All experimental protocols were reviewed and approved by the Ethical Committee of Wenzhou Medical University (Wenzhou, China).

Primary cultures of hippocampus neurons were obtained and cultured according to previously described protocol (12). Briefly, primary rat hippocampus samples were prepared from Sprague-Dawley rat brains at embryonic days 1–3, which were purchased from the Experimental Animal Center of China Medical University (Beijing, China) and were dissected in calcium- and magnesium-free Hank's balanced salt solution (Beyotime Institute of Biotechnology, Haimen, China), following incubation with a 0.25% trypsin solution for 30 min at 36°C in order to obtain primary hippocampus neuron cells. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/high glucose, horse serum containing 10% fetal bovine serum, 1% L-glutamine (3.6 mM), and 1% penicillin antibiotics and were grown in a 5% CO₂ atmosphere at 37°C. The primary rat hippocampus cells were cultured on plates which were coated in the fetal bovine serum (Beyotime Institute of Biotechnology) and were cultured at 37°C in humidified air. Two-thirds of the growing medium was changed every 2–3 days and the cells were subcultured roughly once a week. On day 12 of culturing, the incubation media were replaced with media with H₂O₂ (3, 30 and 300 µmol/l) to achieve oxidative stress injury. Following incubation for 30 min, NAC was added to the media at concentrations of 1, 10, 100 or 1,000 µmol/l.

Cell viability was measured using the MTT assay (Beyotime Institute of Biotechnology), which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases (13). MTT is absorbed by viable cells and then converted to formazan by the enzyme, succinate dehydrogenase in the mitochondria. The quantity of produced formazan thus correlates with the number of living cells. Cells were seeded in 24-well polystyrene plates with ~3×10³ cells per well. Plates were incubated at 37°C for 24 h to allow the cells to attach. After treatment with H₂O₂ for 0.5 h at 37°C followed by NAC for 24 h, the same volume of medium was added to the control cultures. Cell viability was determined using an MTT toxicity assay by adding 10 µl of 5 mg/ml MTT to each well. After 4 h of incubation at 37°C in humid air, formazan crystals were solubilized in 200 ml dimethyl sulfoxide. The optical density was measured at a wavelength of 570 nm with background correction at 655 nm using a Bio-Rad microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The mean averages of optical density from six replicate wells were used for each experimental sample and the control sample. Cell viability was calculated with a reference to the absorbance of control wells not challenged with H₂O₂ (assumed as 100% protection). Analyzed by light microscopy, viable cells displayed normal nuclear size and dark brown granules, whereas toxic cells exhibited condensed chromatin. The number of residual viable cells was counted.

To assess the enzymatic activity of GSH-peroxidase and lipid peroxide in primary hippocampus neuron culture after H₂O₂ injury, the cultures were washed with ice-cold phosphate-buffered saline (PBS) and then pooled and homogenized in 0.1 mol/l PBS containing 0.05 mmol/l ethylenediaminetetraacetic acid according to previous protocol (14). GSH-peroxidase activity was assessed using a GSH assay kit (Beyotime Institute of Biotechnology) by quantifying the rate of oxidation of reduced GSH to oxidized GSH. To investigate the effect of NAC on anti-oxidative stress in the AD cell model, the biomarkers of oxidative stress, including GSH and GSH disulfide (GSSG) were assessed. The level of GSH activity by the means of GSH/GSSG ratio. GSSG was obtained by determining the absorbance of 5-thio-2-nitrobenzoic acid produced from the reaction of the reduced GSH with DTNB. The level of maleic dialdehyde (MDA), a product of lipid peroxidation, was measured using an MDA assay kit (Beyotime Institute of Biotechnology) based on the thiobarbituric acid method (15).

The fluorescent probe DCFH-DA was used to monitor intracellular accumulation of ROS (16). Hippocampus neurons were seeded in collagen-coated 24-well plates at a density of 4×10⁵ cells/ml and incubated for 72 h. Cells were incubated with 300 µmol/l H₂O₂, a mixture of 1, 10 or 100 µmol/l NAC, or 300 µmol/l H₂O₂ alone at 37°C for 9 h. The cells were collected and washed with PBS three times. DCFH-DA was diluted in fresh DMEM to a final concentration of 5 µm and incubated with the cells for 30 min at 37°C. The chemicals were then removed and the cells were washed three times with PBS. Fluorescence emission was measured at excitation and emission wavelengths of 485 and 520 nm, respectively using fluorescence microplate. ROS production was expressed as a percentage of the control sample.

The primary rat hippocampus neurons were homogenized in protein extraction solution comprised of 20 mM Tris-HCl (pH 7.4), containing 1 mM NaF, 150 mM NaCl, 1% Triton X-100 and freshly-added protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), and 100 µM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). The supernatant contained total and membrane-enriched proteins. The Bicinchoninic Acid protein determination method was employed for the concentration of the proteins. Then, the proteins (30–50 ug) were separated by 8–12% sodium dodecyl polyacrylamide gels at 80 V for 50 min followed by 120 V for 40 min and electrophoretically transferred to a polyvinylidene fluoride membrane (PVDF) at 300 mA; the duration of electrophoresis depended on the molecular weight of the proteins. The PVDF membrane was blocked with freshly prepared Tris-buffered saline with Tween-20 (0.1%) containing 5% non-fat dry milk for 30–60 min at room temperature with constant agitation. Subsequently, the membrane was incubated with polyclonal rabbit anti-phospho-p38 immunoglobulin G (IgG; 1:1,000; cat. no. 2729, Cell Signaling Technology, Inc., Danvers, MA, USA), monoclonal rabbit anti-phospho-JNK IgG (1:1,000; cat. no. 4671, Cell Signaling Technology, Inc.), polyclonal rabbit anti-phospho-extracellular regulated kinase (ERK; 1:1,000; cat. no. 4370, Cell Signaling Technology, Inc.), polyclonal mouse anti-phospho-tau IgG (1:00; cat. no. 9632, Cell Signaling Technology, Inc.), monoclonal rabbit anti-p38 IgG (1:1,000; cat. no. 8690, Cell Signaling Technology, Inc.), polyclonal rabbit anti-JNK IgG (1:1,000; cat. no. 5136, Cell Signaling Technology, Inc.), polyclonal rabbit anti-ERK IgG (1:1,000; cat. no. 8544, Cell Signaling Technology, Inc.), polyclonal rabbit anti-tau IgG (1:500; cat. no. T7951, Sigma-Aldrich; Merck KGaA) and monoclonal mouse anti-β-actin IgG (1:1,000; cat. no. AA128, Beyotime Institute of Biotechnology) overnight at 4°C. The membrane was then incubated with anti-rabbit or anti-mouse horseradish peroxidase IgGs (1:1,000; A0208 or A0216, respectively, Beyotime Institute of Biotechnology) for 1–2 h at room temperature. Immunoreactive bands were visualized using an Enhanced Chemiluminescent Western Blotting Substrate (cat. no. 32106, Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and quantified using Quantity One software 3.0 (Image Lab, Bio-Rad Laboratories, Inc.).

Data were expressed as the mean ± standard deviation. Comparisons between different groups were performed by one-way analysis of variance followed by least significant difference post-hoc comparisons when appropriate. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA).

The viabilities of primary hippocampus neurons exposed to different concentrations of H₂O₂ (3, 30 or 300 µmol/l) were detected after 24 h of H₂O₂ incubation. The H₂O₂ reduced cell viabilities in a dose-dependent manner (P<0.05 vs. control group; Fig. 1A). The survival rate of the hippocampus neurons was ~78% when the neurons were incubated with 3 µmol/l of H₂O₂ for 24 h. However, the survival rate of neurons reduced to ~31% when treated with 300 µmol/l of H₂O₂ (P<0.01 vs. control group; Fig. 1A). Exposure of cells to NAC (1, 10, 100 or 1,000 µmol/l) significantly improved cell viability (P<0.05 vs. control group: Fig. 1B), although treatment with 1 µmol/l NAC was not significantly different when compared with H₂O₂ alone (P>0.05; Fig. 1B). In addition, 100 µmol/l NAC almost completely saved neurons from 300 µmol/l H₂O₂-induced cell deaths (82% survival rate compared with the control group). Analysis under a light microscope demonstrated that H₂O₂-induced neuron death in 50% of cells and significantly reduced neurite length. The pretreatment of cells with 100 µmol/l NAC tended to overcome these detrimental effects of H₂O₂ incubation (P<0.01; Fig. 2). Treatment with 100 µmol/l NAC significantly reduced H₂O₂-induced cell death (P<0.05; Fig. 2), indicating that NAC treatment elicited a potent protection effect on H₂O₂-induced cell viability.

In the present study, the MDA level as a measure of lipid peroxidation were significantly increased in the H₂O₂ group (300 µmol/l) compared with the control group (P<0.05; Fig. 3A). The MDA levels were significantly reduced in the NAC-low (L; 10 µmol/l, low-concentration of NAC) and NAC-H (100 µmol/l, high concentration of NAC) treatment groups vs. the H₂O₂ group (P<0.05; Fig. 3A). Similarly, no significant difference between the NAC-L and NAC-H groups was identified with regard to reducing the MDA level (P>0.05; Fig. 3A). To investigate the effect of NAC on anti-oxidative stress in the AD cell model, the biomarkers of oxidative stress, including GSH and GSH disulfide (GSSG) were assessed. The level of GSH activity by the aid of GSH/GSSG in the H₂O₂ group (300 µmol/l) was significantly decreased compared with the control group (P<0.05; Fig. 3B and C). Additionally, treatment with NAC significantly alleviated GSH activity compared with the H₂O₂ group (P<0.05; Fig. 3B and C). Furthermore, the NAC-H group demonstrated significantly increased GSH activity compared with the rats receiving NAC-L (P<0.05; Fig. 3B and C).

Oxidative stress is crucial in the pathogenesis of AD. In the current study, by using ROS fluorescent dye, DCFH-DA, the results demonstrated that intracellular DCF fluorescence was significantly increased in the presence of 300 µmol/l H₂O₂ compared with the control sample, which was abolished by treatment with 10 and 100 µmol/l NAC (P<0.05; Fig. 4), but not 1 µmol/l NAC (P>0.05; Fig. 4). Taken together, these data indicate that the neuroprotective effects of NAC against H₂O₂-induced neurotoxicity involve limiting oxidative stress injury.

Enhanced levels of phosphorylated (p)-p38 were detected in the presence of 300 µmol/l H₂O₂ injury (P<0.05 vs. control; Fig. 5A), while treatment with 100 µmol/l NAC caused the decline of p-p38 levels (P<0.05 vs. H₂O₂ incubation alone; Fig. 5A). Thus, the protective effects of NAC involved the attenuation of p38 protein phosphorylation. Furthermore, the levels of JNK protein phosphorylation were analyzed in primary hippocampus neurons in the absence or presence of NAC. p-JNK levels were significantly increased in cells exposed to H₂O₂ (P<0.05 vs. control; Fig. 5B) and significantly decreased following the addition of 100 µmol/l NAC to the culture (P<0.05 vs. H₂O₂ alone; Fig. 5B). Similarly, p-ERK expression levels were increased in H₂O₂-induced hippocampus neurons (P<0.05 vs. control; Fig. 5C), indicating that ERK activity had increased. In addition, NAC (100 µmol/l) significantly reduced the induction of p-ERK following H₂O₂ incubation when compared with the control group (P<0.05; Fig. 5C).

In the present study, western blot analyses demonstrated that an increased level of p-tau was observed in the presence of 300 µmol/l H₂O₂ compared with control hippocampus neurons (P<0.05; Fig. 5D), while the expression levels of p-tau were decreased in the NAC (100 µmol/l) treatment group compared with the H₂O₂ alone group (P<0.05; Fig. 5D). This clearly indicated that H₂O₂ resulted in tau phosphorylation in the hippocampus neurons and that NAC ameliorates the levels of p-tau.