Pu-Erh tea and GABA attenuates oxidative stress in kainic acid-induced status epilepticus

Abstract

Background: Pu-Erh tea is one of the most-consumed beverages due to its taste and the anti-anxiety-producing effect of the gamma-aminobutyric acid (GABA) if contains. However the protective effects of Pu-Erh tea and its constituent, GABA to kainic acid (KA)-induced seizure have not been fully investigated.

Methods: We analyzed the effect of Pu-Erh tea leaf (PETL) and GABA on KA-induced neuronal injury in vivo and in vitro.

Results: PETL and GABA reduced the maximal seizure classes, predominant behavioral seizure patterns, and lipid peroxidation in male FVB mice with status epilepticus. PETL extracts and GABA were effective in protecting KA-treated PC12 cells in a dose-dependent manner and they decreased Ca(2+) release, ROS production and lipid peroxidation from KA-stressed PC12 cells. Western blot results revealed that mitogen-activated protein kinases (MAPKs), RhoA and cyclo-oxygenase-2 (COX-2) expression were increased in PC12 cells under KA stress, and PETL and GABA significantly reduced COX-2 and p38 MAPK expression, but not that of RhoA. Furthermore, PETL and GABA reduced PGE(2) production from KA-induced PC12 cells.

Conclusions: Taken together, PETL and GABA have neuroprotective effects against excitotoxins that may have clinical applications in epilepsy.

Figure 1

H&E stain of KA-stressed FVB mice cortex. Kainic acid (KA, 10 mg/kg) caused neuronal damage. After 5 h KA-induced SE of FVB mice, the cortex was observed with cell shrinkage and long shape (B). PETL 10 mg/kg (C) or GABA 1 mg/kg (D) significantly reduced KA-induced neuronal damage in cortex of the FVB mice as compared to control (A). (20x)

Figure 2

DAPI and TUNEL staining of hippocampus form KA-stressed mice. KA induced apoptosis (green fluorescence) of hippocampus neurons on vehicle control mice (B). The TUNEL staining showed that 10 mg/kg PETL (C) and 1 mg/kg GABA (D) significantly reduced KA-induced apoptosis in hippocampus of the FVB mice brain as compared to control (A). (200x)

Figure 3

Effect of PETL extract and GABA on cell viability and cytotoxicity of KA-stressed PC12 cells. Cells were treated with KA (150 μM) alone or with various concentrations of PETL extract (1, 10 μ g/ml) or GABA (0.1, 1, 10 μM) for 24 h. LDH (A) release was decreased and cell viability (B) was increased by PETL extract and GABA. *P < 0.01 as compared to KA control.

Figure 4

Effect of PETL extract and GABA on Ca²⁺ generation from KA-treated PC12 cells. Cells were treated with KA (150 μM) alone or with various concentrations of PETL extract or GABA for 24 h. PETL and GABA were effectively reducing the release of Ca²⁺ under KA stress. *P < 0.01 as compared to the KA control.

Figure 5

Effect of PETL extract and GABA on ROS generation in PC12 cells under KA stress. PETL extract (1, 10 (j,g/ml) and GABA (0.1, 1, 10 uM) were effectively reducing the ROS production from PC12 cells induced by KA stress (150 uM) at 120-min. *P < 0.01 as compared to the KA control.

Figure 6

In vitro and in vivo effect of PETL extract and GABA on the KA-induced oxidative stress. KA-induced lipid peroxidation of PC12 cells and brain neuron tissue of FVB mice were determined by ELISA and spectrophotometry, respectively. PETL or GABA was effectively reducing lipid peroxidation of PC12 cells by under 24-h KA stress (A) and in mice with 2-h KA-induced SE (B). *P < 0.01 as compared to the KA control.

Figure 7

Kainic acid-induced caspase-3 activation. Cells were treated with KA (150 μM) alone or with PETL extract and GABA in various concentrations for 24 h. Both PETL and GABA decreased the caspase-3 activity significantly, *P < 0.01 as compared to the KA control.

Figure 8

Effect of PETL extract and GABA on KA-activated signaling pathway. COX-2, JNK, ERK, p38 MAP kinases, and RhoA in PC12 cell under KA stress for 30-min was determined by Western blot assay. Values represent the mean from three independent experiments. *P < 0.05 as compared to the KA control.

Figure 9

Effect of PETL extract and GABA on PGE₂ production. PETL extract and GABA, significantly reduced the PGE₂ production of KA-induced PC12 cells. *P < 0.01 as compared to the KA control.