It has been well established that oxidative agents, such as H₂O₂, can induce cell death [21]. In order to investigate the influence of ASTA on immunitary cell viability, we treated U937 cells with H₂O₂, and examined its effects. Since a concentration of 100 µM H₂O₂ and an incubation time of 12 h were previously identified as optimal time and concentration (data not shown) for the induction of deleterious effects on U937 cell viability, these conditions were selected for the rest of experiments. The viability of cells exposed to 100 µM H₂O₂ for 12 h was 61.5 ± 2.8% vs. 96.0 ± 2.8% of the control value, while the viability of cells that were pre-treated for 24 h with ASTA at a concentration of 10 μM prior to 12 h exposure to H₂O₂ increased significantly to 78.1 ± 1.9% (p < 0.01) (Table 1). These results indicate that the viability of H₂O₂-treated cells decreased significantly, but that the ASTA exerted a protective effect against the H₂O₂-induced cytotoxicity.

Oxidative stress has been shown to promote the production of several cytokines, including pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. U937 cells were treated with either H₂O₂ or ASTA to investigate the characteristics of pro-inflammatory cytokines production. The single treatment with H₂O₂ (100 µM) was made for 12 h of culture, whereas the single treatment with ASTA (10 µM) was performed during the 24 h culture. In the combined treatment experiment, U937 cells were treated with 10 µM of ASTA for the first 24 h and then with only 100 µM H₂O₂ for 12 h. The IL-1β, IL-6 and TNF-α secretion was intensely induced by stimulating the cells with H₂O₂ compared with the control, but was significantly lower when pre-incubated for 24 h with ASTA before H₂O₂ stimulation (Figure 1).

Reactive Oxygen Species (ROS) may activate nuclear factors, such as NFkB, leading to the production of pro-inflammatory cytokines, which in turn enhance inflammation and, therefore, the generation of other reactive species [22]. In most cases, NF-κB exists in a heterodimeric form composed of p65 (or RelB) and p50. Furthermore, phosphorylation of the p65 subunit at Ser 536 is associated with activation of NF-κB [23,24]. To identify the NF-κB activation and nuclear translocation after H₂O₂ stimulation, the phosphorylation levels of p65 subunit were measured by western blot analysis using a phospho-p65-specific Ab anti p-Ser-536 in U937 cells nuclear protein extracts after H₂O₂ treatment. Also, to test whether the ASTA affect the H₂O₂ induced activation of NF-κB, U937 cells were pre-treated with ASTA and then stimulated with H₂O₂ for Western blot analysis using a phospho-p65-specific pAb. Figure 2 showed that nuclear translocation of NF-κB containing the p-p65 subunit was strongly induced H₂O₂ treatment, but blocked by ASTA pre-treatment. These data suggest NF-κB involvement in ASTA reduction of pro-inflammatory cytokines secretion induced through H₂O₂ treatment.

Several reports showed inhibitory effect for SHP-1 phosphatase activity on cytokines secretion [25]. In order to investigate a role for SHP-1 in eliciting ASTA effects in U937 cells, western blot using specific anti-SHP-1 pAb experiments were performed. As shown in Figure 3A treatment with H₂O₂ strongly down-regulated SHP-1 protein expression. Pre-incubation with ASTA restored SHP-1 basal levels, showing a negative correlation between p65 nuclear translocation and SHP-1 expression. Following this, in order to test whether SHP-1 is involved in the ASTA-mediated inhibition of NF-κB activation, western blot experiments for p-p65 on U937 nuclear extracts protein were performed in the presence of a specific inhibitor of SHP-1 (sodium stibogluconate, SS, 10 µM). Pre-incubation with SS blocked the ASTA-mediated inhibition of NF-κB activity (Figure 3B).

Moreover, pre-incubation with selective SHP-1 inhibitor, blocked ASTA down-regulation of H₂O₂ induced cytokines release (Table 2). These data indicate that ASTA blocked the H₂O₂ mediated activation of NF-κB and its downstream cytokines secretion through modulating SHP-1 expression.

U937 mononuclear cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in a 5% CO₂ atmosphere in RPMI 1640 medium (GIBCO, Invitrogen) containing 10% fetal calf serum, 100 ng/mL streptomycin, 100 U/mL penicillin and 2 mM L-glutamine. Cells derived from the same freeze-down batch were thawed, grown and seeded (at 2 × 10⁵ cells per well) onto six-well tissue culture plates and cultured in medium with and without 10 µM astaxanthin (ASTA), with and without sodium stigluconate (SS) and treated with H₂O₂ (100 µM). ASTA was dissolved in dimethyl sulfoxide (DMSO) and diluted with the medium. The final concentration of DMSO in the medium was 0.5%. Control groups not contained ASTA and/or H₂O₂. Cell viability was determined by trypan blue dye exclusion and MTT assay (Biotium, Hayward, CA, USA).

The primary antibodies used for Western blotting were rabbit anti-SHP-1, anti NF-κB and anti-p-NF-κB (p65) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO, USA). Astaxanthin was obtained by Sigma-Aldrich. Sodium stibogluconate was purchased from Merck (Darmstadt, Germany).

U937 cells were pretreated with ASTA for 24 h. After pre-incubation with ASTA, H₂O₂ was added to the wells. In other experiment pre-incubation was performed with ASTA and SS. The supernatants were collected and assayed using the Searchligth Elisa kit according to the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL, USA.).

U937 cells were washed once in cold phosphate-buffered saline (PBS; 0.5 mol/L sodium phosphate, pH 7.5) and harvested by gentle scraping, and used to prepare total protein or nuclear extracts. Total protein extracts were prepared by treating cells with lysis buffer [50 mmol/L Tris–HCl pH 7.5, 0.4% Nonidet P-40 (NP-40), 120 mmol/L NaCl, 1.5 mmol/L MgCl₂, 2 mmol/L phenylmethylsulphonyl fluoride (PMSF), 1 µg/mL leupeptin, 3 mmol/L NaF and 1 mmol/L dithiothreitol] for 30 min at 4 °C. Nuclear extracts were prepared according to Osborn et al. [24]. Cells were pelleted, frozen in dry ice⁄ethanol, resuspended in 75 µL of Buffer A (10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 0.5 mmol/L EDTA, 1.5 mmol/L MgCl₂, 0.2% NP-40 and 0.5 mmol/L PMSF) and placed on ice for 10 min to allow lysis. Nuclei were pelleted by centrifugation at 3500 g for 10 min at 4 °C, resuspended in 1 mL of Buffer B (20 mmol/L HEPES pH 7.9, 400 mmol/L NaCl, 1.5 mmol/L MgCl₂, 0.5 mmol/L EDTA, 25% glycerol and 0.5 mmol/L PMSF) and placed on a rocking platform for 30 min at 4 °C. The nuclear lysates were then clarified by centrifugation at 14,000 g for 20 min at 4 °C and the supernatants (nuclear extracts) collected. The protein concentrations of the extracts were determined using the Bradford method (Bio-Rad protein assay, Hercules, CA, USA). For Western blot analysis, 50 µg of protein per lane was separated on a 4–12% NuPAGE gradient gel (Gibco Invitrogen), electro-transferred on to a nitrocellulose membrane and blocked with 10% skimmed milk in PBS containing 0.1% Tween-20. Blots were probed and incubated overnight at 4 °C with the rabbit polyclonal IgG anti-SHP-1, the rabbit polyclonal IgG anti-pNF-κB and NF-κB (p65) all at 0.2 µg/mL in Tris-buffered saline (TBS)/0.1% Tween-20. A rabbit antihuman monoclonal antibody recognizing the human β-actin was used as control in all experiments. Blots were then washed and incubated for 1 h with goat antirabbit–horseradish peroxidase (Pierce Biotechnology, Rockford, IL, USA) diluted 1:10,000 in TBS/0.1% Tween-20. Immunoblot signals were developed using the Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology). The blot images were analyzed with a gel analysis software package (Gel Doc 1000; Bio-Rad, Milan, Italy). Data are expressed as mean ± SD intensity of optical density.

The results are expressed as mean ± SD. Statistical analysis was performed using analysis of variance (ANOVA). The probability of null hypothesis of <5% (p < 0.05) was considered statistically significant. The comparison H₂O₂ vs. H₂O₂ + ASTA and H₂O₂ + ASTA vs. H₂O₂ + ASTA + SS was performed using post hoc test with the alpha level at 0.05.