Figure 1.
Phloroglucinol ameliorates 6-OHDA induced motor deficits in rats.
(A) All of the animals of the four groups performed seven trials of the accelerated rota-rod test at 2 weeks after administration of vehicle, phloroglucinol, 6-OHDA or 6-OHDA plus phloroglucinol. Animals were placed on the rota-rod treadmill at an accelerating speed from 6 round/min to 30 round/min in 3 min. The latency to fall was measured and three training sessions were performed before each test. Animals were tested with seven trials on a given day. (ANOVA, post-hoc by Duncan, * p<0.05). (B) All of the animals of the four groups performed the apomorphine-induced rotation test at 3 weeks after the administration of vehicle, phloroglucinol, 6-OHDA or 6-OHDA plus phloroglucinol. Apomorphine was subcutaneously injected at 0.5 mg/kg, and the rotation was monitored for 60 min using the apparatus described by Ungerstedt and Arbuthnott [9]. The results were expressed as contralateral or ipsilateral net turns/60 min. (ANOVA, post-hoc by Duncan, ** p<0.01, ***p<0.001).
Figure 2.
Phloroglucinol attenuates 6-OHDA-induced loss of dopaminergic neurons and synapses in the midbrain.
(A) (C) Immunoreactivity of TH (red), synaptophysin (green) and DAPI (blue) was evaluated in ipsilateral (A) and contralateral sides (C) of the midbrain of each animal group by IHC, resspectively. All of the slides were examined on a LSM510 confocal microscope. (B) Quantitative analysis of TH immunoreactivity was performed and marked as a percentage of the control group. (D) The protein levels of TH and synaptophysin in the midbrain region were assessed with Western blotting. The relative densitometric value of TH or synaptophysin vs. that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown in graph. (ANOVA, post-hoc by Duncan,*p<0.05, **p<0.01, *** p<0.001).
Figure 3.
Phloroglucinol exerts protective effects against 6-OHDA in SH-SY5Y cells.
(A) Cell viability was measured by an MTT assay. SH-SY5Y cells were treated with a range of different concentrations of 6-OHDA (0, 25, 50, 100, and 200 µM of 6-OHDA). The IC50 was determined to be 90 µM 6-OHDA. (B) The cells were pre-treated with phloroglucinol (5, 10, 20, and 40 µg/ml) 1 h before treatment with 6-OHDA. The percentage of cell viability was measured compared to control. (ANOVA, post-hoc by Duncan, * p<0.05, **p<0.01). (C) Apoptotic bodies (arrows) were observed in cells stained with Hoechst 33342 dye and quantified by fluorescence microscopy. (ANOVA, post-hoc by Duncan, * p<0.05, **p<0.01).
Figure 4.
Phloroglucinol reduces the intracellular ROS caused by treatment with 6-OHDA in SH-SY5Y cells.
(A) Cells were seeded in a 96-well plate at 1.5×104 cells/well. After16 h plating, the cells were treated with phloroglucinol at 10 µg/ml. After 1 h, 90 µM of 6-OHDA was added to the plates. The cells were incubated for an additional 24 h at 37°C. After the addition of 25 µM of DCF-DA solution for 10 min, the fluorescence of the DCF was detected using a PerkinElmer LS-5B spectrofluorometer. (B) The intracellular ROS level was evaluated using a flow cytometry as previously described. (C) Microscopic images were collected using laser scanning confocal microscope 5 PASCAL. (ANOVA, post-hoc by Duncan, * p<0.05, **p<0.01).
Figure 5.
Phloroglucinol down-regulates lipid peroxidation, protein carbonylation and DNA base modification induced by 6-OHDA treatment in SH-SY5Y cells.
(A) Lipid peroxidation was assayed by determination of 8-isoprostane levels. 8-Isoprostane levels were determined in the culture medium by use of a commercial enzyme immunoassay and were performed according to the manufacturer's instructions. (B) The amount of carbonyl formation in protein was determined using an ELISA kit and expressed as nM. (C) The 8-hydroxyguanine content in DNA was determined using a Bioxytech 8-OHdG-ELISA kit purchased from OXIS Health Products and was performed according to the manufacturer's instructions. Cellular DNA was isolated using the DNAzol reagent and quantified using a spectrophotometer. (ANOVA, post-hoc by Duncan, * p<0.05, **p<0.01).
Figure 6.
Phloroglucinol attenuates the 6-OHDA-mediated loss of antioxidant enzymes in SH-SY5Y cells and rat brains.
(A) 50 µg of protein was added to 50 mM phosphate buffer (pH 7) containing 100 mM H2O2. The reaction mixture was incubated for 2 min at 37°C and the absorbance was monitored at 240 nm for 5 min. The change in absorbance over time was proportional to the breakdown of H2O2. The catalase activity was expressed as units/mg protein and one unit of enzyme activity was defined as the amount of enzyme required to breakdown 1 µM of H2O2. (B) Protein levels of catalase were evaluated with Western blotting in SH-SY5Y cells. (C) The harvested cells were suspended in 10 mM phosphate buffer (pH 7.5). The cells were centrifuged at 12,000× g for 30 min at 4°C to remove the tissue debris. Glutathione peroxidase activity was determined using the FR 17 assay kit according to the manufacturer's protocol. The enzyme reaction was assessed by adding the substrate, tert-butyl hydroperoxide and was recorded at 340 nm. The rate at which the absorbance (340 nm) decreased is directly proportional to the activity of glutathione peroxidase (expressed in mU/ml). (D) The protein levels of glutathione peroxidase in SH-SY5Y cells were evaluated with Western blotting in SH-SY5Y cells. (E) The protein levels of catalase in the ipsilateral midbrain region were assessed with Western blotting. (F) The protein levels of glutathione peroxidase in the ipsilateral midbrain region were assessed with Western blotting. (ANOVA, post-hoc by Duncan, * p<0.05, **p<0.01).
Figure 7.
Phloroglucinol attenuates the 6-OHDA-mediated loss of Nrf2 and p-Nrf2 in the nuclear fraction and p-Akt in SH-SY5Y cells and rat brains.
(A) The protein levels of Nrf2 and p-Nrf2 in the ipsilateral midbrain region were assessed with Western blotting. The blot is the representative of the two independent experiments. TATA box binding protein (TBP) was used as a loading control for nuclear fraction. (B) Cells were transfected with an ARE-luciferase construct (1 µg per well). After overnight, cells were treated with phloroglucinol or 6-OHDA, cell lysates were mixed with a luciferase substrate, and the luciferase activity was measured by a luminometer. (ANOVA, post-hoc by Duncan, * p<0.05, **p<0.01). (C) The protein levels of p-Akt and Akt in the ipsilateral midbrain region were assessed with Western blotting. The blot is the representative of the two independent experiments. The intensity of the bands was measured relative to the amount of Akt in each sample. The relative densitometric value of p-Akt vs. that of Akt is shown in graph.