飲食因子--大蒜有機硫成分與含硫氨基酸--調節π屬麩胱甘肽硫轉移酶表現之研究

Dietary factors--garlic allyl sulfides and sulfur amino acids--regulate the gene expression of the π class of glutathione S-transferase

蔡佳文(Chia-Wen Tsai)

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/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00090

摘要

生物體為了維持生命與健康必須由飲食中獲得氨基酸、碳水化合物、脂肪酸、礦物質及維生素等多種營養因子，它們可提供能量、修補體內組織、維持生長、調節酵素或賀爾蒙活性，近年來，證據顯示各類營養因子在基因表現的調控上，可能扮演比原先預期更重要的角色。除了營養因子之外，近年來由於養生觀念的盛行，食物中各式各樣phytochemicals (比如phenolic compounds和allyl sulfides等)所展現的多樣生理活性也成為注意焦點，許多證據更是指出它們的生理機能性與調節基因表現有著密切關係。雖然大蒜上調解毒酵素--π屬麩胱甘肽硫轉移酶(π class of Glutathione S-transferase, GSTP)表現已有不少研究，但其調節機轉則仍未知；至於氨基酸狀態是否影響此一phase II解毒酵素表現，也是一值得討論議題。基於以上理由，本研究結合分子生物技術，進一步探討飲食因子--大蒜有機硫成分(DAS、DADS和DATS)與含硫氨基酸調節GSTP的效應與機轉。

本研究以初代肝細胞及Clone 9 肝細胞株為細胞模式，首先將初代肝細胞培養於DAS、DADS和DATS (50-200 μM)之培養基24小時後，觀察這些大蒜有機硫成分對於GSTP酵素活性、mRNA和蛋白質表現的效應；至於在Clone 9肝細胞部分，以DAS、DADS和DATS (5-200 μM)處理後，除了上述實驗之外，我們亦進行轉殖作用(transfection)，並探討對於訊號傳遞的影響。結果顯示，除DAS之外，DADS和DATS皆以劑量關係誘發GSTP酵素活性、mRNA和蛋白質表現。大鼠GSTP基因的5’端啟動區(全長約2713 bp)利用質體建構(plasmid construction)方式做成GSTP luciferase reporter gene，並進行5’deletion以觀察大蒜含硫成分對於改變GSTP基因轉錄速率作用的可能位置，轉殖後，結果顯示：DADS和DATS 的luciferase活性相較控制組分別增加2.8和3.9倍，若將位於5’端啟動區-2.7至-2.6 kb的GSTP enhancer I (GPE I)刪除，則破壞DADS和DATS的誘發效應，然而GPE II的刪除並不影響DADS和DATS的作用，可知GSTP 5’端啟動區的GPE I是DADS和DATS增加GSTP轉錄作用所必需。由於GPE I上擁有一對含AP-1結合位置的12-O-tetradecanoylphorbol-13-acetate response element (TRE)，因此進一步利用Electrophoretic mobility shift assay (EMSA)分析在DADS或DATS處理下，細胞核內AP-1與DNA結合力的變化，結果指出：DADS及DATS的確增加AP-1活化與TRE的結合，如同時加入SP600125 (JNK抑制劑)及PD98059 (MEK抑制劑)不但減少此結合作用，同時也降低DADS及DATS誘發GSTP的表現。以上結果可知，DADS和DATS這兩種脂溶性大蒜硫成分可能藉由JNK及ERK活化AP-1，進而透過TRE 的結合，增加GSTP酵素活性、mRNA和蛋白質的表現。

在氨基酸實驗部分，當細胞培養液中甲硫氨酸與半胱氨酸濃度同時低至0.1 mM時，大鼠初代肝細胞GSTP蛋白質表現含量與酵素活性均明顯高於培養在高含硫氨基酸(0.5 mM甲硫氨酸和0.2 mM半胱氨酸)的細胞，且此一效應與胰島素或甲基脫氫皮質醇(dexamethasone)無關。探討此一誘發效應是否也可出現在其他氨基酸缺乏時，結果顯示L-lysine、L-leucine、L-isoleucine和L-phenylalanine等必須氨基酸限制並未能改變GSTP表現，再進一步比較其他GST異構酶表現，GSTA和GSTM的表現均不受含硫氨基酸影響。綜合這些結果，氨基酸限制對GSTP基因表現的誘發現象可能具含硫氨基酸專一性。在調節機轉上，目前仍進行中，也尚未獲得結論，但證據顯示限制含硫氨基酸上調GSTP基因表現的途徑可能有oxidative stress-dependent pathway與amino acid response element-dependent pathway，進一步實驗仍屬必要。

關鍵字

解毒酵素 ； π屬麩胱甘肽硫轉移酶基因調節 ； 大蒜有機硫成分 ； 含硫氨基酸

並列摘要

Nutrients including amino acids, carbohydrates, fatty acids, minerals, and vitamins have multiple functions, in addition to maintain normal cell growth, they build body tissue, influence energy metabolism and enzyme activity.
Nutrients are also able to interact with gene regulation which allowing organisms to adapt to the changes of nutritional environment. In addition to nutrients, dietary phytochemical compounds (such as phenolic compounds and allyl sulfides) are also reported to regulate gene expression. Several studies showed that garlic up-regulates the π class of glutathione S-transferase (GSTP) gene, but the molecular mechanisms are not fully understood. The GSTP gene regulation is also not noted by amino acids.Based on these reasons, the main objective of this study was to investigate the molecular effect of garlic allyl sulfides (DAS、DADS and DATS) and sulfur amino acids on the expression of GSTP gene.

First, hepatocytes isolated from male Sprague-Dawley rats were cultured with 50-200 μM of DAS, DADS, or DATS for 24 h to explore the effect of GSTP enzyme activity, mRNA and protein level. In addition to primary hepatocytes, Clone 9 liver cells were treated with 5-200 μM of DAS, DADS, or DATS for 24 h. Besides analysising above experiments, we transfected the GSTP luciferase reporters gene into the Clone 9 cells, and further investigated the signal pathway involved in garlic allyl sulfides up-regulation of this detoxification enzyme. As results indicated, DADS and DATS induce GSTP enzyme activity, mRNA and protein expression. In contrast, DAS lacks of the effect on this phase II drug-metabolizing enzyme expression. In Clone 9 liver cells, the pTA-luciferase reporter assay showed luciferase activity in DADS- and DATS-treated cells to be 2.8- and 3.9-fold higher than that in control cells, respectively. Deletion of -2.7 to -2.6 kb in the GSTP promoter region, which contains the GSTP enhancer I (GPE I) element, abolished the up-regulation of GSTP transcription by DADS and DATS. Deletion of GPE II, however, did not affect the induction of reporter activity. It suggested that the GPE I was responsible for such up-regulation. GPE I have two 12-O-
tetradecanoylphorbol-13-acetate response elements (TREs), which are regulated by activator protein-1 (AP1). Electrophoresis mobility shift assay (EMSA) showed AP-1 binding to TRE is increased with DADS and DATS treatment, and such an enhancement is blocked in the presence of SP600125 (JNK inhibitor) and PD98059 (MEK inhibitor). Therefore, the up-regulation of DADS and DATS on GSTP gene expression is likely dependent on JNK- and ERK-mediated pathway, which activates AP-1 binding to TRE.

In amino acids study, we noted the expression of the GSTP is higher in hepatocytes incubated with a low L-methionine and L-cysteine concentrations (0.1 mM) medium than those cultured in high sulfur amino acid medium (0.5 mM L-methionine and 0.2 mM L-cysteine). Moreover, the induction of GSTP by L-methionine and L-cysteine restriction was not significantly influenced by insulin and dexamethasone. Such an up-regulation of GSTP expression was not noted on L-lysine, L-leucine, L-isoleucine or L-phenylalanine restriction. In contrast with the induction of GSTP, the expression of the GSTA and GSTM was not changed by amino acid restriction. Results suggest that the modulation on the expression of GSTP gene is sulfur amino acids-specific. To elucidate the molecular mechanisms of sulfur amino acids on GSTP gene transcription, research is currently on progress.

並列關鍵字

detoxification enzyme ； π class of glutathione S-transferase ； gene regulation ； garlic allyl sulfides ； sulfur amino acid

參考文獻

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