A total of 60 male BALB/c mice (age ~6 weeks, weight ~20 g) were purchased from Shanghai SIPPR-BK Laboratory Animal Co., Ltd. (SCXK2013-0016). All animal experiments were approved by the Institutional Animal Care and Use Committee at Nanjing University of Traditional Chinese Medicine, and were conducted in accordance with institutional guidelines for the care and use of laboratory animals. The experimental animals were randomly housed in mouse cages (5 mice/cage) for 1 week under the conditions of constant temperature (~23°C) and humidity (~50%), and a 12-h light/dark cycle, with free access to food and water prior to the experiment.

The chronic restraint stress model was established as previously described (23). Each mouse was single-housed for the whole experimental procedure and placed in a 50-ml centrifuge tube with several ventholes for 6 h daily (from 9:00 to 15:00) for 3 weeks. Following chronic restraint stressing, the mice were returned to their original cages. Additionally, overnight illumination was randomly performed on all mice twice-weekly. The mice in the control group were group-housed under standard conditions (n=8). The body weights of all mice were recorded every week.

Apigenin was obtained from Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (Nanjing University of Traditional Chinese Medicine), and subsequently dissolved in normal saline with 0.5% w/v Tween-80 prior to administration via gavage (20, 40 and 60 mg/kg; n=10/group). Further, 25 mg/kg fluoxetine hydrochloride (Tokyo Chemical Industry Co., Ltd.) was dissolved in normal saline and administered via gavage (n=10). The mice in the control group were treated with normal saline with 0.5% w/v Tween-80 via gavage (n=8). The administration was started on the 22th day of modeling (modeling was carried out from the day one) and lasted for 14 days (once a day). The drug doses were optimized according to our previous optimal dose-response-relationship studies (data not shown).

The sucrose preference test was performed every 7 days throughout the experimental period. Mice were single-housed, and then each mouse was presented with two bottles filled with 2% sucrose water for 3 consecutive days. After an 18-h deprivation of both water and food, each mouse was presented with two bottles for 2 h with the same appearance: One filled with clear water and the other with 2% sucrose water. Sucrose preference was calculated using the following formula: Sucrose preference (%)=sucrose solution consumption (g)/total consumption (g).

The anxiety-like behavior and physical condition of the mice were analyzed in the open field test, which was conducted as described previously (24). On the 36th day of the experimental process, each mouse was subjected to the open field test in a bright open area (~300 lux, 40×40 cm). The mice were softly placed in the test area and allowed to explore freely for 5 min. Digitized images of the active orbit of each mouse were recorded. The total distance travelled and the time spent in the center were analyzed using ANY-maze software (version 4.3; Stoelting Co.) to evaluate the locomotor activity and anxiety-like behavior of mice. The experimental apparatus was washed with 70% ethyl alcohol between consecutive tests.

Following a 2-week intervention administration, each mouse were gently placed into a 5-l cylindrical transparent glass tank with clear water (~23°C) and forced to swim for 6 min. The immobility time was measured during the final 4 min of the 6 min from the video recorded using ANY-maze software. Following the test, the mice were dried with an electric hair dryer and returned to their cages.

The tail suspension test is employed to evaluate depressive-like behavior and the response to antidepressant treatments in mice at day 36 (25). The test was performed by an ANY-maze system that recorded 6 animals at a time. Each mouse was suspended by the tail at 50 cm above the floor with adhesive tape affixed 1 cm from the tip of the tail. The entire test required 6 min to complete, and animals were considered to be mobile or immobile. The final 4 min of the total 6 min were analyzed using ANY-maze software to quantify the immobility time.

All mice were euthanized by cervical dislocation after the behavioral tests, and the hippocampus samples were promptly collected and placed on ice. The samples were then placed into radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) with an enzyme inhibitor (Beyotime Institute of Biotechnology) at 5°C and rapidly homogenized for western blotting, as previously reported (21,26). Protein concentration was determined colorimetrically by BCA assay (Pierce; Thermo Fisher Scientific, Inc.). Protein lysates (30 µg) were separated by 10% SDS-PAGE electrophoresis and were transferred onto polyvinylidene difluoride (PVDF) membranes. The primary antibodies, incubated at 4°C for 12 h, included: Rabbit anti-AMPKɑ (1:1,000; 2532S), rabbit anti-phosphorylated (p)-AMPKɑ (Thr172; 1:1,000; 2535S), anti-p-Unc-51 like autophagy activating kinase-1 (ULK1; Ser317; 1:1000; 12753S), rabbit anti-p-mTOR (1:1,000; 2971S), rabbit anti-mTOR (1:1,000; 2792S; all obtained from Cell Signaling Technology, Inc.), rabbit anti-microtubule-associated protein light chain 3 (LC3)-II/I (1:1,000, 14600-1-AP), rabbit anti-ULK1 (1:1,000; 20986-1-AP), rabbit anti-p62/sequestosome 1 (SQSTM1; 1:1,000; 18420-1-AP) and rabbit anti-GAPDH (1:3,000; 10494-1-AP; all purchased from ProteinTech Group, Inc.). The secondary antibody, incubated at room temperature for 2 h, was horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:3,000; SA00001-2; ProteinTech Group, Inc.). Densitometry was conducted and analyzed using ImageJ software (version 1.52a, National Institutes of Health). Blots were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). The loading amounts in each lane were normalized to GAPDH. All experiments were performed three times.

For cytotoxicity assays, cells were grown in Dulbecco's Modified Eagle's medium (Gibco; Thermo Fisher Scientific Inc.) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc.) in an atmosphere of 5% CO₂ and 95% air for 24 h at 37°C. HT22, SH-SY5Y and N2a cells (obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.) were seeded into 60 wells of a 96-well plate (6,000 cells/well), with the remaining wells holding media. Following incubation for 24 h, cells were treated with various concentrations of apigenin (0, 3.125, 6.25, 12.5, 25, 50 and 100 µM dissolved in 0.1% DMSO) for 48 h. Thereafter, MTT was added into each well with a volume of 20 µl and incubated for 4 h. DMSO was added to each well and the absorbance was measured at 492 nm using a microplate reader (Thermo Fisher Scientific, Inc.). The CC₅₀ values were calculated as the concentration of apigenin resulting in 50% reduction of absorbance compared to untreated cells. All experiments were performed three times.

All data were expressed as the mean ± standard error of the mean. Differences among groups were analyzed using one-way ANOVA with Bonferroni correction to adjust for multiple testing, and P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed with GraphPad Prism 6.0 software (GraphPad Software, Inc.).

A significant difference in sucrose preference was observed in the chronic restraint stress group (vehicle group) compared with the control group (P<0.05; Fig. 2A). A significant effect of treatment on sucrose preference was observed (F_(5,9)=2.646, P=0.0375); as presented in Fig. 2A, the deficits induced by chronic restraint stress were significantly ameliorated following treatment with 60 mg/kg apigenin for 14 days (P<0.05 vs. vehicle group). Additionally, a significant effect of treatment on body weight was observed (F_(5,44)=11.55, P<0.0001); specifically, both 20 and 60 mg/kg apigenin significantly increased body weight compared with the vehicle group (20 mg/kg, P<0.01; 60 mg/kg, P<0.001; Fig. 2B).

In the open field test (Fig. 3A), the time spent in the center of the experimental area was not significantly affected by treatment group (F_(5,31)=2.014, P=0.1041); however, all doses of apigenin demonstrated a non-significant trend towards prolonging the time spent in the center, which was markedly reduced by chronic restraint stress. The total distance travelled (Fig. 3B) was significantly affected by animal treatment (F_(5,42)=3.684, P=0.0075); however, no significant differences were observed between the control and vehicle groups, or the vehicle and apigenin groups (P>0.05). Reduced time spent in the center is indicative of anxiety-like behavior in mice, and the total distance indicated that apigenin or chronic restraint stress did not alter the motor ability of mice.

As presented in Fig. 4, treatment significantly affected the immobility time in the tail suspension (F_(5,37)=3.162, P=0.0178) and forced swim tests (F_(5,39)=5.282, P=0.0008). The prolonged immobility time induced by chronic restraint stress (P<0.05 vs. control group) in the tail suspension test was rescued by the administration of 60 mg/kg apigenin (P<0.01 vs. vehicle group; Fig. 4A). In the forced swim test (Fig. 4B), the increased immobility time induced by chronic restraint stress (P<0.001 vs. control group) was significantly reduced following 40 mg/kg apigenin treatment (P<0.05 vs. vehicle group). The alleviation of these behavioral test deficits indicated the antidepressive actions of apigenin in depressive-like mice.

The expression levels of LC3-II/I and p62/SQSTM1 were assessed in order to measure the degree of autophagy in the hippocampus; treatment significantly affected the expression of LC3-II/ (F_(4,22)=5.621, P=0.0028) and p62 (F_(4,17)=7.041, P=0.0016; Fig. 5). As presented in Fig. 5, the levels of autophagy were found to be reduced in the vehicle group, as determined by the significantly downregulated expression of LC3-II/I (P<0.01 vs. control group) and increased level of p62/SQSTM1 (P<0.05 vs. control group). Conversely, apigenin significantly enhanced the levels of LC3-II/I (20 mg/kg, P<0.05; 40 and 60 mg/kg, P<0.01 vs. vehicle group) and downregulated the expression of p62 (20 mg/kg, P<0.01; 40 mg/kg, P<0.05; 60 mg/kg, P<0.001 vs. vehicle group) in hippocampal samples. These findings indicated that apigenin regulated the degree of autophagy in chronic restraint stress mice.

Treatment significantly affected the phosphorylation of AMPK (F_(4,21)=4.298, P=0.0107; Fig. 6A); the expression levels of p-AMPK were rescued following administration of apigenin (20 mg/kg, P<0.01; 60 mg/kg, P<0.05 vs. vehicle group). ULK1-Ser317 levels were not significantly affected by treatment (F_(4,15)=2.907, P=0.0578; Fig. 6B). Fig. 6C and D present the significant effects of treatment on the p-AMPK/AMPK (F_(4,21)=17.83, P<0.0001; Fig. 6C) and ULK1-Ser317/ULK1 ratios (F_(4,15)=8.596, P=0.0008; Fig. 6D). The decreased levels of p-AMPK/AMPK (P<0.001 vs. control group) and ULK1-Ser317/ULK1 (P<0.05 vs. control group) induced by chronic restraint stress were significantly reversed following the administration of apigenin (all doses, P<0.001 vs. vehicle group; Fig. 6C; 60 mg/kg, P<0.05 vs. vehicle group; Fig. 6D). The results indicated that the AMPK/ULK1 pathway may be a target of apigenin in the hippocampus of depressive-like mice.

The phosphorylation of mTOR normalized to GAPDH (F_(4,22)=7.425, P=0.0006) and total mTOR (F_(4,22)=10.75, P<0.0001) was significantly affected by treatment group (Fig. 7). As presented in Fig. 7A, the increase in the levels of p-mTOR induced by chronic restraint stress in the vehicle group was significantly reversed following treatment with apigenin (20 mg/kg, P<0.001 vs. vehicle group). The expression levels of p-mTOR/mTOR were significantly increased by chronic restraint stress (P<0.05 vs. control group; Fig. 7B); however, following administration of apigenin, the p-mTOR/mTOR ratio was significantly reduced (20 and 60 mg/kg, P<0.001; 40 mg/kg, P<0.01 vs. vehicle group). These results suggest that the activity of mTOR was involved in apigenin-mediated autophagy level.

To determine the cytotoxic potential of apigenin, its effects on the viability of different cell lines were evaluated (Fig. 8A). Apigenin induced a significant increase in the viability of HT22 cells at concentrations of ≤12.5 µM (P<0.001); however, it induced cytotoxic effects in all cell lines at high concentrations (F_(6,49)=8,653, P<0.0001 for N2a; F(6,49)=2,445, P<0.0001 for HT22; F_(6,49)=1,533, P<0.0001 for SH-SY5Y). As presented in Fig. 8B, it was revealed that the CC₅₀ of apigenin in SH-SY5Y, HT22 and N2a cells were 42.97, 74.96 and 50.06 µM, respectively. The data indicated that apigenin exerted dose-dependent cytotoxic effects on neuronal cells.