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. 2015 May 6;26(7):416-23.
doi: 10.1097/WNR.0000000000000363.

The protective role of (-)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation

Qianqian He  1 , Lei Bao, Jeffrey Zimering, Kun Zan, Zuohui Zhang, Hongjuan Shi, Jie Zu, Xinxin Yang, Fang Hua, Xinchun Ye, Guiyun Cui
Affiliations

The protective role of (-)-epigallocatechin-3-gallate in thrombin-induced neuronal cell apoptosis and JNK-MAPK activation

Qianqian He et al. Neuroreport. .

Abstract

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has anti-inflammatory and antioxidant properties and provides neuroprotection against central nervous system diseases. Yet, it is not known whether EGCG may be neuroprotective against intracerebral hemorrhage. In this study, we used a simplified in-vitro model of thrombin neurotoxicity to test whether EGCG provides neuroprotection against thrombin-associated toxicity. Exposure of primary cortical neurons to thrombin (100 U/ml) caused dose-dependent and time-dependent cytotoxicity. Cell Counting Kit 8 and lactate dehydrogenase were used to monitor cell viability after exposure of neurons to thrombin or EGCG and after EGCG pretreatment. Flow cytometric analysis and western blotting demonstrated that thrombin-induced neuron degeneration occurs through apoptosis. A concentration of 25 μM EGCG significantly abolished thrombin-induced toxicity and prevented apoptosis by suppressing c-Jun-N-terminal kinase (JNK) phosphorylation, and the JNK inhibitor SP600125 reduced thrombin-induced caspase 3 activation and apoptosis. These data suggest that EGCG may have protective effects against thrombin-induced neuroapoptosis by inhibiting the activation of JNK, leading to caspase 3 cleavage. EGCG is a novel candidate neuroprotective agent against intracerebral hemorrhage-induced neurotoxicity.

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Figures

Fig. 1
Fig. 1
Assessment of neuron purity. (a) β3-Tublin-marked neurons showed red fluorescence. (b) Nuclei counterstained with DAPI showed blue fluorescence. (c) Colocalization of cytoplasm β3-tublin and nuclear DAPI (scale bar, 50 μm). DAPI, 4',6-diamidino-2-phenylindole.
Fig. 2
Fig. 2
CCK8 and LDH assays were used to detect cell viability. (a, b) Neuron cell viability after exposure of neurons to thrombin or EGCG with a different concentration gradient (*P<0.05 vs. 5 U/ml thrombin, ***P<0.001 vs. control group). (c, d) Neuron cell damage increased in the thrombin group and EGCG pretreatment significantly increased cell viability (*P<0.05 vs. control group). Columns represent the mean±SE. n=4. CCK8, Cell Counting Kit 8; EGCG, (−)-epigallocatechin-3-gallate; LDH, lactate dehydrogenase; TM, thrombin.
Fig. 3
Fig. 3
Activation of JNK and caspase 3 in neuron cells. (a) The time dependence of JNK/caspase 3 activation in thrombin-induced neurons (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 vs. control group, ##P<0.05 vs. thrombin group). (b) EGCG pretreatment significantly decreased JNK and caspase 3 activation. (c) SP600125 pretreatment significantly decreased caspase 3 activation (*P<0.05 vs. thrombin group). Columns represent the mean±SE. n=3. DMSO, dimethyl sulfoxide; EGCG, (−)-epigallocatechin-3-gallate; JNK, c-Jun-N-terminal kinase; TM, thrombin.
Fig. 4
Fig. 4
Fluorescence activated cell-sorting Annexin V–FITC/propidium iodide (PI) kit was used to detect cell apoptosis. (a, b) EGCG and SP600125 pretreatment significantly decreased cell apoptosis. *P<0.05 versus the thrombin group, **P<0.01 versus control group, ##P<0.01 versus thrombin group. Each column represents the mean±SE. n=3. DMSO, dimethyl sulfoxide; EGCG, (−)-epigallocatechin-3-gallate; TM, thrombin.
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