探討飲食中不同含硫化合物對大鼠肝細胞pi屬麩胱甘肽硫轉移酶表現及其分子機制

鄭伊評

doi:10.6834/CSMU.2009.00159

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探討飲食中不同含硫化合物對大鼠肝細胞pi屬麩胱甘肽硫轉移酶表現及其分子機制

Effect and mechanism of dietary sulfur compounds up-regulate the gene expression of the pi class of glutathione S-transferase in rat liver Clone 9 cells.

鄭伊評 , Masters　　Advisor：劉承慈;李宗貴　　

繁體中文 DOI： 10.6834/CSMU.2009.00159

含硫化合物； Clone 9肝細胞株； GSTP ； ERK/AP-1 ； PI3K/Akt ； sulfur compounds ； pi class of glutathione S-transferase (GSTP) ； activator protein-1 (AP-1) ； mitogen-activated protein kinases (MAPK) ； Clone 9 liver cells

Abstract │ Reference (141) │ Altmetrics

Abstract 〈TOP〉

許多研究皆指出含硫化合物對人類疾病有不等程度的預防作用，並具有抗腫瘤與誘發phase II生物轉換酵素的特性。過去本實驗室已發現存於蔥蒜中的有機硫化物 (organosulfur compounds, OSCs)包括diallyl disulfide (DADS)和diallyl trisulfide (DATS)可以上調大鼠Clone 9細胞pi屬麩胱甘肽硫轉移酶 (glutathione S-transferase, GSTP)基因的表現，然而其他非屬大蒜的含硫化合物是否也有相同的調控機轉則是未知的，因此本實驗即要比較其他非屬大蒜的含硫化合物硫辛酸 (α-lipoic acid, LA)、還原型硫辛酸 (dihydrolipoic acid, DHLA)和蘿蔔硫素 (sulforaphane, SFN)對於調控GSTP表現的作用機轉。本實驗以大鼠Clone 9肝細胞株為實驗模式，結果顯示，將Clone 9細胞分別培養於不同濃度LA (50、200和600 μM)、DHLA (50、200和600 μM)、SFN (0.2、1和5 μM)之培養基，皆以劑量關係誘發GSTP的蛋白質、mRNA和酵素活性表現。接著我們繼續研究LA 、DHLA和SFN調控 GSTP的分子機轉。以Western blot分析，若處理DADS (50 μM)、DATS (50 μM)、LA (600 μM)、DHLA (600 μM)和SFN(5 μM)皆可促進extracellular signal-regulated kinase (ERK)和phosphatidylinositol 3-kinase (PI3K) /Akt的磷酸化作用，但不影響 c-Jun NH2-terminal kinase (JNK)和p38蛋白質。預處理20 μM PD98059 (ERK抑制劑)亦能抑制DADS、DATS、LA、DHLA和SFN的p-ERK和GSTP的蛋白質表現，但預處理1 μM wortmannin (PI3K抑制劑)後雖然可以抑制p-Akt但GSTP並無受到抑制。另外由Electromobility gel shift assay (EMSA)結果顯示，細胞處理LA (600 μM)、DHLA (600 μM)和SFN (5 μM)會增加AP-1活化，而使用PD98059則會破壞此AP-1的結合作用。結論：不同含硫化合物DADS、DATS、LA、DHLA、和SFN上調GSTP基因表現與ERK/AP-1訊號途徑有關。

Parallel Abstract 〈TOP〉

Several sulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione 
S-transferase (GSTP) in Clone 9 liver cells, but whether the other sulfur compounds have the same ability are unknown. In the present study, we explore the modulatory of three sulfur compounds including α-lipoic acid (LA), dihydrolipoic acid (DHLA) and sulforaphane (SFN) on the gene expression of GSTP. As results indicated, LA (50, 200 and 600 μM), DHLA (50, 200 and 600 μM), and SFN (0.2, 1 and 5 μM) increased GSTP protein expression, mRNA and enzyme activity in a concentration-dependently manner. Then we further investigated the signal pathway. In the presence DADS (50 μM), DATS (50 μM), LA (600 μM), DHLA (600 μM), and SFN (5 μM) increase extracellular signal-regulated kinase (ERK) and phosphatidylinosotol 3-kinase/Akt phosphorylation in 5 min, but not c-Jun NH2-terminal kinase (JNK) and p38. Pretreatment of cells with PD98059 (ERK inhibitor) or wortmannin (PI3K inhibitor) suppressed the induction of ERK and Akt activation by sulfur compounds, respectively. And we finally determined whether the up-regulation of GSTP protein expression by sulfur compounds was mainly inhibited by PD98059, not by wortmannin. Electromobility gel shift assay (EMSA) showed that upon treatment with LA, DHLA, and SFN, the DNA binding activity of AP-1, were started to induce at 15 min. After pretreatment with the PD98059, however, the increase in AP-1 binding to DNA were abolished. In conclusion, the effectiveness of three sulfur compounds: LA, DHLA, and SFN is likely related to the ERK/AP-1 signaling pathway in Clone 9 liver cells.

Reference ( 141 ) 〈TOP〉

行政院衛生署：97年度死因統計 (2008)
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