METH was obtained from Criminal Investigation Institute of Liaoning Province. OT was obtained from Key Organics. Phosphate buffer saline (PBS) was obtained from Hyclone. Atosiban, Dimethyl sulfoxide (DMSO), 4′, 6-diamidino-2-phenylindole (DAPI), Poly-l-lysine and MTT were obtained from Sigma-Aldrich. B27 supplement, Neurobasal Medium, Fetal Bovine Serum (FBS), 0.25% Trypsin-EDTA, DMEM/F12 medium and penicillin/streptomycin were obtained from Gibco. Mitochondrial membrane potential assay kit with JC-1, ROS Assay Kit, BCA protein assay kit, Alexa Fluor 488, Goat Anti-Rabbit lgG and Goat Serum were obtained from Beyotime Biotechnology. Horseradish peroxidase (HRP)-goat anti-mouse IgG and HRP-goat anti-rabbit IgG were obtained from Nakasugi Golden Bridge. Anti-Oxytocin Receptor and anti-beta III Tubulin antibodies were obtained from Abcam. Bax, CREB, Anti-Phospho-CREB (P-CREB), Anti-P44/42MAPK (ERK1/2), Anti-Phospho-P44/42MAPK (P-ERK1/2), Anti-Caspase-3 and anti-Cleaved Caspase-3 antibodies were obtained from Cell Signaling Technology. Anti-Bcl-2 Polyclonal antibody was obtained from Proteintech. Anti-beta-actin antibody was obtained from Santa Cruz Biotechnology. Anti-GAPDH antibody was obtained from Hangzhou Xianzhi Biological Technology Co., Ltd.

Male and female Sprague Dawley rats (250–280 g) were used to obtain neonatal rats were from Liaoning Changsheng Biotechnology Co., Ltd. Primary hippocampal neurons were prepared as previously described with minor modifications (Zhang et al., 2010). Briefly, hippocampal regions were removed from Sprague Dawley rats which were neonatal within 24 h in Ca²⁺ and Mg²⁺ free Hank’s balanced salt solution under a stereo microscope in a sterile environment. After the hippocampus tissue was cut into small pieces by ophthalmological scissors, an appropriate amount of 0.25% Trypsin-EDTA was added to the shredded hippocampus and then digested in a 37°C water bath for 15 min. Trypsin digestion was then stopped by adding an appropriate amount of DMEM/F12 medium containing 10% FBS and discard the supernatant. Add fresh DMEM/F12 containing 10% FBS culture medium and gently blow the hippocampus tissue into a single cell with a hot-polished plastic pipette. After standing for about 1 min, the supernatant cell suspension was filtered through a 200 mesh cell sieve to prepare a single cell suspension. Aspirate the mixed cell suspension for cell counting. The cell density was adjusted to 5×10⁵ cells/mL and inoculated into a culture plate coated with 0.01% poly-lysine in advance, gently mixed and cultured in a humidified 95% air, 5% CO₂ incubator. After 4 h of culture, the culture medium was replaced with a medium containing 86% Neurobasal medium, 10% FBS, 2% B27, 1% glutamine stock solution, and 1% double antibody (penicillin, streptomycin). After that, half of the culture solution was replaced every 2 d, and neurons cultured for 7–10 d were selected for subsequent experiments. The rats were used in this study and the cultivation protocol of primary hippocampal neurons were reviewed by the Animal Ethics Committee of Shenyang Pharmaceutical University, and the regulations were consistent with the Guideline for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Neuron purity were identified as described previously (Zhang et al., 2010). Adding appropriate amount of 4% PFA fixed hippocampal neurons for 20 min. Then cells were permeabilized with 0.03% Triton X-100 for 10 min, block with 5% goat serum for 1 h at room temperature. Incubate with 0.1% beta-Tubulin III diluted in PBS at 4°C overnight and rinse with phosphate buffer saline with 0.1% Tween-20 (PBST). 0.5% Alexa Fluor 488-labeled goat anti-rabbit secondary antibody was added and incubated for 1 h at room temperature in the dark, then rinsed with an appropriate amount of PBST. Add DAPI staining in the dark for 10 min, discard the stain, and add a proper amount of PBST to rinse. After adding an appropriate amount of PBST to each well, the cells were observed using an inverted fluorescence microscope.

The primary hippocampal neurons were then randomly divided into six groups. The control cells were cultured for 24 h in a medium without drug, and the cells in OT (0.01–1 μM) group, METH (1–8 mM) group and ATO (0.01–1 μM) group were cultured for 24 h in a drug-containing medium. OT + METH group and ATO + OT + METH group were pre-incubated with optimal concentration of OT (1 μM) and/or ATO (1 μM) for 2 h before treatment with METH, and then treated with METH (4 mM) for 24 h. After drug treatment, cells in each group were tested for cell viability by MTT assay. ROS, mitochondrial membrane potential, Cleaved Caspase-3 and p-ERK1/2 were detected by immunofluorescence. Bcl-2, Bax, Cleaved Caspase-3, Caspase-3, CREB, P-CREB, ERK1/2, P-ERK1/2 and OTR were detected by western blotting.

Hippocampal neurons were cultivated in 48-well plates for 7 d. Assay for cell viability refer to the literature (Chang et al., 1998). Hippocampal neurons cultured for 7 d were incubated with drugs for a certain period of time at 37°C, and four replicate wells were set in each group. Fresh culture medium and a final concentration of 0.25 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution were added during the test, and the cells were incubated in a 37°C incubator (95% air, 5% CO₂) for 4 h. The medium was then aspirated, an equal volume of DMSO solvent was added and mixed with a micro-vibrator for 10 min. Absorbances were read at 570 nm using a microplate reader. The experiment was repeated for 6 times. Viable cells (cell viability, CV) were measured in percentage with respect to the following formula: C V ( % ) = ( OD  ( sample ) ) / ( OD  ( Control ) ) × 100 %

Fluorescence intensity changes of Dichloro-dihydro-fluorescein diacetate (DCFH-DA) could quantitatively detect intracellular ROS levels. DCFH-DA itself has no fluorescence and could pass through the cell membrane freely. After entering the cell, it could be hydrolyzed by intracellular esterase to form DCFH. DCFH does not penetrate the cell membrane, making it easy for the probe to be loaded into the cell. DCFH could be converted to DCF after exposure to ROS. By measuring the fluorescence of DCF, the level of intracellular ROS could be known, and the fluorescence intensity is directly proportional to the intracellular ROS level. Rosup is a drug that raises intracellular ROS. Rosup (100 μM) was added to the untreated cell culture plates and incubated for 30 min in a 37°C incubator. The drug-treated medium was discarded, and fluorescent probe DCFH-DA (10 μM) was added to the drug-treated, Rosup-treated cells in a 37°C incubator for 20 min in the dark. The loaded cells were washed with serum-free medium to remove residual probes. Then an appropriate amount of cell culture medium was added to the cells, observed with a confocal microscope (Nikon, C2 Plus).

When the mitochondrial membrane potential is high, 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) aggregates in the matrix of mitochondria in the form of polymer, which could produce red fluorescence; when the mitochondrial membrane potential was low, JC-1 could not accumulate in the matrix of mitochondria, only exist in the cytosol, JC-1 was a monomer at this time, which could produce green fluorescence. The relative proportion of commonly used red-green fluorescence was a measure of the proportion of mitochondrial depolarization. Decreased mitochondrial membrane potential was a hallmark feature of early apoptosis. CCCP (10 μM, a drug that lowers mitochondrial membrane potential) was added to untreated cells and incubated for 20 min in a 37°C incubator. The drug-treated and CCCP-treated hippocampal neurons were gently washed once with PBS and then mixed with the culture solution with JC-1 staining solution and the cells were incubated for 20 min in a 37°C incubator. After the incubation, the culture solution was aspirated and washed twice with pre-cooled JC-1 staining buffer. Then an appropriate amount of cell culture medium was added to the cells, observed with a confocal microscope (Nikon, C2 Plus).

The cells were lysed with an appropriate amount of ice-cold RIPA lysis buffer (97% RIPA strong lysate, 1% benzylsulfonyl fluoride, 1% NaF, 1% Na₃VO₄), then the cells were disrupted with a 1 ml syringe and allowed to stand at low temperature for 30 min. After the sample was cryopreserved, the BCA method was used to determine the total protein concentration, and then RIPA was used to adjust the total protein concentration of each group to the same concentration. Add loading buffer to each cell lysate, vortex and centrifuge to mix well, then heated at 95–100°C for 10 min to fully denature the protein. Proteins of each group of samples were separated using 10% SDS-polyacrylamide gels (SDS-PAGE). The separated proteins were transferred onto polyvinylidene fluoride membranes. After blocking in 5% skim milk, the membranes were incubated with primary antibodies including anti-Caspase-3 antibody (Cell Signaling, 9664T), anti-Cleaved Caspase-3 antibody (Cell Signaling, 9661S), Bax antibody (Cell Signaling, 2772T), CREB antibody (Cell Signaling, 9197S), Anti-Phospho-CREB (Cell Signaling, 9198S), Anti-ERK1/2 (Cell Signaling, 4695S), Anti-Phospho- ERK1/2 (Cell Signaling, 4370S), Anti-Oxytocin Receptor Antibody (Abcam, ab217212), BCL-2 Polyclonal Antibody (Proteintech, 12789-1-AP), Beta-actin Antibody (Santa Cruz Biotechnology, sc-47778), GAPDH Antibody (Hangzhou Xianzhi Biological Technology Co., Ltd., AB-MM 001). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) at room temperature. Finally, place the membranes on the blackboard and add an appropriate amount of ECL illuminant to expose it on the imager (Bio-Tanon, Tanon-2500R). The experiments were replicated three times.

All data were statistically analyzed using SPSS 21.0 software. Western blot images and fluorescent images were processed using image J software and IPP 6.0 software. The data was plotted using GraphPad Prism 6.02 software. All values were expressed as means ± SEM. One-Way ANOVA was used to determine the overall significant difference between groups. The LSD test was used when the variance was equal, and the Dunnett-t test was used when the variance was not observed. A value of p < 0.05 was considered as statistically significant.

Primary hippocampal neurons cultured in vitro for 7 d were subjected to purity analyses using immunofluorescence staining with beta-Tubulin III antibody and DAPI. Purity of hippocampal neurons was 90% (Figure 1A), which could be used in subsequent experiments as described (Xu et al., 2014). As shown in Figure 1C, hippocampal neurons were treated with METH (1–8 mM) and the cell viability gradually decreased along with the increase of METH concentration. Compared with the control, 4 mM METH significantly reduced the viability of hippocampal neurons, which are similar to METH concentration reported in the literature (Qiao et al., 2014). Therefore, 4 mM METH was selected as the subsequent model concentration. MTT assay indicated that OT (0.01–1 μM) did not exhibit a toxic effect on the neurons (Figure 1B). Afterwards, the neurons were pretreated with OT (0.01–1 μM) for 2 h, followed by a 24 h’ stimulation with METH (4 mM). Figure 1D showed that OT (0.1–1 μM) significantly attenuated the decrease in the vitality of hippocampal neurons induced by METH.

It has been reported that ATO can antagonize the neuroprotective effect of OT on an equal dose (Ceanga et al., 2010; Kaneko et al., 2016). In order to clarify the role of oxytocin receptor, we carried out an experiment designed as follow: ATO (0.1 μM) and OT (0.1 μM) were pre-incubated for 2 h, and then incubated with METH (4 mM) for 24 h. MTT assay indicated that ATO (0.01–1 μM) did not show a toxic effect (Figure 2B). As shown in Figure 2A, OT could inhibit the enlargement of the cell body and the shrinkage of the nucleus induced by METH and this effect could be prevented by ATO. Similar results could also be obtained from MTT experiment (Figure 2C). We tested whether this effect was reflected in other cytotoxicity indicators of METH, including ROS levels and mitochondrial membrane potential levels. As result, OT pre-incubation prevented the decrease of mitochondrial membrane potential and the increased of ROS in hippocampal neurons induced by METH. Pre-incubation with ATO significantly prevented these changes (Figures 2D–G). These results indicate that oxytocin receptor participated in the prevention of METH-induced cytotoxicity in hippocampal neurons.

As shown in Figures 3A,B, METH increased the expression of Cleaved-Caspase-3 and decreased Bcl-2/Bax in hippocampal neurons. Pre-treatment with OT significantly attenuated the up-regulation of Cleaved-Caspase-3 expression and the down-regulation of Bcl-2/Bax expression induced by METH.

As shown in Figures 4A–C, OT pre-incubation attenuated hippocampal neuronal nuclear shrinkage partial nuclear fragmentation and the up-regulation of Cleaved-Caspase-3 expression induced by METH. Pre-incubation with ATO significantly prevented these effects of OT. Similarly, Pre-incubation with ATO significantly reduced the expression of Bcl-2/Bax compared to METH + OT group (Figure 4D). These results indicate that OT could significantly attenuate METH-induced apoptosis via oxytocin receptor in hippocampal neurons.

In order to explore how OT influences on oxytocin receptor in the injured hippocampal neurons, we investigated the changes of the density of oxytocin receptor via western blotting. As shown in Figures 5A,B, OT and ATO had no significant effect on the density of oxytocin receptor in hippocampal neurons in our experiment condition. Figures 5C,D showed that METH (4 mM) significantly decreased the density of oxytocin receptor. Pre-incubation with OT (0.1–1 μM) prevented the decrease of oxytocin receptor density induced by METH. Pre-incubation with ATO significantly prevented this effect.

METH significantly increased the expression of p-ERK1/2 in hippocampal neurons compared with the control group, whereas pre-administration of OT and ATO could not attenuate the increase of p-ERK1/2 expression induced by METH (Figures 6B,C). Pre-treatment with OT significantly attenuated the decrease of p-CREB induced by METH (Figure 6D). Pre-treatment with ATO could prevent this effect (Figure 6D). These results indicate that OT could attenuate METH-induced p-CREB expression reduction through the oxytocin receptor in hippocampal neurons.