Unless otherwise stated, all chemicals were obtained from Sigma Chemical Co., St. Louis, MO, USA. Mouse monoclonal anti-rat transferrin receptor 1 (TfR1) and goat anti-mouse IgG-Alexa 488 were purchased from Invitrogen, Carlsbad, CA, USA; rabbit polyclonal anti-rat divalent metal transporter 1 (DMT1, SLC11A2) and rabbit polyclonal anti-ferritin-heavy-chain (Ft-L) from Proteintech, Chicago, IL, USA; rabbit polyclonal anti-ferritin-hight-vhain (Ft-H) from Bioworld Technology Inc., Louis Park, MN, USA; and rabbit polyclonal anti-mouse ferroportin 1 (Fpn1) from Novus Biologicals, Littleton, CO, USA. Goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody was bought from Li-Cor, Lincoln, NE, USA; TRIzol reagent from Life Technologies, Carlsbad, CA, USA; and AevertAid First Strand cDNA Synthesis Kit and BCA Protein Assay Kit both from Thermo Scientific, Waltham, MA, USA. Microtubule associated protein 2 (MAP2) and astrocyte marker glial fibrillary acidic protein (GFAP) were obtained from Chemicon International, Inc. Temecula, CA, USA; the in situ cell death detection kit, TMR red from USA Roche, Nutley, NJ, USA. The Health Department of Hong Kong and Shanghai Government and the Animal Research Ethics Committee of The Chinese University of Hong Kong and Fudan University approved the experimental procedures of this study.

Primary cortical neurons were prepared from embryonic day 16–17 Sprague-Dawley rats as previously described (Ke et al., 2006). Dissociated cortical cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Invitrogen Life Technologies) with antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml) and then seeded on culture plates pre-coated with poly-L-lysine at a density of 1.5 × 10⁶ cells/ml. The cultures were maintained at 37°C in a humidified environment with 5% CO2 in a CO2 incubator (NAPCO 5400). After 24 h, the cultural medium was replaced with neurobasal medium supplemented with 2% B-27 and antibiotics (penicillin 100 U/ml, streptomycin 100 μg/ml). The cultured neurons were maintained in the neurobasal B27 medium for 6 days. The purity of the cultures was assessed by staining with the neuron-specific antibodies against MAP2 and the astrocyte marker GFAP. A purity of at least 98.5% was achieved.

Ad-hepcidin was constructed as described previously (Du et al., 2011). Hepdicin protein encoding region (Genebank NM-053469) was cloned from the rat cDNA by PCR (Du et al., 2009). The recombinated adenovirus was named “ad-hepcidin.” Propagation and purification of recombinated adenovirus were conducted by sequential centrifugation in CsCl step gradients and dialysis against 10% glycerol in phosphate-buffered saline (PBS). Finally, the virus with the titer of 2 × 10E11 plaque forming units [pfu]/μL was obtained for the infection of the brain in vivo. GFP-expression adenoviruses (ad-blank) were used as negative control.

Total RNA extraction and cDNA preparation were performed using TRIzol reagent and AevertAid First Strand cDNA Synthesis Kit in accordance with the instructions of the manufacturers, respectively. Real-time PCR was carried out using faststart universal SYBR Green master and lightcycler96. The specific pairs of primers were: hepcidin, forward primer: 5′-gaaggcaagatggcactaagca-3′; reverse primer: 5′-tctcgtctgttgccggagatag-3′ and β-actin, forward primer: 5′-gaaatcgtgcgtgacattaaagag-3′; reverse primer: 5′-gcggcagtggccatctc-3′ (Du et al., 2012). The hepcidin mRNA level of each sample was normalized to that of the β-actin mRNA. Relative mRNA level was presented as 2 [Control Ct (β-actin-Hepcidin)—Expt. Ct (β-actin-Hepcidin)]. The specific band of hepcidin was confirmed by DNA gel electrophoresis.

The cells were washed and homogenized and then sonicated as described previously (Chang et al., 2005; Ke et al., 2005). Protein content was determined using the BCA protein Assay kit. The primary antibodies used are: mouse monoclonal anti-rat TfR1 (1:500); rabbit polyclonal anti-rat DMT1 (1:1,000), rabbit polyclonal anti-mouse Fpn1 (1:1,000), rabbit polyclonal anti-mouse Ft-L (1:1,000), and rabbit polyclonal anti-mouse Ft-H antibody (1:1,000). After being washed three times, the blots were incubated with goat anti-rabbit or anti-mouse IRDye 800 CW secondary antibody (1:5,000) for 1-h at room temperature. The intensity of the specific bands was detected and analyzed by Odyssey infrared image system (Li-Cor, Lincoln, NE, USA). To ensure even loading of the samples, the same membrane was probed with mouse monoclonal anti-actin antibody at a 1:2,000 dilution.

Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) staining was performed by use of the in situ cell death detection kit, TMR red according to the manufacturer's instruction. Tissue sections were analyzed with fluorescence microscopy (Zhao et al., 2008; Wu et al., 2011) and positive cells counting were done with Image pro plus 6.0 software.

Cell injury was quantitatively assessed by the measurement of LDH released from damaged cells. The quantity of LDH release in the medium was determined by the decrease in absorbance at 340 nm for NADH disappearance within 3 min (He et al., 2008; Du et al., 2010). Briefly, 500 μl of supernatant was collected from each well and mixed with 1.3 ml of NADH (0.217 mmol/l) and 1.3 ml of sodium pyruvate (1.77 mmol/l) in the modiWed Krebs–Henseleit buffer (118 mmol/l NaCl, 4.8 mmol/l KCl, 1 mmol/lKH₂PO₄, 24 mmol/l NaHCO₃, 3 mmol/l CaCl₂, 0.8 mmol/l MgPO₄, pH 7.4) for 30 s at 37°C. The activity was spectrophotometrically measured by the use of an ELX-800 microplate assay reader (Elx800, Bio-tek, USA).

Cell iron was determined using a graphite furnace atomic absorption spectrophotometer (GFAAS, Perkin-Elmer, Analyst 100) as described previously (Zhu et al., 2016). Cells were diluted with HEPES buffer and homogenized with a sonicator (MSE Soniprep 150 Ultrasonic Disintegrator, MSE Scientific Instruments, England). A 50-μl portion of the homogenate was added to an equal volume of ultra-pure nitric acid in a 0.5 ml polypropylene microfuge tube, digested for 48 h at 50°C, and diluted with 3.12 mmol/L nitric acid for iron analysis. Standard curves (0–40 ppb) were prepared by diluting iron standard with blanks prepared from homogenization reagents in 0.2% HNO3. Standards and digested samples were read in triplicate by injecting 50 μl aliquots including 0.05 mg Mg(NO3)2 as matrix modification into graphite furnace. Absorbance readings at 248.3 nm, slit at 0.2 nm, pretreatment temperature at 1400°C, atomization temperature at 2400°C were recorded.

Statistical analyses were performed using SPSS software for Windows (version 15.0; SPSS, Inc., Chicago, IL). Data were presented as mean ± SEM. The difference between or among the means was determined by Kruskall-Wallis test followed by Mann-Whitney test for multiple comparisons or One-Way or Two-way ANOVA in appropriate experiments followed by Newman-Keuls post-hoc test. A probability value of P < 0.05 was taken to be statistically significant.

We first examined the effects of treatment with ad-hepcidin on the expression of hepcidin mRNA in primary cortical neuron. It was found that infection with ad-hepcidin induced a significant increase in the expression of hepcidin mRNA in cultured neurons (Figure 1). The levels of hepcidin mRNA in neuronal cells treated with 50 or 100 MOI of ad-hepcidin both were significantly higher than those in control cells, being 226.5 ± 28.6-fold (MOI 50) and 466.6 ± 32.13-fold (MOI 100) of the control values; respectively. Infection of the cells with ad-blank had no effect on the expression of hepcidin mRNA, no difference being found between the neurons in Ad-Blank and control groups (Figure 1).

We then investigated the effects of different concentrations of ad-hepcidin on neuronal integrity or viability by incubating cultured neurons with 0, 25, 50, 100, and 200 MOI of ad-hepcidin for 40-h respectively and then examining the contents of LDH release from neurons. Treatment with 200 MOI of ad-hepcidin induced a significant increase in LDH release from neurons (Figure 2A). The contents of LDH release from control neurons were significantly lower than those from the neurons treated with 200 MOI of ad-hepcidin, but not different from those from neurons treated with 25, 50, and 100 MOI of ad-hepcidin. This indicated that ad-hepcidin at the lower concentrations (i.e., 50–100 MOI) had no marked-effects on neuronal integrity or viability, and 100 MOI of ad-hepcidin was hence chosen to be used in the following experiments.

We also observed the effects of different concentrations of hemin on neuronal integrity or viability by incubating the cultured neurons with 0, 7.5, 15, 30, and 60 μM of hemin for 24-h respectively and then examining the contents of LDH release from neurons. It was found that the contents of LDH release from neurons progressively increased with the concentrations of hemin added (Figure 2B). The amounts of LDH release from neurons treated with 30 and 60 μM of hemin were 39.30 ± 1.76 and 71.77 ± 2.07%; respectively, implying that these concentrations of hemin could induce neuron injury under our experimental conditions.

To find out whether hepcidin was able to protect neurons from hemin-induced damage, the cells were treated with ad-hepcidin (100 MOI), or ad-blank for 16-h and then with 30 or 60 μM of hemin for 24-h or incubated with hepcidin peptide (200 nM) plus 30 or 60 μM of hemin for 24-h. The contents of LDH released from neurons in groups of Ad-hepcidin100 + Hemin30 (27.07 ± 2.25%) and Hepcidin200 + Hemin30 (26.63 ± 1.96%) or Ad-hepcidin100 + Hemin60 (49.21 ± 3.25%) and Hepcidin200 + Hemin60 (48.17 ± 2.97%) were significantly lower than those from neurons in groups of Hemin30 (38.66 ± 2.11%) or Hemin60 (71.76 ± 2.22%); respectively (Figure 2C). The findings indicated that ad-hepcidin and hepcidin peptide both had a significant role to protect neurons from hemin-induced damage.

To further confirm the neuro-protective role of hepcidin, we then assessed effects of ad-hepcidin or hepcidin peptide on hemin-induced apoptosis by TUNEL staining analysis. The neurons were treated with ad-hepcidin (100 MOI) or ad-blank for 16-h and then with 30 μM of hemin for 24-h or incubated with hepcidin peptide (200 nM) plus 30 μM of hemin for 24-h. The findings showed that very few TUNEL-positive cells could be detected in the control, and also in ad-blank, ad-hepcidin and hepcidin peptide groups (Figure 3). However, treatment with hemin or hemin plus ad-blank was found to induce a significant increase in the numbers of TUNEL-positive cells or apoptotic cells (% of total cells). The numbers of TUNEL-positive cells were markedly higher in Hemin and Hemin+ad-blank groups than those in the controls, and also in Hemin+ad-hepcidin and Hemin+hepcidin peptide groups. This demonstrated that pre-treatment with ad-hepcidin or hepcidin peptide could significantly suppress the hemin-induced increase in the numbers of apoptotic cells and reveled that ad-hepcidin or hepcidin peptide have the ability to protect neurons from hemin-induced apoptosis.

In order to answer to the question of why hepcidin could protect neurons from hemin-induced injury and apoptosis, we investigated the effects of ad-hepcidin and hepcidin peptide on cell iron contents and ferritin levels in neurons treated with hemin. Iron was investigated here because the cell toxicity of hemin is thought to be due to iron that is liberated when hemin is degraded (Owen et al., 2016). No differences in iron contents were found among the neurons in the groups of control, ad-blank, ad-hepcidin, and hepcidin peptide. However, iron contents in neurons treated with hemin or hemin plus ad-blank were showed to be significantly higher than those in the controls, while iron contents in neurons treated with ad-hepcidin or hepcidin peptide plus hemin were markedly lower than those in neurons treated with hemin only (Figure 4A). This evidenced a inhibiting role of hepcidin on the hemin-induced increase in cell iron.

Western blot analysis showed that there were no differences in Ft-L (Figure 4B) and Ft-H (Figure 4C) contents between the control and ad-blank-treated neurons. The same as its effect on cell iron, treatment with hemin only also led to a significant increase in both Ft-L and Ft-H contents in neuronal cells, while pre-treatment with ad-hepcidin and co-incubation with hepcidin peptide both suppressed the expression of Ft-L and Ft-H proteins in neurons no matter treated with or without hemin. There were no differences in Ft-L and Ft-H contents between neuronal cells treated with hemin and hemin plus ad-blank.

In order to find out the mechanisms by which hepcidin restores the hemin-induced changes in cell iron contents, we further examined effects of hepcidin on the expression of two major iron importers TfR1 and DMT1 and the only-known iron exporter Fpn1. In contrast to its effect on cell iron and ferritin contents, treatment with hemin resulted in a remarkable decrease not only in iron importers TfR1 (Figure 5A) and DMT1 (Figure 5B) but also iron exporter Fpn1 (Figure 5C). And pre-treatment with ad-hepcidin and co-incubation with hepcidin peptide both suppressed the expression of all three proteins in neurons treated with hemin as well as without hemin. There were no differences in expression of TfR1, DMT1, and Fpn1 between the control and ad-blank-treated or hemin-treated and hemin+ ad-blank-treated neurons.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.