Effects of Green Tea Extracts on the Excitability of Primary Cultured Rat Embryonic Neuron

從綠茶中萃取出來的多酚化合物主要有四種成份：(–)-epigallocatechin gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epicatechin (EC)。這些多酚已經被證實能夠抑制癌細胞的生長，防止神經退 化性疾病的形成，甚至能減少許多疾病的產生風險，像是中風、肥胖、心血管疾 病和老化。為了探討綠茶多酚對初級培養大鼠胚胎神經細胞的興奮性質有何影 響，我們利用全細胞膜片箝制技術去記錄離子通道電流和動作電位。研究結果顯 示ECG 減慢了電壓調控型鈉離子通道的不活化速率，而EC50 為12.88 ± 0.73 μM，同時此反應是可逆轉的；然而EGCG 卻沒有同樣的作用。鈉離子通道的不 活化恆穩態曲線也被ECG從-41.67 ± 0.08 毫伏特向負極移動到 -45.15 ± 0.05 毫 伏特。另外鈉離子通道從關閉狀態的恢復速率時間常數也從3.91 ± 0.08 毫秒延 長到 7.74 ± 0.43 毫秒。利用全細胞膜電流箝制技術記錄動作電位，ECG 會將電 作電位的強度和半寬分別從原先的96.5 ± 0.5 毫伏特和3.17 ± 0.13 毫秒增加到 103.9 ± 0.3 毫伏特和7.07 ± 0.12 毫秒，但是動作電位產生的頻率卻從16.4 ± 1.2 赫茲降到 6.6 ± 0.8 赫茲。這些研究結果顯示ECG 會經由影響鈉離子通道的性質 進一步改變神經細胞動作電位的產生，因而改變神經細胞的興奮性質。ABSTRACT Polyphenols extracted from green tea (Camellia sinensis) is composed of four major chemicals: (–)-epigallocatechin gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG), and (–)-epicatechin (EC). These polyphenols have been shown to inhibit the growth of tumor cells, prevent neurodegenerative diseases, and reduce the risk of a series of illnesses, such as stroke, obesity, cardiovascular diseases, and aging. To study the effects of polyophenols on the excitability of primary cultured rat embryonic cortical neurons, whole-cell patch-clamp recording was used to monitor the ionic currents and action potential firing. The result showed that ECG reversibly slowed the inactivation of voltage-gated sodium channels with an EC50 of 12.88 ± 0.73 μM; however, EGCG did not have any effect on Na+ current. The steady-state inactivation of Na+ channel was negatively shifted from -41.67 ± 0.08 to -45.15 ± 0.05 mV and the time constant required for the recovery of Na+ channel to close state was prolonged from 3.91 ± 0.08 to 7.74 ± 0.43 ms by ECG. When action potentials were elicited under current clamp mode, the amplitude and half-width were increased from 96.5 ± 0.5 to 103.9 ± 0.3 mV and 3.17 ± 0.13 to 7.07 ± 0.12 ms, respectively; but frequency was decreased from 16.4 ± 1.2 to 6.6 ± 0.8 Hz. The results suggest that ECG may modulate excitability of neurons through altering sodium channels.CONTENTS Ⅰ. ABSTRACTS Ⅰ.1 Chinese abstract………………………………………………………………1 Ⅰ.2 English abstract……………………………………………………………….2 Ⅱ. INTRODUCTION Ⅱ.1 Benefits of green tea polyphenols……………………………………………3 Ⅱ.2 Inactivation of voltage-gated sodium channel…………………………….....4 Ⅱ.3 Action potential in excitable cell......................................................................5 Ⅱ.4 Our aims………………………………………………...................................6 Ⅲ. METHODS AND MATERIALS Ⅲ.1 Solution and chemicals……………………………………………………....7 Ⅲ.2 Reagents……………………………………………………………………..7 Ⅲ.3 Primary neuronal cell culture..........................................................................8 Ⅲ.4 Electrophysiology...………………………………………………………….8 Ⅲ.5 Date analysis…………………………………………………………………9 Ⅳ. RESULTS Ⅳ.1 ECG slows inactivation of VGSC……………………..................................10 Ⅳ.2 Cadmium blocks slowed inactivation of VGSC by ECG…………………...11 Ⅳ.3 Slowed inactivation of VGSC by ECG doesn’t require calcium influx…….11 Ⅳ.4 ECG suppresses steady-state inactivation curve ……………………..…….11 Ⅳ.5 ECG slows recovery rate from inactivation………………………………...12 Ⅳ.6 ECG alters frequency, amplitude, half-width of action potential…………...12 Ⅳ.7 ECG modulates action potential pattern of neurons according to their intrinsic firing frequency…………………………………………………………13 Ⅴ. DISCUSSION……………………………………………………………………14 Ⅵ. REFERENCE…………………………………………………………………...17 Ⅶ. Scheme…………………………………………………………………………...22 Ⅷ. Table………………………………………...……………………………………23 Ⅸ. Figures…………………………………………………………………………...2