Figure 1.
Cytotoxicity screening of mangosteen extract in SK-N-SH cells.
SK-N-SH cells were treated with mangosteen extract at the concentrations of 25-800 µg/ml. After 24-hour incubation, cytotoxicity was determined by MTT assay. Data were presented as mean ± SEM (n=8 for each group). ***P < 0.001 compared to control.
Figure 2.
Effects of ME on chemical-induced cytotoxicity.
(A) Cytotoxic effects of H2O2 on SK-N-SH cells after 24-h incubation. (B) Effects of ME on H2O2-induced cytotoxicity. (C) Cytotoxic effects of PCB-52 on SK-N-SH cells after 24-h incubation. (D) Effects of ME on PCB-52-induced cytotoxicity. SK-N-SH cells were preincubated with ME for 3 h before the treatment of H2O2 and PCB-52 for 24 h. Cell viability was determined by MTT assay and expressed as percentage compared to untreated cells. Data were reported as mean ± SEM (n=10 independent experiments for H2O2). *P < 0.05, ** P < 0.01, *** P < 0.001 compared to untreated control; # P < 0.05, ### P < 0.001 compared to cells treated with corresponding chemicals concentration with no ME preincubation.
Figure 3.
Effects of ME on oxidative stress and apoptosis in vitro.
(A, B) Intracellular levels of ROS in SK-N-SH cells treated with H2O2 (A) or PCB-52 (B) for 24 h with or without prior ME incubation. ROS levels were determined using DCFH-DA assay. The fluorescence was measured and expressed as percentage compared to untreated cells (n=3 independent experiments for each bar). (C, D) Caspase-3 activity in SK-N-SH cells treated with H2O2 (C) or PCB-52 (D) with or without ME preincubation. Caspase-3 activity was measured using colorimetric assay. Data were expressed as percentage of the activity compared to untreated cells (n=5 independent experiments for each bar). *P < 0.05, ** P < 0.01, *** P < 0.001 compared to untreated control; # P < 0.05, ##P < 0.01 compared to cells treated with corresponding chemicals concentration with no ME preincubation.
Figure 4.
Effects of ME on AChE activity.
AChE activity was determined in SK-N-SH cells treated with ME or Donepezil (Aricept®) (A) at different concentration as stated on the X-axis for 24 h. The activity was measured using Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit and expressed as percentage compared to untreated cells. Data were reported as Mean ± SEM (n=3 independent experiments for each bar). *** P < 0.001 compared to untreated control.
Figure 5.
Effects of ME on spatial memory in mice.
(A) Effects of ME on escape latency during 3 test trials of day1, 2 and 3 of the treatment using Morris water maze test. ME (50 or 100 mg/kg) or water (control) was orally administered to the mice 45 min before the trials (n=6). The escape latency (sec) was expressed as Mean ± SEM. * P < 0.05 compared to control in the same test trial on the same test day; # P <0.05 compared to the trial 1 of the same test condition in the same test day. (B) Effects of ME on time in right quadrant (probe trial) on day 1-3 and 14-16 of the treatment using Morris water maze test. The mice were orally administered with water (control) or ME (50 and 100 mg/kg) at 45 minutes before the test. The escape latency (sec) was expressed as Mean ± SEM (n=6). * P < 0.05 compared to control on the same test day; # P < 0.05 compared to 50 mg/kg ME-treated group on the same test day.
Figure 6.
Effects of ME on scopolamine-induced memory impairment in mice.
Mice were orally administered with water (control) or 100 mg/kg ME once daily for 14 days. On day 14, the mice were subcutaneously injected with water or 1 mg/kg scopolamine 15 min after water/ME administration. The mice were subsequently subjected to Morris water maze test (A) or passive avoidance test (B) at 30 min after the injection. Latency time (sec) was expressed as Mean ± SEM (sec) (n=6). * P < 0.05 compared to control on the same test day; # P < 0.05 compared to scopolamine-treated group on the same test day.
Figure 7.
Effects of ME on reactive oxygen species (ROS) level and caspase-3 activity in mouse brain homogenates.
ROS levels and caspase-3 activity were determined in brain homogenates of water/scopolamine-treated mice with/without prior ME treatment for 14 days. Data were presented as percentage compared to untreated cells and reported as Mean ± SEM (n=6). * P < 0.05 compared to the control; # P < 0.05 compared to scopolamine- treated group.
Figure 8.
Western blot analysis of karyopherin β1 (KPNB1) in mouse brain homogenates.
Scopolamine-treated mice with or without prior ME treatment were obtained from passive avoidance test. At the end of the test, whole brain from the mice was subjected to protein extraction. A total 30 µg proteins derived from brain extracts (n=3) were resolved by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking non-specific bindings, the membrane was incubated with mouse monoclonal anti-KPNB1 (1:1,000 in 1% skim milk/PBS) and then incubated with rabbit anti-mouse IgG conjugated with horseradish peroxidase (1:2,000 in 1% skim milk/PBS). β-actin served as the loading control. The immunoreactive bands were visualized by chemiluminescence and autoradiography. Data was presented as KPNB1 band intensity relative to β-actin band. KPNB1 (97 kDa) level was significantly decreased after treatment with scopolamine but could be successfully preserved at its basal level by mangosteen extract. Data were reported as Mean ± SEM (n=3). * P < 0.05 compared to the control.