Sev, AICAR, Annexin V-FITC Apoptosis Detection Kit and Hoechst 33342 were purchased from Sigma-Aldrich (Merck KGaA). Fetal bovine serum (FBS) was purchased from Gibco (Thermo Fisher Scientific, Inc.). The following primary antibodies were purchased from Abcam: Anti-actin (cat. no. ab179467), anti-total-(t-) AMPK (cat. no. ab80039), anti-p-AMPK (cat. no. ab133448), anti-SIRT1 (cat. no. ab189494). AMPK (cat. no. bf30053) and SIRT1 ELISA kits (cat. no. bf90658) were purchased from Northern Institute of Biotechnology of China.

PC12 cells, originally derived from a pheochromocytoma of rat adrenal medulla, were used to evaluate the involved mechanism of Sev-induced cell apoptosis. PC12 cells were obtained from Shanghai Institute for Biological Science Cell Bank, CAS. The cells were cultured in DMEM with 5% FBS in an incubator at 37°C with 5% CO₂. For Sev exposure, the cell culture plates were placed in the airtight incubator connected to an anesthesia machine that was used to supply Sev into the incubator. PC12 cells were exposed to 3% Sev for 24 h (18). For the intervention studies, AICAR, an AMPK activator, was used to treat PC12 cells at a concentration of 0.5 mM for 1 h at 37°C prior to Sev treatment.

The cell apoptosis was evaluated by flow cytometry analysis using Annexin V-FITC/PI apoptosis analysis, according to the manufacturer's instructions (Shanghai Yeasen Biotechnology Co., Ltd.). Briefly, PC12 cells were exposed to Sev for 24 h, and harvested using trypsin. The harvested cells were resuspended in 1X binding buffer (Shanghai Yeasen Biotechnology Co., Ltd.) at a concentration of 1×10⁶ cells/ml. A total of 5 µl of Annexin V-FITC and 10 µl PI staining solution was subsequently added to the suspensions. The suspensions were vortexed gently, and 400 µl of 1X binding buffer was added and incubated for 5 min at room temperature in the dark. The fluorescence of FITC and PI was analyzed by flow cytometry. Both early apoptotic (Annexin V-FITC⁺/PI⁻) and late apoptotic (Annexin V-FITC⁺/PI⁺) cells were included in cell apoptosis determinations.

A total of 6×10⁵ PC12 cells were seeded into 6-well plates and exposed to Sev at 37°C for 24 h. A total of 1 ml of 4% paraformaldehyde was added to each well for 10 min for fixation and washed twice with PBS. A total of 10 mg/l Hoechst 33342 was added and stained for 10 min at room temperature. Cell apoptosis was observed under a fluorescence microscope. Apoptotic cells were characterized by rounded cell morphology, half-moon nuclei with condensed chromatin. The apoptotic cells were counted the average no. of cells/mm² in 10 fields.

After incubation with Sev for 24 h, PC12 cells were collected, rinsed twice with cold PBS and lysed by RIPA buffer (Thermo Fisher Scientific, Inc.) containing 1 mM PMSF on ice for 30 min, followed by centrifugation at 12,000 × g for 5 min at 4°C. A total of 40 µg of total protein was loaded in each well and resolved by 10% SDS-PAGE. After electrophoresis, samples were transferred onto polyvinylidene fluoride (PVDF) membranes, which were subsequently blocked with 5% skimmed milk for 1 h and incubated with anti-t-AMPK (1:800), anti-p-AMPK (1:500), anti-SIRT1 (1:1,000) and anti-actin (1:2,000) primary antibodies at 4°C for 12 h. The membrane was washed three times with PBST (0.1% Tween-20), and then incubated with the second antibody (goat anti-rabbit 1:2,000 in blocking solution) at 37°C for 2 h. The membrane was then washed three times with PBST. Then, the signals were visualized with chemiluminescence reagents (Thermo Fisher Scientific, Inc.). Results were semi-quantified via Quantity One software (version 4.4; Bio-Rad Laboratories, Inc.).

AMPK and SIRT1 levels in cell culture medium and hippocampus homogenate were measured by commercially available ELISA kits.

A total of 20 male Sprague-Dawley (SD) rats (28±2 days old; weight 100±10 g) were used. Animals were housed with free access to food and water at the temperature of 22±2°C, humidity of 55±5% and a 12-h light/dark cycle. The rats were randomly assigned to the control (Con) and Sev groups. The Con group was not exposed to Sev. Sev group received inhalation of 3% Sevin O₂/N₂ (fraction of inspired oxygen 50%) for 2 h daily for 5 days consecutively. The dose of Sev was determined as previously described (19). For the intervention studies, AICAR, an AMPK activator, was administered to the rats via an intraperitoneal injection for 2 h at a dose of 500 mg/kg prior to Sev treatment. The health and behavior of animals were monitored every day. The present study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All efforts to improve animal welfare were taken, including to minimize suffering and distress, use of an aesthetics or special housing conditions.

Rats were trained in a 1.5 m diameter open-field water maze filled with water (26°C) and made opaque with latex liquid. Prominent extra-maze visual cues around the room remained in fixed positions throughout the experiment. During behavioral testing, animals were required to locate a hidden submerged platform 10 cm in diameter (1.5 cm below the surface), which remained in the same position across trials for individual animals but was counterbalanced across animals. The animals were given four trials per day for 4 days. Trials began as the rat was placed in the pool facing the side wall at a start position and ended once the animal had found the platform; if the rat had not found the platform within 120 sec, it was guided there by hand. A video camera mounted to the ceiling directly above the center of the maze was used in conjunction with the animal tracking system. Test animals underwent Sev exposure for 5 days followed by the water maze experiment for 5 days. On day 11, the animals were sacrificed to explore further mechanisms under anesthesia with mebumal sodium (50 mg/kg) by intraperitoneal injection.

After the last water maze was completed, the rats were anesthetized and euthanized by mebumal sodium. The hippocampus was removed and the rats were verified dead. Then, the hippocampus was weighed, transferred into a glass homogenizer and five volumes of precooled PBS (0.02 mol/l, pH 7.0–7.2) was added. The tissue was fully ground and then treated with ultrasonic crushing. After centrifugation of the prepared homogenate at 10,000 × g for 5 min at 4°C, the hippocampal homogenate was found in the supernatant.

t-tests and two-way ANOVA, followed by a Bonferroni post hoc analysis, were performed for comparisons between groups. Statistical analysis was conducted on a standard software package (SAS^® version 10.0; SAS Institute, Inc.) was used. All data are presented as the mean ± SEM. Each experiment was repeated >4 times. P<0.05 was considered to indicate a statistically significant difference.

Cell apoptosis was assessed by Hoechst 33342 staining and Annexin V-FITC/PI staining. To investigate the effects of Sev on neuronal apoptosis, the percentage of apoptotic cells was measured using Annexin V/PI staining. The results showed that the percentage of apoptotic cells was significantly increased after treatment with Sev (Fig. 1A and B). Furthermore, Hoechst 33342 staining also showed that Sev caused neuronal apoptosis (Fig. 1C and D).

The AMPK and SIRT1 protein expression levels were assessed by western blot analysis. As shown in Fig. 2A and B, p-AMPK and SIRT1 protein expression levels were significantly decreased after Sev treatment. The t-AMPK protein expression had no significant difference between the two treatment groups. The ratio of p-AMPK/t-AMPK decreased in the group treated with Sev. Next, AMPK and SIRT1 activity was examined in cell culture medium by ELISA. There was a significant inhibitory effect of Sev on AMPK and SIRT1 activity (Fig. 2C and D). These results indicated that inhibition of AMPK/SIRT1 signaling was related to neuronal apoptosis in response to Sev.

To examine the role of AMPK/SIRT1 signaling in Sev-induced neuronal apoptosis, the neurons were treated with the AMPK activator-AICAR at a concentration of 0.5 mM, and the percentage of apoptotic cells was evaluated using Annexin V/PI staining. As shown in Fig. 3A and B, AICAR was able to attenuate the neuronal apoptosis induced by Sev. AICAR also blocked the decline inSIRT1 activity induced by Sev (Fig. 3C). These results suggested that neuronal apoptosis caused by Sev was dependent on the AMPK/SIRT1 signaling pathway.

To examine the role of AMPK/SIRT1 signaling in Sev-induced cognition impairment, the rats were treated with the AMPK activator-AICAR at 500 mg/kg, and cognition was evaluated using the water maze test. As shown in Fig. 4A-C, Sev increased the escape latency time and distance in the water maze. The escape latency time and distance were notably decreased in the AICAR-treated rats. Furthermore, Sev decreased the AMPK and SIRT1 activity in the hippocampus. When AICAR was administered to rats, the AMPK and SIRT1 activity in the hippocampus was significantly increased (Fig. 4D). These results suggested that AMPK activation could improve the cognitive impairment caused by Sev.