Introduction

Stroke is the leading cause of death and serious, long-term disability. To date, clinical strategies that help to stop the neurodegeneration following anoxic/ischemic insults remain limited. [(D-Ala2, D-Leu5) enkephalin] (DADLE) is a synthetic opioid peptide and a highly selective delta opioid receptor (DOR) agonist1,2. Since the neuroprotective effects of DOR activation were first described1, it has attracted a great deal of attention from researchers worldwide. Accumulating evidence has suggested that stimulating DOR with DADLE elicits potent neuroprotection against anoxic/ischemic injury1,3,4,5. Therefore, DADLE may be a promising novel therapeutic candidate for treating neurodegeneration in stroke.

After the neuroprotective effects of DOR activation were recognized, subsequent studies focused on the underlying mechanism of DORs, and great progress has been made so far. The DOR is a Gi protein-coupled receptor that functions through the cAMP signaling pathway6. Although the details remain unclear, it is generally accepted that the mechanisms of DOR neuroprotection fall into two categories: DORs can trigger endogenous protective pathways4,7,8,9 and maintain ionic homeostasis10,11,12,13. In particular, it has been confirmed by multiple studies that the PKC/MAPK pathway plays a crucial role in the neuroprotective functions of DORs4,7,8,14. Moreover, DOR activation might elicit neuroprotection via the PI3K/Akt signaling pathway15,16.

Our previous studies have investigated the neuroprotective effects of DADLE using a NaN3-induced mitochondrial dysfunction model. Much to our surprise, we found that stimulating DOR with DADLE for both a short (minutes) and long (days) time showed notable protective effects on primary rat cortical neuronal cultures8,17. Further investigation revealed that short-term DADLE stimulation increases neuronal survival through the PKC-mitochondrial ERK pathway in the NaN3 model8. However, the mechanism underlying the protective effects of DADLE during prolonged exposure remains unclear. In this study, we will explore the possible downstream signaling pathways mediating the neuroprotective effects of sustained DOR stimulation with DADLE in the NaN3-induced neuronal injury model.

Materials and methods

Primary culture of rat cortical neurons

Primary cortical neuron cultures were prepared as previously described8,17. Briefly, after Sprague-Dawley rats (embryonic d17–18; Shanghai Experimental Animal Center, Chinese Academy of Science, Shanghai, China) were sacrificed on gestation d 17−18, the cortical neurons were carefully collected. Then, the cells were resuspended in neurobasal medium (Gibco-BRL, Life Technologies, Rockville, MD, USA) supplemented with B-27, glutamine (0.5 mmol/L), penicillin (100 U/mL) and streptomycin (100 μg/mL) and plated onto poly-L-lysine (100 μg/mL, Sigma, St Louis, MO, USA) pre-coated dishes at a density of 1×106 cells/mL. The cells were ready for drug administration on day 8. Animal experiment protocols complied with the guidelines of the Animal Care and Use Committee of Fudan University. All efforts were made to minimize the suffering of the animals.

Drug administration

Freshly prepared NaN3 (80 μmol/L) was applied to primary cultured neurons to induce mitochondrial dysfunction. DADLE (0.1 μmol/L) or naltrindole (NTI, 0.1 μmol/L) was administered to selectively activate or inhibit DOR activity8,17. LY296004 (20 μmol/L) is a selective PI3K inhibitor, and Akt inhibitor VIII (5 μmol/L) is a specific Akt inhibitor18. All drugs were purchased from Sigma (St Louis, MO, USA).

Lactate dehydrogenase (LDH) assay

The activities of LDH in the cultivated medium were measured with an LDH kit (Sigma, St Louis, MO, USA) and detected with a DU-640 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA, USA) as previously described8,17. The absorbance data were normalized to those of the control group.

Cell viability assay

Cell viability assays were performed by adding cell counting kit-8 reagent (CCK-8, Dojindo Laboratories, Japan) directly to the cell cultures. After incubation at 37 °C for 1 h, the absorbance was read by a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm and was proportional to the number of living cells. Each experiment was repeated three times.

Cytosolic and nuclear protein preparation

Cytosolic and nuclear proteins were extracted using a nuclear extraction kit (Chemicon, Temecula, CA, USA) according to the manufacturer's protocol. Briefly, neurons were collected after drug exposure and incubated in cytoplasmic lysis buffer for 15 min. The cells were pelleted by centrifugation at 250×g for 5 min and resuspended in cytoplasmic lysis buffer. A syringe with a small gauge needle was used to break up the cytoplasmic membrane. The supernatant was collected after centrifugation at 8000×g for 20 min and used as the cytosolic fraction. The remaining nuclear pellet was resuspended in nuclear extraction buffer for 30 min, and a fresh syringe was used to disrupt the nuclei. Following centrifugation at 16 000×g for 5 min, the supernatant was collected and used as the nuclear extract. Each step was performed on ice at 4 °C.

Immunoblotting analyses

Neurons were harvested after drug exposure, and the cell lysates were prepared as described before8. Protein samples were electrophoresed on 10%−12% SDS-PAGE gels. The antibodies used were as follows: rabbit polyclonal antibodies against pp38, p38, pERK, ERK, pJNK, JNK, pAkt, Akt, and H3 (1:1000; all from Cell Signaling Technology, Danvers, MA, USA), rabbit antibodies against pp85, p85, p65, and Bcl-2 (1:1000; all from Chemicon, Temecula, CA, USA), mouse antibody against β-actin (1:2000; Sigma, St Louis, MO, USA), and sheep antibody against Cyto c (1:1000; Chemicon, Temecula, CA, USA).

RNA extraction, RT-PCR and real-time PCR

Total RNA was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA (2 μg) was reverse-transcribed by using the PrimeScript RT reagent kit (Takara, Japan), and single stranded cDNA was amplified by quantitative real-time PCR with the SYBR Premix Ex Taq Kit (Takara, Japan) on a Mastercycler ep realplex real-time PCR system (Eppendorf, Hamburg, Germany). The fold change was calculated using the ΔΔCt method (2-ΔΔCt) with GAPDH as the internal control. The following primers were used: Bcl-2, 5′--3′ and 5′--3′; GAPDH, 5′--3′ and 5′--3′. Data are presented as the means±SEM of 4 independent experiments with 3 real-time PCR replicates.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed using a ChIP assay kit (Millipore, Billerica, MA, USA) according to the manufacturer's protocol. In brief, after drug treatment, neuronal cells were cross-linked with formaldehyde, lysed and sonicated. Immunoprecipitation was performed with antibodies to NF-κB (Cell Signaling Technology, Danvers, MA, USA), acetyl-H3 and acetyl-H4 (both from Chemicon, Temecula, CA, USA). Additionally, samples were immunoprecipitated with rabbit IgG as a negative control. Immunoprecipitated DNA was subjected to semi-quantitative and quantitative real-time PCR analyses using primers specific for the Bcl-2 promoter (5′--3′ and 5′--3′). After 30 cycles of amplification, 20 μL of the PCR product was analyzed on 2% agarose gels. Real-time PCR was performed as described above.

Statistical analysis

The data are shown as the mean±SEM and were subjected to statistical analysis using one-way ANOVA. The level of statistical significance was set at P<0.05.

Results

Prolonged DADLE exposure selectively activates the PI3K/Akt/NF-κB pathway in NaN3-treated neurons

To examine the influence of prolonged DADLE stimulation on MAPK and Akt activity in NaN3-treated neurons, primary rat cortical neuron cultures were treated with 80 μmol/L NaN3 and DADLE or NTI for 2 d. The phosphorylation levels and the total levels of ERK, JNK, p38 and Akt were detected by immunoblotting after drug treatment. Neither the phosphorylation levels nor the total levels of ERK, p38 or JNK were affected by NaN3 (Figure 1A); however, the levels of pAkt were reduced by NaN3 (0.4-fold higher than those of the controls), and the total Akt levels were not affected (Figure 1C). DADLE not only reversed the NaN3-induced inhibition of Akt activation but also further increased the levels of pAkt (1.72-fold higher than those of the controls); these effects were abolished by NTI (Figure 1C).

Figure 1
figure 1

Sustained DOR stimulation with DADLE selectively activates the PI3K/Akt/NF-κB pathway in NaN3-treated neurons. Primary rat cortical neuron cultures were treated with 80 μmol/L NaN3 and with DADLE (0.1 μmol/L) or NTI (0.1 μmol/L) for 2 d to activate or inhibit DOR activity. The phosphorylation levels and total levels of ERK, JNK, p38, the p85 subunit of PI3K, Akt, and the p65 subunit of NF-κB in the nucleus were detected by immunoblotting after drug treatment. DADLE had no effect on the levels of phosphorylated p38 (pp38), phosphorylated ERK (pERK) or phosphorylated JNK (pJNK) (A) but further increased the phosphorylation levels of the regulatory p85 subunit of PI3K (pp85) (B) and Akt (pAkt) (C) and promoted the nuclear translocation of p65 (D) in neurons treated with NaN3 for 2 d; these effects were abolished by the presence of NTI, LY296004 (PI3K inhibitor; 20 μmol/L) or Akt inhibitor VIII (5 μmol/L). * P<0.05 compared to Ctrl; # P<0.05, ## P<0.01 compared to the NaN3 group. n=5.

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Next, the phosphorylation levels and total levels of the p85 subunit of PI3K were determined by immunoblotting after drug treatment. As shown in Figure 1B, the levels of pp85 were significantly decreased by NaN3, and DADLE not only reversed the NaN3-induced decrease in pp85 levels but also further elevated PI3K activity; these effects were abolished by NTI. Moreover, the total levels of p85 were not altered in any group after drug exposure. LY296004, a selective PI3K inhibitor, also prevented Akt activation in the neurons exposed to DADLE and NaN3 (Figure 1C), indicating that PI3K acts as a downstream signal of DOR stimulation and is required for Akt activation in NaN3-treated neurons.

To explore the impact of sustained DADLE stimulation on the activity of NF-κB in NaN3-treated neurons, translocation of the p65 subunit of NF-κB into the nucleus was detected by immunoblotting after drug exposure. The levels of p65 in the nucleus were remarkably decreased by NaN3, and this effect was attenuated by DADLE. The effect of DADLE was abolished by NTI, LY296004 and Akt inhibitor VIII, implying that DOR stimulation reverses NaN3-induced NF-κB inhibition via the PI3K/Akt pathway.

The data above suggest that sustained DOR stimulation with DADLE selectively activates the PI3K/Akt/NF-κB pathway but not MAPKs in the NaN3 model.

Prolonged DADLE treatment reduces cell injury in NaN3-treated neurons via the PI3K/Akt pathway

To determine whether the protective effect of sustained DADLE stimulation is mediated by the PI3K/Akt pathway, neuronal damage was determined by measuring LDH release into the culture medium after drug exposure, and cell viability was detected by using a CCK-8 kit. DADLE completely suppressed the increase in LDH release induced by NaN3 and restored cell viability; these effects were abolished by LY296004 and VIII (Figure 2). These data imply that sustained DOR stimulation reduces NaN3-induced neuronal injury via the PI3K/Akt pathway.

Figure 2
figure 2

Sustained DOR stimulation with DADLE reduces cell injury in NaN3-treated neurons via the PI3K/Akt pathway. Neuron cultures were treated with NaN3 and DADLE, LY296004 or Akt inhibitor VIII. LDH release into the culture medium was measured after drug exposure for 2 d (A), and cell viability was tested with a CCK-8 kit (B). DADLE manifestly reduced the NaN3-induced increase in LDH release and restored cell viability; these effects were reversed by co-treatment with LY296004 (20 μmol/L) or Akt inhibitor VIII (5 μmol/L). * P<0.05 compared to Ctrl; # P<0.05 compared to the NaN3 group. n=5.

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Prolonged DADLE exposure enhances Bcl-2 expression and attenuates Cyto c release in NaN3-treated neurons through the PI3K/Akt pathway

NaN3 is a specific complex IV inhibitor widely used to induce mitochondrial dysfunction and Cyto c release19,20. To explore the effect of sustained DADLE stimulation on mitochondrial Cyto c release in NaN3-treated neurons, the levels of cytosolic Cyto c were tested by immunoblotting after drug exposure. As seen in Figure 3A, Cyto c levels in the cytosolic fraction were robustly increased by NaN3; this effect was suppressed by DADLE. The effect of DADLE was totally abolished by NTI, LY296004 and VIII, implying that sustained DOR stimulation in NaN3-treated neurons prevents Cyto c release through the PI3K/Akt pathway.

Figure 3
figure 3

Sustained DOR activation with DADLE attenuates Cyto c release and enhances Bcl-2 expression in NaN3-treated neurons through the PI3K/Akt pathway. Neuron cultures were treated with NaN3 and DADLE, NTI, LY296004 or Akt inhibitor VIII for 2 d. The protein levels of cytosolic Cyto c (A) and Bcl-2 (B) in neurons were measured by immunoblotting after drug exposure, and the mRNA levels of Bcl-2 were also determined (C). Cytosolic Cyto c levels were robustly increased in NaN3-treated neurons, whereas the mRNA levels and protein levels of Bcl-2 were both remarkably decreased. DADLE suppressed the downregulation of Bcl-2 and the increase in cytosolic Cyto c levels; these effects were abolished by the co-administration of NTI, LY296004 or Akt inhibitor VIII. * P<0.05, ** P<0.01 compared to Ctrl; # P<0.05 compared to the NaN3 group. n=5.

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Bcl-2 is a well-known anti-apoptosis protein that prevents Cyto c release21. To investigate the impact of sustained DOR activation on Bcl-2 expression in NaN3-exposed neurons, Bcl-2 levels were determined by immunoblotting after drug exposure. As shown in Figure 3B, Bcl-2 levels were considerably reduced by NaN3, and this effect was reversed by DADLE; furthermore, the effect of DADLE was entirely prevented by NTI, LY296004 and VIII. Next, the mRNA levels of Bcl-2 were determined after drug treatment. Bcl-2 mRNA levels were significantly decreased by NaN3; this effect was reversed by DADLE (Figure 3C). Again, the effect of DADLE was completely inhibited by NTI, LY296004 and VIII, indicating that sustained DOR stimulation with DADLE in NaN3-treated neurons restores Bcl-2 protein levels by attenuating its transcriptional dysfunction via the PI3K/Akt pathway.

These results indicate that sustained DOR stimulation with DADLE decreases cell damage in NaN3-treated neurons by promoting Bcl-2 expression and preventing Cyto c release through the PI3K/Akt pathway.

Prolonged DOR stimulation with DADLE enhances the NF-κB binding activity and histone acetylation of the Bcl-2 promoter in neurons treated with NaN3

NF-κB has been reported to promote the transcriptional activity of Bcl-222; therefore, we further investigated whether the upregulation of Bcl-2 transcription is modulated by NF-κB during sustained DOR activation. According to ChIP assays, our results showed that the levels of NF-κB bound to the Bcl-2 promoter were remarkably reduced by NaN3; this effect was completely blocked by DADLE (Figure 4). The effect of DADLE was abolished by NTI, which indicates that sustained DOR activation enhanced NF-κB binding to the Bcl-2 promoter in NaN3-treated neurons.

Figure 4
figure 4

Sustained DOR activation with DADLE promotes NF-κB binding to the Bcl-2 promoter in NaN3-treated neurons. Neuron cultures were treated with NaN3 and DADLE or NTI for 2 d, and chromatin immunoprecipitation (ChIP) analyses were performed with an antibody to NF-κB after drug treatment. DNA samples without immunoprecipitation served as the input. Representative semi-quantitative PCR analyses and quantitative real-time PCR analyses of the ChIP samples show that the levels of NF-κB bound to the Bcl-2 promoter were remarkably reduced by NaN3; this effect was completely blocked by DADLE. The effect of DADLE was abolished by NTI. * P<0.05 compared to the NaN3 group; # P<0.05 compared to the DADLE+NaN3 group. n=5.

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ChIP was also used to determine the effect of DADLE on the histone acetylation level of the Bcl-2 promoter in NaN3-treated neurons. As shown in Figure 5, the levels of acetylated H3 and H4 for the Bcl-2 promoter were both remarkably reduced by NaN3; these effects were completely blocked by DADLE. The effects of DADLE were abolished by NTI. These data suggest that sustained DOR activation increased the histone acetylation levels of the Bcl-2 promoter in neurons treated with NaN3.

Figure 5
figure 5

Sustained DOR stimulation with DADLE increased the histone acetylation levels of the Bcl-2 promoter in NaN3-treated neurons. Neuron cultures were exposed to NaN3 and DADLE or NTI for 2 d, and chromatin immunoprecipitation (ChIP) analyses were performed with antibodies to acetyl-H3 or acetyl-H4 after drug treatment. Representative semi-quantitative PCR analyses and quantitative real-time PCR analyses of the ChIP samples show that the acetylation levels of both H3 and H4 for the Bcl-2 promoter were decreased by NaN3; these effects were both completely inhibited by DADLE. The effects of DADLE were totally reversed by co-treatment with NTI. ** P<0.01 compared to acetyl-H3 in the NaN3 group; ## P<0.01 compared to acetyl-H4 in the NaN3 group. n=5.

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Discussion

So far, there are no effective treatments to stop or reverse neurodegeneration after a stroke. Despite the encouraging results seen in preclinical studies, most cytoprotective drugs have failed in clinical trials. One of the main reasons for this is that those drugs showed protection at only the very early stage of neurodegeneration, and their effects did not last as the disease developed.

Our earlier work found that stimulating DOR with DADLE for both a short (minutes) and long (days) time resulted in significant neuroprotective effects in the NaN3 model8,17. These results imply that DADLE provides immediate protection at the early stage of neuronal injury and that its protective effects would last for days if the toxin is not removed. Our further investigations of the underlying mechanisms revealed that at least two downstream signaling pathways may be sequentially activated and responsible for the neuroprotective effects of DADLE against mitochondrial injury. While short-term DADLE stimulation increases neuronal survival through the PKC-mitochondrial ERK pathway8, sustained DOR activation with DADLE in the present study had little effect on MAPK activity in NaN3-treated neurons but selectively activated the PI3K/Akt/NF-κB pathway; these actions might mediate the neuroprotective effects of prolonged DOR activation.

Though the intricacies of the role of DOR in neuroprotection are not yet well-established, several endogenous neuroprotective pathways have been reported to be involved. Activating DOR via hypoxia preconditioning can balance the pERK and pp38 levels through the PKC/MAPK pathway; this action elicits neuroprotective effects in a severe hypoxia model in vitro 4. Narita et al found that the neurogenesis and anti-apoptosis effects of DOR stimulation may rely on Trk phosphorylation, which is associated with PI3K, MEK, and CaMKII activation in normally cultured neural stem cells and in H2O2-treated cells23. DOR activation also protects NG108-15 cells from pertussis toxin insult through activating the PI3K/Akt signaling pathway15. This evidence further indicates that DOR activation causes potent neuroprotection through distinct pathways, which makes DOR a potential therapeutic target.

The role of DOR in neuronal transcription and gene expression has not been well studied until recently. It has been reported that DADLE induces transcription inhibition, especially in apoptotic pathways, which may result in cellular metabolism arrest and provide neuroprotective effects24,25. Nevertheless, this effect of DADLE is probably DOR-independent. Sen et al found that DADLE upregulates NGF by activating the PI3K/Akt/NF-κB pathway26. Our present work showed that DADLE upregulates Bcl-2 expression and exhibits anti-apoptotic effects, which are very likely mediated by DOR. These findings indicate that DADLE may bi-directionally regulate gene expression in neuronal cells; thus, the role of DOR remains unclear and needs further investigation.

This is the first study to demonstrate that prolonged DADLE exposure could epigenetically promote Bcl-2 expression in NaN3-treated neurons. Bcl-2 is an important anti-apoptosis protein and is tightly associated with cell survival regulation. Evidence has shown that downregulating Bcl-2 via siRNA leads to increased apoptotic markers and ROS levels27, whereas overexpressing Bcl-2 protects neurons from glutamate insult28 and focal cerebral ischemia9. In our present study, we found that Bcl-2 levels were downregulated during NaN3 exposure; this effect was reversed by the co-administration of DADLE. This change in Bcl-2 protein levels is consistent with that in mRNA levels and was inhibited by LY296004 and VIII, indicating that sustained DADLE stimulation may restore Bcl-2 protein levels mainly by attenuating its transcriptional dysfunction via PI3K/Akt signaling.

The anti-apoptotic functions of Bcl-2 are not only associated with its protein levels but also regulated by its phosphorylation. It has been suggested that in a mitotic arrest-induced model, multi-site phosphorylation of Bcl-2 within an unstructured loop inhibits its anti-apoptotic functions29 . In contrast, the phosphorylation of Bcl-2 at S70 prevents apoptosis in cytokine-dependent cell lines30. Therefore, further studies are needed to determine how the phosphorylation status of Bcl-2 is affected by NaN3 and DADLE treatment.

NF-κB is an important transcriptional factor with five different subunit isoforms that are ubiquitously expressed; NF-κB participates in the regulation of the expression of many genes, and some of these genes are involved in the process of apoptosis31. Several studies showed that translocation of the NF-κB p65 subunit into the nucleus is activated via the PI3K/Akt pathway; there, it participates in regulating anti-apoptotic gene expression32,33, possibly through enhancing acetylation efficiency in the p65 binding region34. In the present study, we revealed that DOR stimulation reversed NaN3-induced p65 inhibition via the PI3K/Akt pathway and promoted Bcl-2 transcription by increasing the histone acetylation of the Bcl-2 promoter. These results are supported by other studies showing that NF-κB might directly bind to the Bcl-2 promoter and promote its transcription35,36, as well as the fact that sustained DOR activation enhanced p65 binding to the Bcl-2 promoter in neurons treated with NaN3 in this study. Therefore, upon sustained DADLE stimulation in NaN3-induced mitochondrial injury, the epigenetic upregulation of Bcl-2 expression might play an important role in the downstream signaling of DOR neuroprotection. Since Bcl-2 levels could be affected by both the expression and degradation processes, whether NaN3 or DADLE affect Bcl-2 protein degradation still needs further investigation.

In conclusion, we showed here that the mechanism underlying sustained DOR activation by DADLE is very different from the mechanism underlying its short-term stimulation in NaN3-treated neurons. A different signaling pathway (PI3K/Akt/NF-κB) is activated, and it further promotes Bcl-2 expression through epigenetic regulation. Bcl-2 upregulation suppresses Cyto c release and rescues neuronal cells (Figure 6). The present work is an essential supplement to our earlier studies and implies that DADLE may provide a distinct therapeutic opportunity for stroke treatment.

Figure 6
figure 6

Scheme showing the pathways that mediate the neuroprotective effects of sustained DOR stimulation with DADLE against NaN3 insult.

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Author contribution

Jing-chun GUO and Cui-qing ZHU designed the study; Min ZHU and Ming LIU performed the research; Min ZHU and Qi-lin GUO analyzed the data; and Min ZHU, Ming LIU and Jing-chun GUO wrote the paper.