Ethyl vanillin (EVA), nerve growth factor (NGF), Aβ_1-42, Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS) and horse serum (HS) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). TUNEL Detection Kit (#11684817910) was purchased from Roche (Basel, Switzerland). The kits for malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and lactate dehydrogenase (LDH) detection were provided from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ROS assay kit and mitochondrial membrane potential with JC-1 kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). Polyclonal antibodies against cleaved caspase-3 (#9661), apoptosis regulator Bcl-2 (#2876s) and apoptosis regulator Bax (#2772) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase conjugated goat anti-rabbit antibodies (#GB23303) and bovine serum albumin (#G5001) were obtained from Servicebio, Inc. (Woburn, MA, USA).

Undifferentiated PC12 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured with 5 ng/ml NGF in DMEM containing 10% HS, 5% FBS and 1% penicillin-streptomycin in a CO₂ incubator (37°C and 5% CO₂). The medium was changed every other day. The cells were seeded at a density of 1×104 cells/ml and allowed to grow for 24 h prior to experimentation.

To assess the protective effect of EVA against Aβ_1-42-induced cytotoxicity, cell viability was detected by the MTT reduction assay, as previously described. Aβ_1-42 aggregates were prepared as previously described (30). PC12 cells were cultured at a density of 1×104 cells/well in poly-L-lysine-coated 96-well microplates for 24 h at 37°C. The cells were pretreated with various doses of EVA (0, 10, 30 and 100 µM) for 12 h and then exposed to 20 µM of Aβ_1-42 for 12 h. The EVA group was treated with 100 µM of EVA for 24 h. At the end of the drug treatment, 20 µl of MTT solution (5 mg/ml) was added to each well and the cells were incubated at 37°C for 4 h. The supernatants were subsequently replaced with 150 ml of dimethylsulfoxide. Cell viability was determined at a wavelength of 570 nm using an ELISA reader (Varioskan™ Flash Multimode Reader; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The LDH activity is an in vitro cellular toxicity marker that was determined in the present study using a commercial assay kit. In brief, PC12 cells were cultured at a density of 1×104 cells/well in poly-L-lysine-coated 96-well microplates for 24 h at 37°C. The cells were pretreated with various doses of EVA (0, 10, 30 and 100 µM) for 12 h and then exposed to 20 µM of Aβ_1-42 for 12 h. At the end of the drug treatment, 100 µl of the incubation medium was collected for the extracellular LDH activity assay. The adherent cells were washed with phosphate buffered saline (PBS) three times and subsequently homogenized. The homogenate was centrifuged at 4,000 × g for 30 min at 37°C and the supernatant was collected for the intracellular LDH activity assay. The absorbance of each sample was measured at a wavelength of 490 nm using a microplate reader according to the manufacturer's protocol. LDH release was calculated as follows:

Apoptotic cells were observed by TUNEL assay. Briefly, PC12 cells were cultured and treated on circular glass coverslips. The cells were pretreated with 100 µM of EVA for 12 h and then exposed to 20 µM Aβ_1-42 for 12 h. At the end of drug treatment, the cells were fixed in 4% paraformaldehyde for 15 min at 4°C and washed with PBS. The samples were further incubated in a blocking solution (obtained from the assay kit) for 20 min at 37°C and permeabilizing solution (0.1% Triton X-100 in 0.1% sodium acetate) for 30 min at 4°C. Subsequently, the cells were stained by TUNEL reaction solution for 1 h at 37°C, followed by cultured with 3% H₂O₂ solution for 15 min and rinsed with PBS for 10 min. The cells were finally treated with 3,3′-diaminobenzidine (DAB) substrate to produce a dark brown precipitate. Coverslips were stained with hematoxylin for 1 min at 37°C and mounted with neutral balsam. The number of TUNEL-positive cells was counted at 6 high-power (magnification, ×400) fields per slide.

The MDA content, an index of lipid peroxidation, was detected using a commercial assay kit according to the manufacturer's protocol. Briefly, PC12 cells were cultured at a density of 4×105 cells/well in 6-well microplates for 24 h at 37°C. The cells were pretreated with various doses of EVA (0, 10, 30 and 100 µM) for 12 h and then exposed to 20 µM Aβ_1-42 for 12 h. At the end of the drug treatment, the cells were washed with PBS and lysed in radio immunoprecipitation assay (RIPA) lysis buffer. The mixture was centrifuged at 12,000 × g for 30 min at 4°C. The collected supernatant samples were used for analysis of MDA concentration.

The accumulation of intracellular ROS was monitored using the fluorescent dye DCFH-DA as a probe. Briefly, PC12 cells were cultured at a density of 4×105 cells/well in 6-well microplates for 24 h at 37°C. The cells were pretreated with various doses of EVA (0, 10, 30 and 100 µM) for 12 h and then exposed to 20 µM of Aβ_1-42 for 12 h. At the end of the drug treatment, 1 ml of 10 µM DCFH-DA solution (part of the ROS kit) was added to the cells and cultured at 37°C for 30 min. In the presence of ROS, DCFH was converted to DCF, as the kit protocol states. Subsequently, the cells were rinsed three times with PBS and the fluorescence intensity was visualized by fluorescence microscopy (Carl Zeiss AG, Oberkochen, Germany). The levels of intracellular ROS of each group were normalized to those of the control group (untreated PC12 cells).

PC12 cells were cultured at a density of 4×105 cells/well in 6-well microplates for 24 h at 37°C. The cells were pretreated with various doses of EVA (0, 10, 30 and 100 µM) for 12 h and then exposed to 20 µM Aβ_1-42 for 12 h consecutive. At the end of the drug treatment, the cells were washed with PBS and lysed in RIPA lysis buffer. The mixture was centrifuged at 12,000 × g for 30 min at 4°C. The collected supernatants were used for the following analyses.

SOD, CAT and GSH-Px activity levels in PC12 cells were measured by commercial assay kits according to the instructions provided by the manufacturer. SOD activity was detected using the xanthine oxidase method at a wavelength of 550 nm using a spectrophotometer. CAT activity was determined using by measuring the absorbance at a wavelength of 405 nm with a spectrophotometer. GSH-Px activity was measured by quantifying the rate of oxidation of reduced glutathione to oxidized glutathione by H₂O₂ at a wavelength of 412 nm. The activity levels were normalized to the protein concentration of each sample.

Mitochondrial membrane potential was detected using the fluorescent JC-1 dye probe. PC12 cells were cultured at a density of 4×105 cells/well in 6-well microplates for 24 h at 37°C. The cells were pretreated with various doses of EVA (0, 10, 30 and 100 µM) for 12 h and then exposed to 20 µM of Aβ_1-42 for 12 h. At the end of the drug treatment, the cells were treated with 1 ml of JC-1 for 30 min at room temperature. Fluorescence intensity alterations were determined using flow cytometry (CXP 2.1, Beckman Coulter, Inc., Brea, CA, USA). The data are presented as the ratio of red to green signal. The changes in mitochondrial membrane potential were calculated as the integral of the decrease in the ratio of JC-1 fluorescence.

IHC analysis was used to evaluate the levels of cleaved caspase-3, Bax and Bcl-2 proteins. Briefly, PC12 cells were cultured and pretreated with 100 µM of EVA for 12 h at 37°C and subsequently exposed to 20 µM of Aβ_1-42 for 12 h at 37°C. At the end of the drug treatment, the cells were fixed in 4% paraformaldehyde for 15 min at 4°C and washed with PBS. Cells were then heated for 10 min in citrate buffer (pH 6.0) for antigen retrieval. The slides were subsequently exposed to 3% H₂O₂ solution for 25 min and blocked in 5% bovine serum albumin at room temperature for 30 min. Fixed cells were incubated with primary antibodies diluted in PBS (anti-cleaved caspase-3, 1:1,000; anti-Bax, 1:1,000; anti-Bcl-2, 1:500) overnight at 4°C. The samples were washed and treated with secondary antibody (goat anti-rabbit, 1:3,000) diluted in PBS at 37°C for 45 min. DAB was used to produce a dark brown precipitate at 37°C for 30 sec and hematoxylin was used to stain the nuclei of the cells at 37°C for 1 min. The cells were observed and images were captured with a fluorescence microscope (magnification, ×400; Carl Zeiss AG). The ratio of brown to total area was analyzed with Image-Pro Plus in 6 fields of view (Media Cybernetics, Inc., Rockville, MD, USA).

Data are presented as the mean ± standard deviation. The differences between two groups were assessed with one-way analysis of variance followed by Dunnett's post hoc test. Analyses were performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Aβ_1-42 can induce both oxidative stress and apoptosis in PC12 cells (13). The viability of Aβ_1-42-treated PC12 cells was assessed in the presence of EVA using the MTT assay (Fig. 2A). Cell viability was reduced to 62.07% in PC12 cells treated with 20 µM Aβ_1-42 compared with the control group in the absence of Aβ_1-42. In contrast to the concentration of 20 µM Aβ_1-42 alone, pretreatment with different doses (10, 30 and 100 µM) of EVA increased cell viability to 66.47, 75.38 and 78.29% of the control value, respectively, in a dose-dependent manner. Furthermore, PC12 cells cultured with 100 µM of EVA exhibited a cell viability of 99.49% compared with that noted in the control group in the absence of Aβ_1-42 at 24 h after the treatment. This result suggested that 100 µM of EVA alone did not reduce the viability of PC12 cells.

The LDH release assay was used to evaluate the effect of treatment with EVA on Aβ_1-42-induced cytotoxicity (Fig. 2B). The LDH release rate was increased to 185.84% in PC12 cells incubated with 20 µM of Aβ_1-42 compared with the control group untreated with Aβ_1-42. Compared with the group treated with 20 µM Aβ_1-42 only, pretreatment with different doses (10, 30 and 100 µM) of EVA decreased the LDH release rate to 175.66, 156.43 and 144.67% of the control value, respectively, in a dose-dependent manner.

To further investigate the protective effect of EVA on the Aβ_1-42-induced PC12 cell damage, the rate of apoptosis of PC12 cells was observed by the TUNEL assay (Fig. 3). The apoptotic cells were characterized by the appearance of intense brown staining. Control PC12 cells were not treated with Aβ_1-42 or EVA. Treatment of PC12 cells with 20 µM of Aβ_1-42 markedly increased the levels of apoptosis compared with those noted in the control cells. This effect was ameliorated by pretreatment of the cells with 100 µM of EVA. The results indicated that Aβ_1-42 inducedPC12 cell injury, and that EVA possessed the ability to inhibit this change induced by Aβ_1-42.

Oxidative stress serves an important role in the induction of PC12 cell apoptosis by Aβ_1-42 (13). The current study investigated the effects of EVA on the MDA levels and the levels of intracellular ROS (Fig. 4) in PC12 cells following treatment with Aβ_1-42. The production of ROS and the MDA levels were increased to 203.91% and 1.79 nmol/mg (control MDA value, 0.87 nmol/mg), respectively in PC12 cells induced with 20 µM of Aβ_1-42, compared with the control group. Pretreatment of Aβ_1-42-treated cells with different doses (10, 30 and 100 µM) of EVA decreased the intracellular ROS levels to 174.93, 160.86 and 139.51%, respectively, and the MDA levels to 1.61, 1.51 and 1.34 nmol/mg/protein, respectively, in a dose-dependent manner.

To determine the effect of EVA on the activities of antioxidative enzymes in Aβ_1-42-treated PC12 cells, the activity levels of SOD (Fig. 5A), CAT (Fig. 5B) and GSH-Px (Fig. 5C) were detected. The results indicated that treatment with Aβ_1-42 reduced the activities of SOD, CAT and GSH-Px to 25.47 U/ml (control SOD value, 44.25 U/ml), 6.13 U/ml (control CAT value, 9.46 U/ml) and 126.01 U/ml (control GSH-Px value, 213.91 U/ml), respectively. However, administration of different doses of EVA (10, 30 and 100 µM) ameliorated the activity levels of SOD (25.07, 33.37 and 36.24 U/ml, respectively), CAT (6.34, 7.71 and 8.11 U/ml, respectively) and GSH-Px (155.13, 177.26 and 198.63 U/ml, respectively). These activity levels of SOD, CAT and GSH-Px following pretreatment with EVA were close to those of the control cells and the protective effect was dose-dependent.

To determine whether EVA could stabilize the mitochondrial function, the mitochondrial membrane potential was measured using the JC-1 assay (Fig. 6). Treatment of PC12 cells with 20 µM Aβ_1-42 for 12 h caused a reduction in the mitochondrial membrane potential to 59.91% compared with the control cells. Pretreatment of PC12 cells with different doses of EVA (10, 30 and 100 µM), caused a significant increase in the mitochondrial membrane potential (69.72, 77.45 and 83.85% compared with the control value, respectively) in a dose-dependent manner.

The relative expression levels of Bcl-2/Bax and caspase-3 serve an important role in the induction of cell apoptosis (30). The effects of EVA on the apoptotic proteins in Aβ_1-42-treated PC12 cells were determined by the investigation of the expression levels of Bax, Bcl-2 and cleaved-caspase-3 proteins (Fig. 7). The results revealed that treatment with Aβ_1-42 reduced the ratio of Bcl-2/Bax to 73.26%. Pretreatment with 100 µM of EVA partially reversed this change in Aβ_1-42-treated PC12 cells. Furthermore, treatment with Aβ_1-42 increased the levels of cleaved-caspase-3 to 159.44% in PC12 cells compared with those of the normal control cells. Pretreatment with 100 µM of EVA inhibited the increase of the expression levels of cleaved-caspase-3 induced by Aβ_1-42. These results suggested that pretreatment with EVA may be effective for the prevention of Aβ_1-42-induced cell apoptosis in PC12 cells.