不同食材萃出物和類黃酮對大鼠初代肝細胞中pi屬麩胱甘肽硫轉移酶表現之探討

Effects of various food extracts and flavonoids on the expression of the pi class of glutathione S-transferase in rat primary hepatocytes

童慧珊(Hui-Shan Tung)

指導教授 ： 李宗貴

中山醫學大學/健康管理學院/營養學研究所/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00108

摘要

許多天然食物的成份所以具有化學保護（chemoprotection）的功效，與其調節生物轉換酵素系統表現、改變生物轉換酵素活性，進而降低外來異物的毒害有關，比如麩胱甘肽硫轉移酶（Glutathione S-transferase, GST）為 phase Ⅱ 生物轉換酵素系統中重要成員之一，負責催化多種親電性外來異物與麩胱甘肽（glutathione, GSH）結合，增加產物親水性能力，加速其排除速率。本實驗分兩部份，第一部份預計探討蓮子（Ls）、蓮子心（Lp）、桑椹（M）及薏仁（As）四種食材之 50％ 乙醇及乙酸乙酯萃出物對大鼠初代肝細胞中 pi 屬GST（GSTP）蛋白質表現、酵素活性以及麩胱甘肽（GSH）含量之影響；第二部份則比較不同類黃酮對 GSTP 表現的誘發效果。
第一部份： SD 大鼠初代肝細胞離體培養 24 小時後，以不影響細胞毒性的濃度下，給予 Ls 與 Lp（0.05∼0.2 mg/ml）、M（0.2∼0.1 mg/ml）和As（0.02∼0.5 mg/ml）處理 48 小時。結果顯示，50％ 乙醇萃出物中，桑椹總酚類含量最高（186.69±23.15 mg GAE/g），依序為蓮子心、薏仁及蓮子（157.97±14.23、25.56±5.63 及 17.32±4.06 mg GAE/g）；而乙酸乙酯萃出物中，亦以桑椹最高（104.32±5.48 mg GAE/g），依序為蓮子心、蓮子及薏仁（28.89±7.68、13.97±10.89 及 2.36±1.99 mg GAE/g）。在類黃酮含量方面，50％ 乙醇萃出物中，蓮子心含量最多（192.43±20.9 mg QE/g），依序為蓮子及薏仁（9.58±1.8 及 9.20±1.2 mg QE/g）；乙酸乙酯萃出物亦是蓮子心最高（98.13±13.5 mg QE/g），依序為蓮子及薏仁（25.30±20.9 及 9.97±0.7 mg QE/g）。免疫西方墨點法指出 Ls、Lp、M 及 As 四種食材之乙醇萃出物及乙酸乙酯萃出物皆能以劑量關係顯著增加 GSTP 蛋白質表現，在0.1 mg/ml劑量時，乙醇萃出物以 As 誘發最好，而乙酸乙酯萃則以 M 誘發 GSTP 蛋白質效果最好，而此誘發結果與總酚類含量及類黃酮含量高低無關。四種食材乙醇萃出物及乙酸乙酯萃出物雖不改變胞內還原態 GSH 含量，卻顯著減少氧化態 GSH（GSSG）（p<0.05），使得 GSH/GSSG 比值顯著提升（p<0.05）。根據蓮子、蓮子心、桑椹及薏仁四種食材在誘發 GSTP 蛋白質表現與改善胞內 GSH 氧化還原狀態所呈現的良好效應，顯示它們在提升解毒及抗氧化能力的應用潛力。
第二部分：探討不同類黃酮（包含apigenin、butein、chrysin、fisetin、hesperidin、kaempferol、luteolin、morin、myricetin、naringerin、quercitrin、quercetin、taxifolin 及 phloretin）對大鼠初代肝細胞內 GSTP 蛋白質表現及可能機轉。SD 大鼠初代肝細胞離體培養 48 小時後，加入 25 μM 類黃酮，再培養 24 小時後取樣，觀察 GSTP 蛋白質與 mRNA 的表現及酵素活性。結果顯示，在初代肝細胞中 14 種類黃酮中，以查酮類的 butein 誘發GSTP 蛋白質表現的能力最強（6.7倍），依序為chrysin（6.4 倍）＞myricetin（5.0倍）＞fisetin（3.8倍）＞quercetin（3.6倍）≧phlortein（3.6倍）≧morin（3.6倍）＞luteolin (3.5倍)＞hesperidin（3.4倍）＞quercitrin （3.0倍）＞naringerin （2.9倍）（p<0.05），而 taxifolin、kaempferol 及 apigenin 與控制組相比則無差異；雖然 GSTP 蛋白質表現增加，但 GSTP mRNA 表現及酵素活性在本實驗卻無差異。進一步探討 GSTP 基因調控區（promoter）上的 GSTP enhancer element I 及 II（GPE I 及 GPE II）在不同類黃酮誘發 GSTP 表現過程中所扮演的角色。三個含不同長度 GSTP promoter 的報導基因，分別是 pTA-2713（含 GPEⅠ 及 GPEⅡ）、pTA-2604（只含 GPEⅡ）及 pTA-2375（不含 GPEⅠ 及 GPEⅡ），短暫轉染（transiently transfect）至大鼠肝臟細胞株 Clone 9 後，再分別處理不同類黃酮 24 小時，結果顯示，轉染含有 GPEⅠ的 pTA-2713 細胞中，類黃酮可誘發含有 GPE I 的 pTA-2713 報導基因的活性，其中以黃酮醇類 fisetin 誘發 luciferase 活性最強（2.5倍），依序為 quercetin＞kamepferol＞chrysin＞butein（2.4∼2.1倍）（p<0.05）， phloretin、naringenin、myricetin 與控制組相比則無差異，而此結果除了 chrysin之外，其餘類黃酮與誘發 Clone 9 GSTP 蛋白質表現趨勢一致；若轉染不含 GPE I的 pTA-2604 或 pTA-2375，則誘發現象隨即消失，顯示出 GPE I 是類黃酮誘發 GSTP 表現的重要調控位置。

關鍵字

蓮子 ； 蓮子心 ； 桑椹 ； 薏仁 ； 類黃酮 ； 麩胱甘肽GSTP enhancer elementⅠ(GPEⅠ) ； pi 屬麩胱甘 月太 硫轉移 酉每 (GSTP) ； 初代肝細胞 ； 大鼠

並列摘要

Many natural foods are known to display potential chemoprotective effects. The modulation of biotransformation enzyme expression and also the change of biotransformation enzyme activity may partly account for the protection of health foods against xenobiotic insult. Glutathione S-transferase (GST), one of the important phase Ⅱ drug-metabolizing enzyme, is responsible for catalyzing the conjugation of glutathione (GSH) to eletrophilic xenobiotics and facilitating their excretion. GST is composed of at least 6 isoforms and the pi class (GSTP) is the most highly inducible form in liver tissues. This study was designed to examine whether food extracts and various flavonoids were capable of modulating GSTP expression in rat hepatocytes. There are two parts in this study: the first one focused on the effects of various extracts of Lotus seed, Lotus plumule, Mulberry and Adlay seed; the second part determined and compared the inducibility of various flavonoids on GSTP protein
expression.
Experiment 1: Twenty-four hours post plating, rat primary hepatocytes were treated with 50% ethanol (Et50) or ethyl acetate (EA) extract of Lotus seed (LsEE, LsEAE) (0.05-0.2 mg/ml), Lotus plumule (LpEE, LpEAE) (0.05-0.2 mg/ml), Mulberry (MEE, MEAE) (0.02-0.1 mg/ml) and Adlay seed (AsEE, AsEAE) (0.02-0.5 mg/ml) for 48 h, respectively. The concentration ranges stated above showed no toxicity to hepatocytes. For ethanolic extracts, Mulberry had the highest total phenolic contents (186.69±23.15 mg GAE/g), following by Lp, As, and Ls (157.97±14.23, 25.56±5.63 and 17.32±4.06 mg GAE/g). For EA extraction, Mulberry again had the highest phenolic contents (104.32±5.48 mg GAE/g), following by Lp, Ls, and As (28.89±7.68, 13.97±10.89 and 2.36±1.99 mg GAE/g). Regarding to flavonoids contents, Et50 extract of Lp had the highest flavonoids contents (192.43±20.9 mg QE/g), following by Ls and As (9.58±1.8, 9.20±1.2 and 17.32±4.06 mg QE/g); a similar pattern was also noted on the EA extracts, Lp (98.13±13.5 mg QE/g) > Ls (25.30±20.9 mg QE/g) > As (9.97±0.7 mg QE/g). Immunoblotting assay showed that ethanol and ethyl acetate extracts of all four foods dose-dependently increased protein expression of GSTP. The maximum induction of GSTP expression was noted for AsEE and MEAE. Although intracellular GSH level was not changed by all extracts, GSSG contents were significantly decreased by both ethanol and ethyl acetate extracts of Lotus seed, Lotus plumule, Mulberry and Adlay seed as compared with control (p<0.05). Decrease of GSSG was accompanied by an increase of GSH/GSSG ratio. Results suggest that Lotus seed, Lotus plumule, Mulberry and Adlay seed effectively induce hepatic GSTP protein expression and also modulate GSH redox status by decreasing of GSSG level. These findings may partly explain the potent activity of Lotus seed, Lotus plumule, Mulberry and Adlay seed in modulation of detoxification and antioxidation capability.
Experiment 2: The effects of 14 flavonoids (included apigenin, butein, chrysin, fisetin, hesperidin, kaempferol, luteolin, morin, myricetin, naringerin, quercitrin, quercetin, taxifolin and phloretin) on GSTP protein expression and activity and also the undergoing mechanism in rat primary hepatocytes were investigated. Forty-eight hours post plating, cells were treated with 25 μM of each flavonoid for an additional 24 h. Immunoblots showed that butein (6.7 fold) had the highest induction of GSTP expression in 14 flavonoids tested, following by chrysin (6.4 fold), myricetin (5.0 fold), fisetin (3.8 fold), quercetin (3.6 fold), phlortein (3.6 fold), morin (3.6 fold), luteolin (3.5 fold), hesperidin (3.4 fold), quercitrin (3.0 fold), and naringerin (2.9 fold). Taxifolin, kaempferol, and apigenin showed no induction on GSTP protein as compared with control. The changes of GST enzyme activity toward ethacrynic acid, however, were not found in this study. To examine whether flavonoids activation of GSTP gene is associated to the GSTP enhancer element (GPE) I and/or GPEⅡ. The pTA-luciferase reporter constructs with full length (pTA-2713, with both GPE I and II), or constructs without GPE I, or without both GPE I and II were transiently transfected into Clone 9 liver cells and then exposed to 10 μM flavonoids for an additional 24 h. Results showed that flavonoid induction of luciferase activity was only noted in cells transfected with pTA-2713 and a 2.1-2.5 fold induction of enzyme activity was noted by fisetin, quercetin, kamepferol, chrysin, and butein. However, phloretin, naringenin and myricetin had no effect as compared with control. The induction pattern in luciferase activity by flavonoids is consistent with changes of GSTP protein expression in Clone 9 cells. Deletion of GPE I abolished the induction by flavonoids. These results suggest that GPE I, an enhancer element in GSTP promoter, is essential for the GSTP gene induction by flavonoids.

並列關鍵字

Lotus seed ； Lotus plumule ； Mulberry ； Adlay seed ； Flavonoids ； pi Class of glutathione S-transferase ； Glutathione ； GSTP enhancer element I ； Primary hepatocytes ； Rat

參考文獻

莊靜如（2002）。是食品也是藥品-桑椹可多吃！。健康世界，201：73-74。

江文章、徐明麗、蘇瑞斌與龐飛（2000）。薏仁加工食品輔助抑制腫瘤功效之評估。醫護科技學刊，2：113-122。

Yang, D., Wang, Q., Ke, L., Jiang, J., and Ying, T. (2007). Antioxidant activities of various extracts of lotus (Nelumbo nuficera Gaertn) rhizome. Asia Pac J Clin Nutr 16 Suppl 1, 158-163.

高德錚、梁純玲（1986）。省產薏苡品質之檢定。台中農業改良場研究彙報，13：11-18。

吳宜娟、江文章、姚賢宗與江孟燦（2003）。糙薏仁及其加工食品對大白鼠血膽固醇濃度及肝臟脂質過氧化作用之探討。台灣農業化學與食品科學，41：(4)：254-262。