Nutr Res Pract.  2013 Aug;7(4):249-255.

Epigallocatechin gallate attenuates L-DOPA-induced apoptosis in rat PC12 cells

• Lee MY
• Choi EJ
• Lee MK
• Lee JJ

Abstract

In this study, the protective effects of EGCG on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced oxidative cell death in catecholaminergic PC12 cells, the in vitro model of Parkinson's disease, were investigated. Treatment with L-DOPA at concentrations higher than 150 microM caused cytotoxicity in PC12 cells, as determined using the 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry detection. The apoptotic ratio was similar in cells treated with 100 microM EGCG plus 150 microM L-DOPA (5.02%) and the control (0.96%) (P > 0.05), and was lower than that of cells treated with L-DOPA only (32.24%, P < 0.05). The generation level of ROS (% of control) in cells treated with EGCG plus L-DOPA was lower than that in cells treated with L-DOPA only (123.90% vs 272.32%, P < 0.05). The optical density in production of TBARS in cells treated with L-DOPA only was higher than that in the control (0.27 +/- 0.05 vs 0.08 +/- 0.04, P < 0.05), and in cells treated with EGCG only (0.14 +/- 0.02, P < 0.05), and EGCG plus L-DOPA (0.13 +/- 0.02, P < 0.05). The intracellular level of GSH in cells treated with EGCG plus L-DOPA was higher than that in cells treated with L-DOPA only (233.25 +/- 16.44 vs 119.23 +/- 10.25, P < 0.05). These results suggest that EGCG protects against L-DOPA-induced oxidative apoptosis in PC12 cells, and might be a potent neuroprotective agent.

Keyword

Epillocatechin gallate; L-DOPA; PC12 cells; apoptosis; oxidative stress

MeSH Terms

Animals
Apoptosis
Catechin
Cell Death
Flow Cytometry
Levodopa
Oxidative Stress
Parkinson Disease
PC12 Cells
Rats
Thiobarbituric Acid Reactive Substances
Catechin
Levodopa
Thiobarbituric Acid Reactive Substances

Figure

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  Fig. 1 Chemical structure of epigallocatechin gallate [16]

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  Fig. 2 Effects of EGCG on viability of PC12 cells. PC12 cells were incubated for 24 h in media containing various concentrations of EGCG (50-200 µM). Each value was calculated as a percentage of the MTT assay in control PC12 cells. The results represent the mean ± SEM of six experiments.

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  Fig. 3 Effects of EGCG on L-DOPA-induced decrease in viability of PC12 cells. After 24 h in culture, PC12 cells were exposed to 50-150 µM L-DOPA or preincubated with 50-100 µM EGCG for 30 min before treatment with L-DOPA. Cell viability was assessed using the MTT method and the results represent the mean ± SEM of six experiments. *P < 0.05 compared with the untreated control group; #P < 0.05 compared with L-DOPA damaged cell groups (ANOVA followed by Tukey's test).

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  Fig. 4 Flow cytometric histograms of PC 12 cells after exposure to 150 µM L-DOPA alone for 24 h or preincubated with 100 µM EGCG for 30 min before treatment with L-DOPA. (A) After incubation, cells were harvested and stained with propidium iodide. Relative DNA content was analyzed by flow cytometry. The X-axis represents DNA content and the Y-axis represents the number of cells. (B) The results shown in present the mean ± SEM of six experiments. *P < 0.05 compared with the untreated control group; #P < 0.05 compared with L-DOPA damaged cell groups (ANOVA followed by Tukey's test).

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  Fig. 5 Effect of EGCG on L-DOPA-induced intracellular accumulation of ROS in PC12 cells loaded with DCFH2-DA. PC12 cells in culture after 1 h were exposed to 150 µM L-DOPA or preincubated with 100 µM EGCG for 30 min before treatment with L-DOPA. Formation of peroxides, detected using 2',7'-dichloroluorescin oxidation and fluorescence, was monitored on a Multi-Well Plate Reader. The results were expressed as a relative percentage of DCF fluorescence. Values are expressed as the mean ± SEM of triplicate determinations in six distinct experiments. *P < 0.05 compared with the untreated control group; #P < 0.05 compared with L-DOPA damaged cell groups (ANOVA followed by Tukey's test).

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  Fig. 6 Effect of EGCG on L-DOPA-induced TBARS formation in PC12 cells. PC12 cells in culture were exposed to 150 µM L-DOPA or preincubated with 100 µM EGCG for 30 min before treatment with L-DOPA. After exposure for 24 h, the increase in TBARS formation was evaluated. The results represent the mean ± SEM of six experiments. *P < 0.05 compared with the untreated control group; #P < 0.05 compared with L-DOPA damaged cell groups (ANOVA followed by Tukey's test).

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  Fig. 7 Decrease in intracellular GSH content by L-DOPA and its inhibition by EGCG. PC12 cells in culture for 24 h were exposed to 150 µM L-DOPA or preincubated with 100 µM EGCG for 30 min before treatment with L-DOPA. After incubation, the total intracellular GSH content was measured using DTNB, as described in the Materials and Methods section. The results represent the mean ± SEM of six experiments. *P < 0.05 compared with the untreated control group; #P < 0.05 compared with L-DOPA damaged cell groups (ANOVA followed by Tukey's test).

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