Dual beneficial effects of (-)-epigallocatechin-3-gallate on levodopa methylation and hippocampal neurodegeneration: in vitro and in vivo studies

Abstract

Background: A combination of levodopa (L-DOPA) and carbidopa is the most commonly-used treatment for symptom management in Parkinson's disease. Studies have shown that concomitant use of a COMT inhibitor is highly beneficial in controlling the wearing-off phenomenon by improving L-DOPA bioavailability as well as brain entry. The present study sought to determine whether (-)-epigallocatechin-3-gallate (EGCG), a common tea polyphenol, can serve as a naturally-occurring COMT inhibitor that also possesses neuroprotective actions.

Methodology/principal findings: Using both in vitro and in vivo models, we investigated the modulating effects of EGCG on L-DOPA methylation as well as on chemically induced oxidative neuronal damage and degeneration. EGCG strongly inhibited human liver COMT-mediated O-methylation of L-DOPA in a concentration-dependent manner in vitro, with an average IC50 of 0.36 microM. Oral administration of EGCG moderately lowered the accumulation of 3-O-methyldopa in the plasma and striatum of rats treated with L-DOPA+carbidopa. In addition, EGCG also reduced glutamate-induced oxidative cytotoxicity in cultured HT22 mouse hippocampal neuronal cells through inactivation of the nuclear factor kappaB-signaling pathway. Under in vivo conditions, administration of EGCG exerted a strong protective effect against kainic acid-induced oxidative neuronal death in the hippocampus of rats.

Conclusions/significance: These observations suggest that oral administration of EGCG may have significant beneficial effects in Parkinson's patients treated with L-DOPA and carbidopa by exerting a modest inhibition of L-DOPA methylation plus a strong neuroprotection against oxidative damage and degeneration.

Figure 1. Human COMT-mediated O-methylation of L-DOPA (left panel), which results in the formation of two monomethylated products.

It is hypothesized that (-)-epigallocatechin-3-gallate (EGCG, structure shown in the right panel) may serve as a naturally-occurring inhibitor of human COMT-mediated O-methylation of L-DOPA in vivo. In addition, owing to its strong antioxidant activity, it is hypothesized that this tea polyphenol may have additional neuroprotective effects in vivo.

Figure 2. Dependence of the in vitro O-methylation of L-DOPA on incubation time (A), cytosolic protein concentration (B), AdoMet concentration (C), and incubation pH (D).

The incubation mixture consisted of 10 µM L-DOPA substrate, 250 µM [methyl-³H]AdoMet (containing 0.2 µCi) or as indicated (C), 0.25 mg/mL of human liver cytosolic protein (HL4C) or as indicated (B), 1 mM dithiothreitol, and 1.2 mM MgCl₂ in a final volume of 1.0 mL Tris-HCl buffer (10 mM) at pH 7.4 or as indicated (D). The incubations were carried out at 37°C for 20 min or as indicated (A). Each value is the mean of duplicate determinations (with average variations <5%).

Figure 3. Inhibition of human liver COMT-mediated O-methylation of L-DOPA by EGCG, the green tea polyphenol (GTP) extract, and the black tea polyphenol (BTP) extract.

The incubation mixture consisted of 10 µM L-DOPA, 250 µM [³H-methyl]AdoMet (containing 0.2 µCi), 0.25 mg/mL of human cytosolic protein, 1 mM dithiothreitol, 1.2 mM MgCl₂, and a dietary inhibitor (concentration as indicated) in a final volume of 0.25 mL Tris-HCl buffer (10 mM, pH 7.4). Incubations were carried out at 37°C for 10 min. Each point is the mean of duplicate determinations (with average variations <5%).

Figure 4. Comparison of the concentrations of the plasma L-DOPA (A), plasma 3-OMD (B), striatal dopamine (C), and striatal 3-OMD (D) in rats.

Blood samples were collected from the tail vein at 0.5, 1, 2, 3, 4 and 6 h after oral administration of L-DOPA (20 mg/kg) alone or L-DOPA + carbidopa (20 +5 mg/kg) (N = 3 for each group). The vehicle group received water administration only. Striatal tissues were collected at indicated time points. Vertical bars indicate the standard deviations (S.D.). * P<0.05 compared to the vehicle group.

Figure 5. Effect of orally-administered EGCG on the methylation of L-DOPA in rats.

A. Plasma L-DOPA concentrations. B. Plasma 3-OMD concentrations. C. Striatal dopamine concentrations. D. Striatal 3-OMD concentrations. Rats (N = 3 for each group) were orally administered 100 or 400 mg/kg of EGCG 2 h before oral administration of L-DOPA + carbidopa (20+5 mg/kg). The vehicle group received water administration only. * P<0.05 compared to the vehicle treatment group. ^# P<0.05 compared to the L-DOPA + carbidopa group.

Figure 6. Protective effects of EGCG against glutamate-induced HT22 cell death.

A. Cell viability. B. Transcriptional activity of NF-κB. C. ROS generation. D. Percentage (%) of H₂-DCF-DA-positive cells. HT22 cells were treated with 5 mM glutamate and/or EGCG at indicated concentrations. Cell viability, transcriptional activity of NF-κB, NF-κB subcellular localization, and ROS accumulation were determined as described in the Materials and Methods. The experiment was repeated at least 3 times. DAPI staining was employed as a nuclear counter stain. Vertical bars indicate standard deviation (S.D., N = 5). * P<0.05 compared to glutamate-treated group.

Figure 7. Effect of orally-administered EGCG on kainic acid-induced rat hippocampal injury.

A. Histopathological and histochemical analyses of the brain tissues. B. Number of degenerating neurons based on the Fluoro-Jade B staining. C. Expression levels of GFAP-immunoreactive (IR) astrocytes in the CA3 region. D. Plasma NO₂ ⁻/NO₃ ⁻ levels. Rats (N = 3 for each group) were orally administered 100 or 400 mg/kg of EGCG 30 min before kainic acid injection. Kainic acid (1 µL of 1 mg/mL solution) was injected into the right lateral ventricle (anterior/posterior, −1.0; rostral, 1.6; dorsal/ventral, 4.5) using a microliter syringe. * P<0.05 compared to kainic acid-treated group.