Nur77 attenuates inflammatory responses and oxidative stress by inhibiting phosphorylated IκB-α in Parkinson’s disease cell model

Nur77 decreased intracellular LDH release and enhanced cell viability in MPP+-treated PC12 cells

To investigate the effects of MPP⁺ and Nur77 on PC12 cells, we exposed cells to MPP⁺(1.0 mM) or CSN-B (10mg/ml) for 24 h and/or siNur77 and examined cell survival using a MTT assay. In the MPP⁺-treated group, the MTT value in Figure 1A decreased significantly from 99.70±4.933% to 24.43±3.368% compared with the controls. In contrast, CSN-B mitigated this reduction and yielded a value of 62.83±6.129%. Interestingly, transfection with siNur77 led to a significant decrease in the mean MTT value of 3.173±0.645%, compared to 24.43±3.368% with MPP⁺ alone. This result suggests that Nur77 exerts a significant protective effect on MPP⁺-treated PC12 cells. Moreover, the inhibition of Nur77 induced PC12 cell death under MPP⁺ stress.

Figure 1. Effects of Nur77 on PC12 cell viability and LDH. (A) Nur77 increase PC12 cell survival and (B) decrease level of Lactate dehydrogenase (LDH) (^***P < 0.001 compared with control group; ^$$P < 0.01, ^$P < 0.05 compared with MMP⁺ group; n=3, mean +/- SEM).

LDH is released from cells after membrane disruption and is therefore used as an indicator of late cell death. In this experiment, increased LDH release in Figure 1B was observed in cultures of MPP⁺-treated PC12 cells (55.17±3.347%) relative to controls (3.87±1.972%), while treatment with CSN-B mitigated this MPP⁺-induced elevation (31.90±5.179%). This finding suggests a significant neuroprotective effect of CSN-B in our in vitro PD model. To confirm that the neuroprotection mediated by CSN-B in this model was associated with Nur77, we treated cells with siNur77 and observed increased LDH release (72.92±2.493%). Accordingly, the observed inhibitory effect of CSN-B appeared to be associated with Nur77.

Nur77 decreased the expression of proinflammatory gene in MPP⁺-lesioned PC12 cells

The pathogenesis of PD is accompanied by the strong expression or activation of various inflammatory factors Figure 2A, including NF-κB, TNF-α, IL-6 and MCP-1 Figure 2B–2E. Changes of inflammatory factors can affect neuronal activity and stress responses in surrounding tissues [19]. We used the classical Nur77 agonist CSN-B to induce strong expression of Nur77 prior to inflammatory factor production. We similarly observed the expression of inflammatory factors after treatment with siNur77. CSN-B and siNur77 treatment alone cann’t change expression of NF-κB, TNF-α, IL-6 and MCP-1. MPP⁺ incubation pronouncedly increased level of NF-κB, TNF-α, IL-6 and MCP-1 compared with controls, while this elevation was significantly abolished following CSN-B treatment. (Supplementary Figure 1). Silencing of Nur77 with siRNA significantly enhanced the expression of NF-κB, TNF-α, IL-6 and MCP-1 in MPP⁺-treated PC12 cells.

Figure 2. Effects of Nur77 on cytokines expression in MPP⁺-treated PC12 cells. (A) Nur77 increase NF-κB, TNF-α, IL-6 and MCP-1. (B–E) CSN-B and siNur77 treatment alone did not effect expression of NF-κB, TNF-α, IL-6 and MCP-1. MPP⁺ incubation pronouncedly increased level of NF-κB, TNF-α, IL-6 and MCP-1, while this elevation was abolished by CSN-B. (^***P < 0.001,^**P < 0.01 compared with control group; ^$$P < 0.01, ^$P < 0.05, NS = not significant. compared with MMP⁺ group; n=3, mean +/- SEM).

Nur77 activates the Nrf2 signaling pathway and increase protein expression of HO-1 and NQO-1 in MPP+-lesioned PC12 cells

In PC12 cells, MPP⁺ treatment was accompanied by mitochondrial dysfunction and oxidative stress. These phenomena inhibited oxidative phosphorylation by inhibiting mitochondrial complex I, thus inducing changes in related oxidative stress indicators. Changes such as alterations in the Keap1/Nrf2 signaling pathway could decrease anti-oxidative stress damage in our PD model and alter corresponding factors, such as HO-1 and NQO-1. Thereby, neuronal survival or apoptosis is regulated by changes in anti-oxidative stress.

Our findings demonstrated a significant reduction in Nrf2 expression under MPP⁺ stress conditions when compared to the control level. However, CSN-B treatment significantly reversed the reduction of Nrf2 caused by MPP⁺, B. Furthermore, siNur77 transfection significantly reduced the expression of Nrf2 under MPP⁺ stress (Figure 3A, 3B). In response to CSN-B, translocation of Nrf2 from the nucleus to the cytoplasm was clearly observed (Figure 3E, 3F).

Figure 3. Regulation of the Nur77 on anti-oxidant stress. (A) CSN-B treatment significantly reversed the reduction of Nrf2 caused by MPP⁺, siNur77 transfection reduced the expression of Nrf2 under MPP⁺ stress. (B–D) CSN-B could significantly increase the expression of HO-1 and NQO-1, while Silencing of Nur77 with siRNA can significantly decrease HO-1 and NQO-1expression.(^***P < 0.001, ^*P < 0.05 compared with control group; ^$P < 0.05.compared with MMP⁺ group; n=3, mean +/- SEM). (E) Immunohistochemistry of Nrf2 in PC12 cells after CSN-B treatment. (F, G) Protein expression and Quantitative analysis of Nrf2 in PC12 cell nucleus.

The anti-oxidative stress index HO-1 and NQO-1 is downstream of Nrf2. In MPP⁺-treated PC12 cells, CSN-B could significantly increase the expression of HO-1 and NQO-1, while Silencing of Nur77 with siRNA can significantly decrease HO-1 and NQO-1expression in Figure 3A, 3C, 3D. After silencing of Nur77, CSN-B couldn’t significantly increase the protein expression of Nrf2, HO-1 and NQO-1 (Supplementary Figure 2). We examined the nuclear-plasma transfer of Nrf2 and found that CSN-B could promote the activated Nrf2 in nuclear, which was also supported by the protein quantitative analysis of Nrf2 nuclear protein in Figure 3E–G.

Inhibition of NF-κB can decrease the TNF-α, MCP-1 and IL-6 expression induced by MPP⁺

To confirm inflammatory response is caused by NF-κB signaling pathway, we used the NF-κB specific inhibitor PDTC to investigate changes of the inflammatory factor (Figure 4A). The expression of TNF-α, MCP-1 and IL-6 significantly increased in MPP⁺-treated PC12 cells compared to control group, while this elevation was significantly inhibited following PDTC treatment (Figure 4B–4E).

Figure 4. The effect of of PDTC on cytokines expression. (A–E) NF-κB specific inhibitor PDTC can decrease the proteins expression of NF-κB, TNF-α, MCP-1 and IL-6 in MPP⁺-treated PC12 cells. (^***P < 0.001, compared with control group; ^$$$P < 0.001.compared with MMP⁺ group; n=3, mean +/- SEM).

Nur77 inhibited the expression of NF-κB by decreasing the phosphorylation of IκB-α in MPP⁺-treated PC12 cells

In recent studies of neurodegenerative diseases, especially AD and PD [21], the regulation of NF-κB is considered a key pathological element. Our study found that phosphorylation IκB-α was significantly increased in MPP⁺-treated PC12 cells, while CSN-B mitigated this increase. Moreover, we also observed significantly elevated levels of phosphorylated IκB-α in siNur77-treated PC12 cells relative to the control group in Figure 5A, 5B.

Figure 5. The effect of cytosporone B and siNur77 on protein expression of phosphorylation of IκB-α. (A, B) MPP⁺ and siNur77 can increase the level of the phosphorylation IκB-α in MPP⁺-treated PC12 cells, while CSN-B reverse this increase. (^***P < 0.001 compared with control group; ^$$$P < 0.001, ^$P < 0.05, compared with MMP⁺ group; n=3, mean +/- SEM).

Nur77 can inhibit inflammatory responses and oxidative stress in primary neurons

To further demonstrate that Nur77 attenuates inflammatory responses and oxidative stress by phospho-IκB-α, primary neurons were treated with MPP⁺, the Nur77 agonist cytosporone B, and/or small interfering RNAs (siRNAs) against Nur77 and siIκB-α. We found that MPP⁺ significant increased the expression of mRNAs encoding TNF-α, IL-6 and MCP-1 and decreased Nrf2, HO-1, NQO-1, while siIκB-α can reverse the changes. The role of CSN-B is the same as siIκB-α, while siNur77 can significantly increase level of TNF-α, IL-6 and MCP-1 and decrease level of Nrf2, HO-1, NQO-1. These results prove that Nur77 can inhibit inflammatory responses and oxidative stress in MPP⁺ treated primary neurons (Figure 6).

Figure 6. Effects of Nur77 on cytokines and oxidant stress in primary neurons. MPP⁺ significant upregulated in the expression of mRNAs encoding (A–C) TNF-α, IL-6 and MCP-1 and downregulated (D–F) Nrf2, HO-1, NQO-1, while siIκB-α can reverse the upregulation and downregulation. The role of CSN-B is the same as siIκB-α, while siNur77 can significantly increase level of TNF-α, IL-6 and MCP-1 and decrease level of Nrf2, HO-1, NQO-1. When IκB-α is silenced by IκB-α siRNA, the regulative role of CSN-B and siNur77 is significantly reduced (^***P < 0.001; compared with control group; ^$$$P < 0.001,^$$P < 0.01,^$P < 0.05, compared with MMP⁺ group; ^#P<0.05 compared with SiNur77+MPP⁺ group; ^ΔP<0.05 compared with CSN-B+MPP⁺ group; n=3, mean +/- SEM).

Cell culture and treatments

PC12 rat adrenal pheochromocytoma cells were maintained routinely in RPMI-1640 medium supplemented with 5% fetal bovine serum, 10% horse serum, 100 U/ml benzyl penicillin and 100 mg/L streptomycin (Gibco, USA). The cells were cultured at 37ºC in a humidified atmosphere containing 5% CO₂. The cells were fed every 2–3 days and sub-cultured once they reached 80–90% confluence. Primary neurons were conducted as previously described [25]. Newborn C57BL/6J mice were obtained within 24 h of birth from HFK Bioscience Co., Ltd. (Beijing, China). Isolated neurons were cultured in DMEM containing F-12 and 1 mM glutamine. The primary culture neurons were maintained at 37°C in an incubator containing 5% carbon dioxide.

The cells were treated with MPP⁺ (Sigma-Aldrich, ≥97%, M7068-10MG,500μM), the Nur77 agonist cytosporone B (CSN-B, Santa Cruz Biotechnology, ≥98%, sc-252653, 10mg/ml), PDTC (Santa Cruz Biotechnology, ≥98%, sc-203224, 300 mM) and/or small interfering RNAs (siRNAs) against Nur77 (siNur77, Santa Cruz Biotechnology, sc-36109) or IκB-α (siIκB-α, Santa Cruz Biotechnology, sc-29361). For all of the experiments, the cells were seeded in 96-well or 6-well plates at a density of 1.0×10⁵ cells/ml and incubated for 24 h.

MTT cell viability assay

PC12 cell viability after MPP⁺, Cytosporone B(CSN-B) and Nur77 siRNA treatment was evaluated using the MTT assay. Briefly, PC12 cells were grown in 96-well plates (100 μl/well) at a density of 1×10⁵ cells/ml. After incubation, the cells were washed once with phosphate-buffered saline (PBS), after which100 μl of serum-free medium containing MTT (final concentration, 0.5 mg/ml) were added to each well. After a 4-h incubation, the supernatant was removed from each well, the cells were washed twice with PBS and the formazan product was dissolved in 100 μl of dimethylsulfoxide (DMSO). The absorbance in each well at a wavelength of 570 nm was read using a microplate reader. The results for each experimental condition are expressed as percentages of the control group results.

LDH assay

Cell viability was also measured by determining the release of LDH into the culture medium following damage to the cellular membranes. Total LDH activity was measured using the Lactate Dehydrogenase Activity Assay Kit (Sigma-Aldrich, MAK066-1KT, USA) according to the manufacturer’s instructions. The absorbance in each culture well at 490 nm was measured using an automatic microplate reader. The data are presented as percentages of LDH in the MPP⁺ group, which was set at 100%.

siRNA silencing

PC12 cells and primary neurons were transfected with siNur77 and siIκB-α in 0.5μg of Lipofectamine 3000 according to the manufacturer’s instructions. For each transfection in a 6-well plate, 2–8 μl of the siRNA duplex (0.25–1 μg or 20–80 pmol siRNA) were diluted in each well. (Supplementary Figure 3)

Immunocytochemistry

PC12 cells were seeded on circular slides with suitable cell processing in a suitable well plate and fixed with paraformaldehyde. The cells were then labeled with a primary antibody specific to Nrf2 (1:200 dilution). After a subsequent incubation with an Alexa Fluor 555-conjugated secondary antibody (1:500 dilution), the labeled cells were observed using an LSM 780 confocal microscope (Zeiss, Germany).

Quantitative real-time RT-PCR

For quantitative real-time RT-PCR, total RNA was extracted from the cells using TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. Subsequently, cDNA was synthesized using the Advantage RT for PCR kit (BD Biosciences, USA) and oligo (dT) primers. Next, target genes were PCR amplified and quantified using the SYBR Green PCR Master Mix (Applied Biosystems, USA), and the results were normalized to the expression of GAPDH mRNA. All experiments were performed in triplicate and repeated at least three times.

Western blotting analysis

After pretreatment with CSN-B and/or MPP⁺ and/or siNur77, the cells were rinsed thrice with ice-cold PBS and incubated in ice-cold lysis buffer for 20 min on ice. The supernatants were then collected and subjected to a bicinchoninic acid assay to determine the protein concentrations. The samples were then mixed with 5× Loading buffer and heated for 5 min at 100ºC. Next, aliquots with equivalent amounts of total protein (30 μg) were separated on NuPageBis-Tris 10% gels (Invitrogen, USA) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4ºC in solutions with primary antibodies (1:2000) against TNF-α, IL-6, MCP-1(Abcam, Cambridge, UK), and (1:2000) against Nrf2, HO-1, NQO-1, NF-κB p65 and Phospho IκB-α (Cell Signaling Technology, Danvers, MA), and primary antibodies (1:5000) against β-Actin (CWBio, Beijing, China), washed thrice and incubated with horse radish peroxidase-conjugated secondary antibodies. Negative controls were prepared by excluding the primary antibodies. Images of the labeled membranes were analyzed using Image J software (National Institutes of Health, USA).

Statistical analysis

The data are expressed as means ± standard errors of the means. The results of MTT and LDH assays and Western blot quantifications of different proteins were analyzed using a one-way analysis of variance, followed by Tukey’s post hoc analysis (SPSS 15.0; IBM, USA). P values of < 0.05 were regarded as statistically significant.