ZnO NPs were obtained from HT Nano Company (Nanjing, China). Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12) was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from PAA (Pasching, Austria). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC), and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). SP600125 and SB203580 were obtained from Beyotime (Shanghai, China). U0126 was obtained from Promega (Madison, WI, USA). Bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL, USA). Annexin V-FITC apoptosis kit was purchased from Abcam (Mountain View, CA, USA). Bcl-2, Bax, JNK, ERK1/2, p38 MAPK, phosphor-JNK, phosphor-ERK1/2, phosphor-p38, and caspase-3 antibodies were purchased from Bioworld (St. Louis Park, MN, USA); poly(ADP-ribose) polymerase-1 (PARP) was purchased from Cell Signaling Technology (Boston, MA, USA); β-actin and secondary antibodies (goat anti-mouse or anti-rabbit IgG-conjugated horseradish peroxidase (HRP)) were purchased from Beyotime (Shanghai, China).
Characterization of ZnO NPs
The shape of ZnO NPs was visualized under a transmission electron microscopy (TEM, CM120, Philips, Amsterdam, the Netherlands) at an accelerating voltage of 100 kV and at least four fields of view.
The average hydrodynamic size of ZnO NPs in water and cell culture medium was determined by dynamic light scattering (DLS; Nano-Zetasizer, 1000 HS, Malvern Instrument Ltd., Worcestershire, UK). Briefly, ZnO NPs was suspended in Milli-Q water and cell culture medium, respectively. The suspensions were sonicated for 30 s at 40 W by ultrasonic processor (VCX130, Sonics & Materials Inc, Newtown, CT, USA).
The surface area of ZnO NPs was determined by multipoint nitrogen adsorption using a Brunauer-Emmett-Teller (BET; ASAP2010, USA).
Primary cell culture and ZnO NPs treatment
The procedure was modified from preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue as described in the methods of Shahar . In brief, neonatal Sprague-Dawley rats (1-day-old) were obtained from Beijing University Medical Laboratory Animal Center. The brain was removed and transferred to a 60-mm Petri dish and then rinsed with a squirt of modified DMEM/F12 culture medium containing 2 mM of glutamine. The cerebral cortex was gently removed from the individual cortical lobes and then immediately placed into a fresh 60-mm Petri dish. The cortices were dissociated into cell suspension. The cell suspensions were plated in 25-cm2 tissue culture flasks at a concentration of 2 × 105 cells/ml. The cells were cultured in DMEM/F12 culture medium in 5% CO2 atmosphere at 37°C. Twenty-four hours after the initial plating, the medium was changed to preserve the adhering astrocytes and to remove the neurons and oligodendrocytes. The medium was changed once every 3 days. The astrocytes were maintained in DMEM/F12 containing 10% fetal bovine serum, 100 IU/ml penicillin, and 10 μg/ml streptomycin. The purity of astrocytes was assessed by GFAP-immunostaining according to the Weinstein method . In these conditions, we can assume that over 95% of the cells were astrocytes. All cell exposure experiments were performed at 80% to 90% of cell confluence with viability of ≥90% as determined by trypan blue staining.
The astrocytes (2 × 105 cells/ml) were first cultured in 96-well plates (Costar, Cambridge, MA, USA) for 24 h prior to treatment. Then the culture medium was replaced with serum-free medium, and the cells were exposed to freshly dispersed ZnO NPs preparations at the final concentrations of 4, 8, or 12 μg/ml for 6, 12, or 24 h, respectively. Under the same conditions, astrocytes were pretreated for 1 h with 5 mM of NAC before a 6-h co-exposure with or without ZnO NPs (12 μg/ml). For the inhibitory effect experiments, the cells were pretreated with inhibitor of JNK, ERK, and p38 MAPK pathway (10 μM of SP600125, U0126, and SB203580, respectively) and then treated with ZnO NPs for the indicated duration and concentration.
Cell viability assay
The effect of ZnO NPs on the viability of astrocytes was measured using the MTT assay according to the method of Deng et al. . After exposure to 4, 8, or 12 μg/ml of ZnO NPs for 6, 12, or 24 h, 10 μl of MTT (5 mg/ml in phosphate buffer solution (PBS)) diluted by 90 μl medium was added to each well and incubated for 1 h at 37°C. The cells were then treated with 100 μl of dimethyl sulfoxide (DMSO). The absorbance was quantified at 570 nm using a microplate spectrophotometer (Thermo MK3, ThermoScientific Instruments, Cambridge, MA, USA). The result was reported as viability with respect to untreated cells.
SEM and TEM observation
Astrocytes were grown on coverslips to a semiconfluent state and then treated with ZnO NPs at the final concentration of 12 μg/ml. After exposure to 12 μg/ml of ZnO NPs for 6 h, the cells were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3), added with 2% sucrose at room temperature for 2 h, then dehydrated using graded ethanol concentrations, critical point-dried in CO2, and gold-coated by sputtering. The samples were then examined by scanning electron microscopy (SEM; JSM-6700 F, JEOL Ltd, Tokyo, Japan).
Astrocytes were grown in 50.4-cm2 culture dishes and then treated with ZnO NPs at the final concentration of 12 μg/ml for 6 h. The cells were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffered (pH 7.4) at 4°C overnight and then postfixed with 1% OsO4 in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C for 2 h. After dehydration with ascending concentrations of ethanol (50% to 100%), the samples were embedded at 60°C for 2 days. Ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate. The sections were examined by transmission electron microscopy (CM120, Philips, Netherlands) at 80 kV.
The level of lactate dehydrogenase (LDH) released from astrocytes was measured to evaluate the cytotoxicity of ZnO NPs. Briefly, astrocytes were treated with 4, 8, or 12 μg/ml of ZnO NPs for 6, 12, or 24 h, respectively. The cell-free supernatant was separated by centrifuge (2,000 rpm, 5 min). The culture supernatants were transferred to clean flat-bottom plate for enzymatic analysis. The activity of LDH in the supernatants was determined using LDH detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. All samples were assayed in duplicates for LDH content by a microplate spectrophotometer (Thermo MK3, MA, USA).
The intracellular levels of ROS were determined by measuring the oxidative conversion of DCFH-DA to fluorescent compound dichlorofluorescin (DCFH) . Briefly, astrocytes were placed in 24-well for 12 h, then treated with 4, 8, or 12 μg/ml of ZnO NPs for 6, 12, or 24 h, incubated with DCF diacetate in culture medium for 15 min, and washed with cold phosphate buffer solution three times. The measurement of green fluorescence (oxidized DCFH) using a microplate fluorometer (LB 941, Berthold Technologies, Bad Wildbad, Germany) with fluorescence intensity (excitation, 488 nm; emission, 530 nm). The total protein concentration was determined using BCA protein assay kits (Pierce, IL, USA). The cell-free wells containing only ZnO NPs and DCFH were used to assess nonspecific particle-induced fluorescence. Fluorescence was reported with respect to unexposed control cells.
Assessment of mitochondrial membrane potential
Mitochondrial membrane potential (MMP) was determined with JC-1, a lipophilic membrane-permeable cationic probe, which is widely used for detecting MMP . After 6, 12, or 24 h exposure to 4, 8, or 12 μg/ml of ZnO NPs, the cells were washed twice in PBS and were incubated for 30 min at 37°C with JC-1, a lipophilic and cationic dye that accumulates in the mitochondria in a potential-dependent manner. The intensity of fluorescence was measured with a fluorescence multi-well plate reader LB 941 (Berthold, Bad Wildbad, Germany) with fluorescence intensity (green: excitation 488 nm, emission 530 nm; red: excitation 535 nm, emission 605 nm). The results were presented as the ratio of intensity of red and green fluorescence.
Nuclear staining with DAPI
Chromatin condensation was determined by 4, 6-diamido-2-phenylindole dihydrochloride (DAPI) staining. Astrocytes were cultured in DMEM containing 10% fetal bovine serum on poly-l-lysine-coated dishes or slides in 5% CO2, at 37°C then treated with 12 μg/ml concentration of ZnO NPs for 6 h. The cells were collected and sequentially washed three times in PBS and then fixed for 15 min in 4% paraformaldehyde in PBS for 30 min at room temperature. After staining with a DAPI solution (10 μg/ml) for 10 min in the dark at room temperature, the stained cells were washed twice with PBS to remove the excess DAPI and the changes of nucleus were examined with excitation of 330 to 380 nm and emission of 420 nm using a fluorescence microscope (Olympus, Tokyo, Japan).
Flow cytometric analysis
Apoptosis was measured using the Annexin V-FITC apoptosis detection kit (Abcam, Mountain View, CA, USA) according to the manufacturer's instructions. After treatment with 4, 8, or 12 μg/ml of ZnO NPs for 6 h, the cells were harvested with trypsin, washed twice with PBS (pH 7.4), and then incubated with 200 μl of binding buffer containing Annexin V-FITC (40 μl/ml) and propidium iodide (PI; 1 μg/ml) for 15 min at room temperature in the dark. The population of Annexin V-positive cells was analyzed by flow cytometry (Epics XL, Beckman Coulter Inc, Pasadena, CA, USA). The early apoptotic cells were located in the lower right quadrant (Annexin V-FITC-positive/PI-negative cells), and the late apoptotic cells were located in the upper right quadrant (Annexin V-FITC-positive/PI-positive cells). The percentages of apoptotic cells (Annexin V-positive cells) were plotted.
Western blot analysis
The expression of Bcl-2, Bax, JNK, ERK1/2, PARP, p38 MAPK, phosphor-JNK, phosphor-ERK1/2, phosphor-p38 MAPK, cleaved caspase-3, and β-actin in whole cell lysates were analyzed by sodium dodecyl sulfate polyacrylaminde gel electrophoresis (SDS-PAGE). The gels were transferred to polyvinylidene difluoride (PVDF) membrane by semi-dry electrophoretic transfer at 20 V for 60 min using the semi-dry transfer system. The PVDF membranes were blocked with 5% nonfat milk at room temperature for 1 h, incubated with the primary antibody (dilution 1:1,000) in Tris/buffered saline/Tween20 (TBST) containing 5% bovine serum albumin overnight in 4°C, and then incubated with the secondary antibody (dilution 1:1,000) at room temperature for 1 h. Immunoreactive bands were detected by an enhanced chemiluminescence detection kit (Millipore Corporation, Billerica, MA, USA) according to the manufacturer's instructions. β-actin was used as loading controls for the total protein content and showed no differences between groups.
Results were presented as mean ± standard deviation (SD) of three representative experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by post hoc comparisons using the Dunnet's multiple comparison test or two-way ANOVA statistical analysis followed by a post hoc test. A probability of value of p < 0.05 was considered as statistically significant.