited the hypoxia-induced decreased cell viability of DRG neurons in a dose-dependent manner (Fig. 2B). The results confirmed that the optimal concentration of Myr was 50 µM.

TUNEL staining was used to assess apoptotic cells. As presented in Fig. 3A, increased TUNEL staining was observed in the hypoxia-induced DRG neurons compared with the normal group. The number of TUNEL-positive cells was reduced in DRG neurons following Myr treatment; however, Myr treatment had no effect on the normal group. Quantification of TUNEL staining revealed that Myr significantly reduced the percentage of TUNEL-positive DRG neurons following hypoxia (Fig. 3B).

To determine the possible involvement of the mitochondrial pathway of apoptosis in hypoxia-induced DRG neurons, protein expression levels of the pro-apoptotic Bax, the apoptosis protease cleaved caspase-3 and the anti-apoptotic Bcl-2 were detected by western blot analysis (Fig 4A). Increased protein expression levels of Bax and cleaved caspase-3, and reduced protein expression levels of Bcl-2, were detected in the hypoxia-induced group compared with the normal group; these effects were significantly abrogated by Myr treatment (Fig. 4B-D). Therefore, Myr may reverse hypoxia-induced apoptosis in DRG neurons by affecting the protein expression levels of apoptosis-associated molecules.

To investigate the effect of hypoxic injury on DRG neurons, the present study analyzed MDA content, LDH release and SOD and GSH-PX activities in the four groups. The MDA content (Fig. 5A) and LDH release (Fig. 5B) in hypoxia-induced DRG neurons was greater compared with the normal group, whereas SOD (Fig. 5C) and GSH-PX (Fig. 5D) activities were reduced. Following treatment with Myr, these hypoxia-induced effects were significantly attenuated. Therefore, Myr exhibited a protective effect against hypoxic injury in DRG neurons.

The levels of CHOP and GRP78 were analyzed by western blot analysis (Fig. 6A) to determine whether ERS was involved in apoptosis of hypoxia-induced DRG neurons. CHOP (Fig. 6B) and GRP78 (Fig. 6C) protein expression levels were significantly increased in DRG neurons treated with hypoxia, and this increase was significantly inhibited by Myr. Similarly, CHOP (Fig. 6D) and GRP78 (Fig. 6E) mRNA expression levels in the hypoxia group were significantly increased compared with the normal group, as assessed by RT-qPCR; however, this was significantly downregulated by Myr. The protein expression levels of cleaved caspase-12 were analyzed as it is a specific marker of ERS-induced apoptosis (Fig. 7A). Western blot analysis indicated that hypoxia significantly increased its protein expression levels; however, this was significantly inhibited by treatment with Myr (Fig. 7B). Immunofluorescence verified this (Fig. 7C). These results suggested that Myr may inhibit hypoxia-ERS-induced apoptosis in DRG neurons.