Protective effect of tetrahydroxystilbene glucoside on 6-OHDA-induced apoptosis in PC12 cells through the ROS-NO pathway

Abstract

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4'-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress.

Figure 1. The chemical structure of TSG.

Figure 2. Effects of TSG and 6-OHDA on cell viability.

Cells were incubated for 24 h in different concentrations of 6-OHDA alone (A) or in different concentrations of TSG alone (B). Cells were preincubated with different concentrations of TSG (C) for 24 h or GSH (D) for 1 h, after which 6-OHDA (75 µM) was added for 24 h. The data are expressed as percentage of untreated control cells ± standard deviation (n = 6). **P<0.01 versus untreated control cells; # P<0.05, # #P<0.01 versus 6-OHDA-treated cells.

Figure 3. Fluorescence images show the nucleic changes of PC12 cells incubated in 6-OHDA with or without TSG.

Cells were stained with the DNA-binding fluorochrome Hoechst 33258. (A) shows normal culture medium nucleic morphology, (B) and (C) respectively show cells cultured in 50 µM TSG or 75 µM 6-OHDA for 24 h. In addition, cells were pretreated with 10 µM (D), 20 µM (E) or 50 µM (F) TSG for 24 h and then incubated in 6-OHDA (75 µM) for an additional 24 h. (G) Histograms showing ratio of condensed nuclei to total nuclei. White arrows represent location of apoptosis cell. Scale bars represent 50 µm. **P<0.01 versus untreated control cells; # #P<0.01 versus 6-OHDA-treated cells.

Figure 4. Cell apoptosis and necrosis detected by flow cytometry.

PC12 cells were incubated in drug-free medium (A) or medium containing 50 µM TSG (B) or 75 µM 6-OHDA (C) for 24 h; or cells were pretreated with 10 µM (D), 20 µM (E) or 50 µM (F) TSG for 24 h and then incubated in 6-OHDA (75 µM) for an additional 24 h. The results shown in (G) are the mean and SE for three independent experiments.

Figure 5. Effect of TSG on 6-OHDA-induced accumulation of intracellular ROS levels.

Cells were pretreated with different concentrations of TSG for 24 h and then incubated in 6-OHDA for an additional 24 h. Intracellular ROS levels were measured using DCFH-DA. Data are expressed as percentage of untreated control cells ± standard deviation (n = 6). **P<0.01 versus untreated control cells; # #P<0.01 versus 6-OHDA-treated cells.

Figure 6. Effect of TSG on 6-OHDA-induced accumulation of intracellular NO (A).

Cells were exposed to 6-OHDA with or without different concentrations of TSG for 24 h. (B) Effect of L-NMMA on 6-OHDA-induced cell viability in PC12 cells. Cells were pretreated with different concentrations of L-NMMA for one hour and then incubated in 6-OHDA for an additional 24 h. Data are expressed as percentage of untreated control cells ± standard deviation (n = 6). **P<0.01 versus untreated control cells; # #P<0.01 versus 6-OHDA-treated cells.

Figure 7. Effect of TSG on 6-OHDA-induced elevation of protein-bound 3-NT in PC12 cells.

PC12 cells were exposed to TSG (10, 20 or 50 µM) for 24 h before 75 µM 6-OHDA was added to the medium for an additional 24 h, and then 3-NT was measured. Data are expressed as percentage of untreated control cells ± standard deviation (n = 5). **P<0.01 versus untreated control cells; # P<0.05, # #P<0.01 versus 6-OHDA-treated cells.

Figure 8. Effect of TSG and 6-OHDA on the expression of nNOS and iNOS.

PC12 cells were exposed to 75 µM 6-OHDA with or without different concentrations of TSG for 24 h, and the nNOS (A) and the iNOS (B) were detected by Western blotting. (C) and (D) show the quantitative analysis of nNOS and iNOS protein levels, respectively. Data obtained from quantitative densitometry were presented as mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 versus untreated control cells; # P<0.05, # #P<0.01 versus 6-OHDA treated cells.