Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS) and antibiotic-antimycotic were purchased from Gibco BRL (Grand Island, NY, USA), astaxanthin (ATX) from Wako (Catalog No. 013–23051, Tokyo, Japan), N-methyl-4-phenylpyridinium (MPP+) ion (No. D048), the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, No. D2926), the HO inducer hemin (ferriprotoporphyrin IX chloride, No. 51280) and 3-[4,5-dimethylthiazol- 2-yl]-2,5- diphenyltetrazolium bromide (MTT) from Sigma-Aldrich (St. Louis, MO, USA), the HO inhibitor tin protoporphyrin IX dichloride (SnPPIX, Cat. No. 0747) from Tocris Bioscience (Abingdon, UK), 4′,6-diamidino-2-phenylindole (DAPI) and 2′,7′-dichlorfluorescein-diacetate (DCFH-DA) from Beyotime Institute of Biotechnology (Shanghai, China), All other chemicals were purchased from commercial sources.
The rat pheochromocytoma cell line (PC12) was cultured in high glucose DMEM, supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. The cell line was grown as undifferentiated cells in a 100-mm2 culture dish at 37°C in a humidified incubator (Forma Scientific, Ohio, USA; Model No. 3130) containing 5% CO2. When the cells were 70% confluent they were harvested and dispersed. The well dispersed cells were then cultured for 24–36 h with an antagonist or ATX in the presence or absence of MPP+. The cultured medium was changed every 2–3 d. In some experiments, cells were pre-treated for 2 h with 20 μM hemin, 10 μM SnPPIX, 10 μM ATX and 1 μM DPI, and stimulated with MPP+ (500 μM) for 24 h. Control cells were cultured without MPP+.
Cell viability assay
MTT, absorbed into the cell and eventually the mitochondria, is broken down into formazan by mitochondria succinate dehydrogenase. Accumulation of formazan reflects the activity of mitochondria directly and the cell viability indirectly. Cell viability was measured by the MTT assay. PC12 cells were seeded on 96-well plates at a density of 8×103 cells/well, cultured, differentiated, and treated according to the above methods. A total of 20 μl of MTT was added at a concentration of 0.5 mg/ml after media (200 μl) was added to each well. The plates were incubated at 37°C for 4 h to dissolve the formazan that had formed. The solution (220 μl) was removed from each well and 150 μl of dimethyl sulfoxide was added. Reduced MTT was measured on an ELISA reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 570 nm. Values for each treatment group are expressed as a percentage of the control value.
Detection of intracellular ROS
The DCFH-DA assay was used to measure ROS production in differentiated PC12 cells treated with MPP+. DCFH-DA is a fluorescent dye that crosses the cell membrane and is enzymatically hydrolyzed by intracellular esterases to non-fluorescent DCFH. The cells were plated at a density of 4×105 cells per 6-well dish. Differentiated PC12 cells were pretreated with DPI (1 μM) and ATX (5, 10, 20 μM) in medium for 2 h, then exposed to MPP+ (500μM) for 24 h. The cells were incubated with DCFH-DA at a final concentration of 10 μM in high glucose DMEM without FBS for 20 min at 37°C and washed three times with DMEM. ROS levels were measured using a flow cytometer (FACScalibur, Becton Dickinson, San Jose, CA, USA) with excitation and emission wavelengths set at 475 and 525 nm, respectively. For each analysis, 10,000 events were recorded. The value for each treatment group was converted to a percentage of the control value.
Western blot analysis
The cells, plated at a density of 4×105 cells per 6-well dish, were treated with various concentrations of antagonist or ATX in media with or without MPP+ (500μM) for 24 h. For whole cell lysates, the cells were washed twice with ice cold PBS, harvested in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, 0.5 mM PMSF), incubated for 10 min on ice, centrifuged at 12,000 × g for 10 min at 4°C and the supernatant, containing cell lysates, collected. Equal amounts of protein (50 μg) from the cell extracts in each treatment condition were separated using 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PGE), then transferred electrophoretically onto polyvinylidene fluoride (PVDF) (Millipore, Carrigtwohill, Ireland). The blots were blocked by incubation in 5% (w/v) non-fat dry milk in PBS with 0.1% Tween 20 (PBS-T) for 4 h. After incubation with a primary antibody [anti-NOX2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1:200 and anti-HO-1 (Stressgen, Ann Arbor, MI, USA) 1:1000] in PBS-T at 4°C overnight, the membranes were washed three times in PBS-T for 10 min. Subsequently, the membranes were incubated for 1 h in PBS-T containing the appropriate horseradish peroxidase-conjugated secondary antibody [anti-mouse IgG and anti-rabbit IgG (Beyotime Institute of Biotechnology, China) 1:2000]. The immunoreactive bands were visualized and quantified using the Luminata Forte Western HRP substrate (Millipore, Billerica, MA, USA). Protein levels were normalized to the housekeeping protein β-actin (Beyotime Institute of Biotechnology, Shanghai, China) 1:1000to adjust for variability of protein loading and expressed as a percentage of the vehicle control.
Immunofluorescence confocal microscopy
PC12 cells were permeabilized and fixed with 4% paraformaldehyde and 0.5% Triton X-100. Slides were blocked with 1% normal donkey serum (Merck, Darmstadt, Germany) in PBS for 30 min at room temperature. Cells were washed with 0.1% BSA (Beyotime Institute of Biotechnology, Shanghai, China)/PBS three times with gentle shaking, then incubated with the primary antibodies diluted (HO-1 1:1000 and NOX2 1:200) in 0.1% BSA/PBS at 4°C overnight. Labeled donkey anti–rabbit IgG or anti-mouse IgG (Invitrogen, Paisley, UK) (1:1000 dilution) were used as the secondary antibody and incubated in the dark for 2 h at room temperature. Specific antibody binding was detected by Alexa Fluor 488- (green label) and Alexa Fluor 594- (red label) conjugated extravidin (Sigma-Aldrich,St. Louis, MO, USA). Confocal microscopy was performed using the Leica SP5 confocal microscopy system (Leica Microsystems CMS GmbH, Mannheim, Germany). Optical sections were taken at 0.5 μm intervals and images were captured and stored digitally for analysis. Fluorescence intensity was quantified from at least three random fields (1024 × 1024 pixels; 310 × 310 μm) per slide, three slides per experimental condition, and repeated three times using separate cell cultures.
Quantitative real-time PCR analysis
Total RNA from PC12 cells was isolated according to the manufacturer’s protocol using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA purity and integrity was confirmed using the ND-1000 NanoDrop (NanoDrop Technologies, Wilmington, USA) and 2100 Bioanalyzer (Agilent, California, USA). RNA (1 μg) was reverse-transcribed into cDNA in a total volume of 20 μl using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany). The cDNA (2 μl) was amplified with a sequence detection system (ABI Prism 7500) in a total volume of 20 μl containing 10 μl of the FastStart Universal SYBR Green Master Mix (ROX) (Roche, Penzberg, Germany) and each primer at 0.3 μM. Forward and reverse primers for spe-cific amplification of HO-1 [F1(5′-CAAGCAGAACCCAGTCTATGC-3′) and [R1(5′-GATGAGTACCTCCCACCTCGT-3′)], NOX2 [F1(5′-CTGCCTCCATTCTCAAGTCTG-3′) and [R1(5′-ATTCATCCCAGCCAGTAAGGT-3′)] and β-actin [F1(5′-CACCCGCGAGTACAACCTTC-3′) and [R1(5′- CCCATACCCACCATCACACC-3′)] were designed, eliminating the possibility of amplifying genomic DNA. Quantitative real-time PCR was performed using the ABI prism 7500 HT sequence detection system (Applied Biosystems, Foster City, CA, USA) based on the 59-nuclease assay  for the various genes indicated and the housekeeping gene GAPDH. Relative expression was calculated using the ΔΔCt method , and passed the validation experiment. The results are expressed as an average of triplicate samples of at least three independent experiments for control and treated cells.
All statistical analyses were carried out using one-way ANOVAs with repeated measures followed by Scheffe’s post hoc tests. A p value below 0.05 was deemed statistically significant.