香菜酒精萃出物誘發大鼠clone 9肝細胞第二型解毒酵素及血基質氧化酶-1表現

摘要香菜是單年生草本食物，原產地為地中海、中東西部以及東南亞，因香菜的莖跟葉具有特殊的香味，常被用於烹調提味之用。香菜富含許多營養素包括類黃酮、多酚類、維生素C、胡蘿蔔素等，都已被證實具有降血糖、降血脂、抗菌、抗氧化等功效，本實驗室在之前的研究也發現，巨噬細胞給予香菜萃出物可以降低細菌內毒素lipopolysaccharide誘發之發炎反應。本研究主要探討香菜萃出物對於大鼠Clone 9肝細胞調控第二型解毒酵素和血基質氧化酶-1 (heme oxygenase 1，HO-1)的功效及相關的調控機制。大鼠Clone 9肝細胞分別給予300μg/mL 95%酒精的萃出物（香菜全株、香菜莖和香菜葉）24小時，結果發現可以增加pi屬麩胱甘肽硫轉移酶 (glutathione S-transferase, GSTP)、HO-1和黃素蛋白(NAD(P)H: quinone oxidoreductase 1, NQO-1) 蛋白質、mRNA表現和酵素活性，其中以香菜莖萃出物誘發的效果最好。此外我們也發現給予香菜萃出物可以增加p38磷酸化表現、增加nuclear factor erythroid 2 related factor 2（Nrf-2）轉錄至細胞核、誘發antioxidant response receptor（ARE） 和GSTP enhancer I（GPE-1）reproter的活性，Electrophoretic Mobility Shift Assay（EMSA）的結果顯示香菜萃出物可以增加ARE 和GPE-1核蛋白與DNA結合之能力。p38抑制劑SB203580抑制香菜萃出物對GSTP、HO-1、NQO-1蛋白質及mRNA的誘發功效。此外，本實驗也以病毒干擾技術抑制Nrf2表現，結果顯示抑制Clone 9 細胞Nrf2表現，使得香菜萃出物無法誘發GSTP、NQO-1蛋白質表現，然而HO-1的表現則不受影響。綜合以上實驗結果可知，香菜萃出物經由Nrf2/ P38訊息傳遞路徑，誘發GSTP、NQO-1蛋白質及mRNA的表現。

Coriandrum sativum belongs to the family Apiaceae widely used in Mediterranean, Middle estern and Southeast Asia as spices and herbal medicine. Coriandrum sativum is rich in phenolic compounds wich have been report to lessen inflammation and oxidative stress. Our previous studies demonstrated the anti-inflammatory property of Coriandrum sativum. Here we examined the effect of Coriandrum sativum ethanol extract on expression of phase II detoxification enzymes and heme oxygenase 1 (HO-1) in rat clone 9 liver cells. Coriandrum sativum ethanol extract singnificantly induced HO-1, pi class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein and mRNA expression and enzyme activities. Furthermore Coriandrum sativum ethanol extract increased phosphorylation of p38 mitogen-activated protein kinase (p38) and nuclear factor erythroid 2 related factor 2 (Nrf-2) nuclear translocation, Nrf-2 nuclear protein DNA binding activity, as well as antioxidant response receptor (ARE) -luciferase activity and GSTP promoter activity. Pretreatment with SB203580, p38 inhibitor, blocked p38 activation as well as HO-1, GSTP and NQO-1 protein expression induced by Coriandrum sativum ethanol extract. Moreover, silencing of Nrf2 expression by shRNA gene knockdown only inhibiting Coriandrum sativum ethanol extract-induced GSTP and NQO-1 protein expression but not HO-1. These results suggested that Coriandrum sativum up-regulated expression of GSTP and NQO-1 through the p38 and Nrf2/ARE pathway.