Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), Pentazocine, Naloxone and MPP⁺ were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Gaithersburg, MD, USA). TUNEL kit was purchased from Roche (Germany). Recombinant Human Dkk1 was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies to β-catenin, PARP, GAPDH and TH were from Abcam (Cambridge, UK). Antibodies to GSK-3β, p-GSK-3β (ser9), cleaved caspase-3, cleaved caspase-8 and β-actin were from Cell Signaling (Beverly, MA, USA). Anti-mouse-HRP IgGanti-rabbit -HRP IgG were purchased from Pierce Biotechnology (Rockford, IL, USA).

SN4741 cells, a mouse embryonic substantial nigra-derived neural cell line, were grown in DMEM containing 1% penicillin, 10% heat-inactivated FBS, 1% glucose and 2 mM glutamine in a humid 5% CO₂ environment at 33°C (Son et al., 1999). The experiments were performed when cells reached 60%–70% confluence. SN4741 cells were exposed to MPP⁺ at various concentrations (0–120 μM) for 24 h. The cells were also pretreated with Pentazocine (0–50 μM) for 24 h before adding MPP⁺. Dkk1 and Naloxone was added prior to Pentazocine.

The cell viability was determined by conventional MTT assay. The SN4741 cells grown in the 96-well microplate were treated with MTT labeling reagents and incubated for 2 h in a humidified incubator at 37°C. Absorbance was determined at 490 nm on a microplate reader (Bio-Rad, Hercules, CA, USA) and cell viabilities were calculated. Results were expressed as percentage of the values of the control cells (control = 100%).

Nuclear fractions of protein were extracted according to protocol of Nucleus and Cytoplasmic Protein Extraction Kit (Borong biology, shanghai, china). Western blot was performed with protein lysates obtained from SN4741 cells. The cells in MPP⁺ group were treated with 90 μM MPP⁺ for 24 h. Meanwhile, cells in control group were treated with equal volume of PBS. Cells in MPP⁺+pentazocine group were pretreated with 20 μM pentazocine for 24 h before adding MPP⁺. Moreover, cells in MPP⁺+pentazocine+naloxone or MPP⁺+pentazocine+Dkk1 group were treated with 10 μM naloxone for 15 min or 100 ng/μl Dkk1 for 2 h before adding pentazocine and MPP⁺. Then the protein was separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA in TBST at room temperature for 2 h and incubated with Antibodies to β-catenin (90 kDa), PARP (116 kDa), GAPDH (36 kDa) and TH (56 kDa; Abcam, Cambridge, UK) and Antibodies to GSK-3β (46 kDa), p-GSK-3β (ser9; 46 kDa), cleaved caspase-3 (19 kDa), cleaved caspase-8 (43 kDa) and β-actin (42 kDa; Cell Signaling, Beverly, MA, USA) at 4°C overnight. After three washes in TBST, the membranes were incubated with HRP-conjugated secondary antibodies. Then the membranes were washed with TBST for three times. At last, the protein bands were visualized using chemiluminescence detection.

The total RNA was extracted using TRIZOL reagent (Code No. 15596018, Invitrogen), and mRNA was reversely transcribed into cDNA (Code No. RR036A, TAKARA). The reagents used in quantitative real-time PCR were purchased from Applied Biosystems™ (Code No. 4472908, Life Technologies). The products were amplified by a quantitative real-time PCR machine. The expression of target gene was normalized to β-actin mRNA level. The primer sequences were shown in Table 1.

SN4741 cells were fixed with 4% paraformaldehyde solution for 10 min. The cells were then blocked with PBS containing 5% bovine serum albumin (BSA) for 2 h and incubated with 0.1% Triton-100 for 30 min. After three washes in PBS, the cells were incubated with β-catenin primary antibody at 4°C overnight. The next day, the cells were labeled with FITC-conjugated anti-rabbit secondary antibodies for 2 h at room temperature. Then we washed the cells with PBS for three times. The nuclei were labeled with DAPI for 10 min at room temperature. Finally, the cells were washed with PBS for three times and imaged with a Nikon fluorescence microscope.

The apoptosis was determined using the one-step TUNEL Apoptosis Kit (Beyotime, JS, China). After each designated treatment, SN4741 cells were fixed, permeabilized, and incubated with TUNEL reaction mixture at 37°C for 1 h according to the manufacturer’s protocol. The cells were then stained with DAPI and observed with a laser scanning confocal microscope (C2 Si; Nikon, Japan) after three times washes with PBS.

The Annexin V-FITC/PI cell apoptosis detection kit (KeyGen Biotech, Nanjing, China) was used to analyzed the cell apoptosis. After washing with PBS, the SN4741 cells were resuspended in 500 μL of binding buffer and then incubated in 5 μL of Annexin V-FITC for at least 10 min. At last, SN4741 cells were washed with PBS and stained with 10 μL of propidium iodide for 20 min at 37°C in the dark. Data acquisition and analysis were performed in a Becton Dickinson FACSCalibur flow cytometer using the Cell Quest software (USA).

SN4741 cells were transfected with TOPFlash/FOPFlash plasmids (200 ng; Millipore, USA) using Lipofectamine 2000 (Invitrogen) in DMEM without FBS. After incubation for 6 h, the primary medium was exchanged for complete DMEM. Cells were exposed to designated treatment 24 h after transfection. One-hundred microliter of lysis buffer was added to the cells which had received designated treatment. Then the cells were transferred to a microcentrifuge tube and incubated at 37°C for 15 min. After 5 s of centrifugation, the supernatant was transferred to a new microcentrifuge which contained luciferase reagent buffer. Luciferase activity was assayed with a dual-luciferase reporter system (Promega).

All the data were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni test. All values are expressed as the mean ± SEM. P < 0.05 was considered as statistically significant.

Pentazocine is an agonist of the opioid receptor (Figure 1A). To evaluate the effect of MPP⁺ on the cell viability, we treated SN4741 cells with various concentrations of MPP⁺ for 24 h and examine the cell viability using MTT. The results indicated that the viability of cells decreased significantly after treated with 90 μM MPP⁺ for 24 h (Figure 1B). Therefore, exposure of 90 μM MPP⁺ for 24 h was used in subsequent experiments. Then we treated the SN4741 cells with 20 μM pentazocine alone for 24 h to investigate whether pentazocine was solely responsible for the change of cell viability. The results showed no difference on cell viability compared to control group. However, the viability of SN4741 cells was increased when the cells were pretreated with different concentrations (10, 20 and 50 μM) of pentazocine for 24 h before MPP⁺ treatment (Figure 1C). All these results indicated that pentazocine could protect cells from MPP⁺-induced cell viability decrease in a dose-dependent manner.

β-catenin protein plays an important role in the canonical Wnt signaling, and many evidences indicate that change of the canonical Wnt signaling is involved in the neuronal degeneration of PD. To investigate whether pentazocine affects the expression of β-catenin protein, the cells were pretreated with pentazocine (20 μM) for 24 h before MPP⁺ treatment. Moreover, we treated the SN4741 cells with 10 μM naloxone for 15 min or 100 ng/μl Dkk1 for 2 h before adding pentazocine and MPP⁺. The β-catenin protein level in each group was measured by immunocytochemistry assay (Figure 2A). The results suggested that MPP⁺ induced a down-regulation in the protein level of β-catenin. However, the β-catenin levels increased when cells were pretreated with pentazocine compared with cells in MPP⁺ group. The adding of naloxone or Dkk1 demonstrated that only Dkk1 was associated with the abrogation of protective effect of pentazocine, whereas naloxone was not. To examine whether the disturbance of β-catenin leads to the change of TCF/LEF related gene expression, we used a TOPFlash/FOPFlash reporter assay. The transfection efficiency of plasmid in SN4741 cells was determined by GFP plasmid (Figure 2B). The TOPFlash/FOPFlash plasmids were transfected into SN4741 cells. Then cells were divided into five groups. MPP⁺ group was treated with 90 μM MPP⁺ for 24 h. Meanwhile, cells from control group were treated with equal volume of PBS. Cells from MPP⁺+pentazocine group were pretreated with 20 μM pentazocine for 24 h before adding MPP⁺. Moreover, cells from MPP⁺+pentazocine+naloxone or MPP⁺+pentazocine+Dkk1 group were treated with 10 μM naloxone for 15 min or 100 ng/μl Dkk1 for 2 h before adding pentazocine and MPP⁺. The results showed that luciferase activity was significantly decreased in the SN4741 cells administrated with MPP⁺. We also observed that pretreatment of pentazocine before adding MPP⁺ significantly promoted the luciferase activity compared with cells in MPP⁺ group. The adding of naloxone or Dkk1 before pentazocine and MPP⁺ treatment also indicated that only Dkk1 was related to the abrogation of protective effect of pentazocine, whereas naloxone was not (Figure 2C).

To test whether the downstream molecules of Wnt/β-catenin signaling waschanged, we used same treatment paradigm. Then we measured the TH, β-catenin, GSK-3β, p-GSK-3β (ser9) and β-catenin levels by Western blot (Figures 3A–D) and quantitative real-time PCR (Figures 3E–G). The specificity of primers were verified in NCBI website (Supplementary Figure S1). The results showed that inhibition of the canonical Wnt/β-catenin signaling was found in MPP⁺ treated group, for the p-GSK-3β (ser9) and β-catenin were down-regulated. Pentazocine can reverse the down-regulation of canonical Wnt/β-catenin signaling and protect the SN4741 cells from MPP⁺ injury, which verified that the down-regulation of canonical Wnt/β-catenin signaling was involved in the MPP⁺-induced neurotoxicity in SN4741 cells. The use of Dkk1 abrogated the effect of pentazocine in the up-regulation of Wnt/β-catenin signaling. However, naloxone showed no influence on Wnt/β-catenin signaling in the SN4741 cells treated with pentazocine and MPP⁺. We also tested the expression of β-catenin in nucleus by Western blot and the data were consistent with the change of β-catenin in total cells. PARP was detected in the nuclear fractions of protein, whereas GAPDH was not. This result indicated that the nuclear fractions were isolated successfully (Figures 4A,B).

TUNEL staining and Western blots were used to evaluate the effect of pentazocine on MPP⁺-induced SN4741 cells apoptosis. The results indicated that the number of TUNEL-positive SN4741 cells increased after MPP⁺ treatment (Figures 5A,B). However, fewer TUNEL-positive SN4741 cells were observed when cells were pretreated with pentazocine for 24 h before adding MPP⁺ compared with the cells treatment with MPP⁺ alone. The protective effect of pentazocine was abrogated when the SN4741 cells were treated with 100 ng/μl Dkk1 for 2 h before adding pentazocine and MPP⁺. Moreover, naloxone showed no influence on the protective effect of pentazocine in the SN4741 cells pretreated with pentazocine and MPP⁺. This finding was further supported by the MTT assay in different groups (Figure 5C).

To strengthen the evidence, SN4741 cells were stained with the Annexin V-FITC/PI. After exposure to MPP⁺ for 24 h, the apoptotic cells were increased compared to that of the control group. Pretreatment of pentazocine for 24 h remarkably abolished this effect. Besides, the protective effect of pentazocine was abrogated when the SN4741 cells were treated with 100 ng/μl Dkk1 for 2 h (Figures 6A,B). The Western blot showed that pentazocine exposure reversed the MPP⁺ induced increase of cleaved caspase-8 and cleaved caspase-3 level in SN4741 cells. To test whether pentazocine exerted protections through Wnt/β-catenin signaling, cells were pretreated with Dkk1 to block the activity of Wnt/β-catenin signaling. Then we found that the protectiveeffect of pentazocine against MPP⁺-induced increase of cleaved caspase-8 and cleaved caspase-3 level was abrogated in cells incubated with 100 ng/μl Dkk1 for 2 h. However, the effect of pentazocine against MPP⁺-induced increase of cleaved caspase-8 and cleaved caspase-3 level could not be abrogated in cells pretreated with 10 μM naloxone for 15 min (Figures 7A–D).