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標題: Molecular mechanism of metallothionein expression regulated by flavonoids and its cytoprotective effect
作者: Chen, Mei-Jung
關鍵字: 人類肝癌細胞;metallothionein;金屬硫蛋白;類黃酮化合物;抗氧化性;Nrf2;MAPK;PI3K;一氧化氮;t-BHP;flavonoid;antioxidant activity;liver cell;nuclear factor E2-related factor;mitogen-activated protein kinase;phosphoinositide 3-kinase;nitric oxide;tert-butyl hydroperoxide
出版社: 食品暨應用生物科技學系
Flavonoids are the members of naturally existing polyphenolic compounds that widely distribute throughout the plant kingdom. It appears to regulate the expression of many genes and and have several pharmacological effects to show multifunctional benefits for human being. Metallothionein (MT), an antioxidant protein, is present in high rates in the liver, which plays an important role in protection against the toxic effect of metals and oxidative stress. However, the signaling mechanisms involved in the activatation of metallothionein by flavonoids are poorly defined. In this study, human hepatoma HepG2 cells were used as a model to investigate the relationship between the antioxidant capacity of eight flavonoids and their effects on the expression of metallothionein. Furthermore, to analyse the signaling pathways involved in quercetin induced metallothionein expression and its cytoprotective effect. The effect of flavonoids on metallothionein expression was evaluated by RT-PCR. The results showed that quercetin, morin, rutin and naringenin significantly (p < 0.05) increased MT-1 and -2 mRNA expression in HepG2 cells. The antioxidant capacity of flavonoids was determined by Trolox Equivalent Antioxidant Capacity (TEAC) assay and Oxygen Radical Absorbance Capacity (ORAC) assay. The data showed that quercetin, morin and rutin represented better antioxidant ability among all flavonoids tested. The correlation coefficient between the influence of flavonoids on the induction of MT-1 mRNA expression and their antioxidant capacity of TEAC and ORAC value were equal to 0.7262 (p < 0.05) and 0.8991 (p < 0.01), respectively, and for MT-2 mRNA were 0.7749 (p < 0.01) and 0.6880 (p < 0.05), respectively.
In the study of the mechanism leading to MT regulation, the effect of quercetin on the expression of MT in HepG2 cells was analyzed. The signaling pathway was determined by western blot. The results showed that quercetin induced MT expression in a time- and dose-dependent manner. Furthermore, quercetin increased the nuclear levels of Nrf2, a transcription factor governing antioxidant response element (ARE). Electrophoretic mobility shift assay indicated that quercetin promoted the binding of Nrf2 to ARE and induced Nrf2-dependent activation of mt promoter.
When investigating the signaling pathways responsible for MT induction in HepG2 cells, we observed that quercetin activated the MAPK and PI3K pathways. Preincubation with JNK specific inhibitor (SP600125), p38 MAPK specific inhibitor (SB203580) and PI3K specific inhibitor (LY294002) abolished quercetin-induced MT expression in HepG2 cells, whereas PD98059, a specific inhibitor of ERK MAPK, showed no significant effect. In order to trace the upstream signaling inducer, the active molecule, nitric oxide (NO), was examined. The data showed that quercetin could increase NO content in a time-dependent manner. These results suggest that the activation of MAPK and PI3K pathways was stimulated by NO. The cytoprotective effect of quercetin induced-metallothionein expression against tert-butyl hydroperoxide (t-BHP) induced toxicity in Clone 9 liver cells was further evaluated. The addition of t-BHP to culture medium resulted in enhancing cytotoxicity as detected by MTT assay and LDH leakage. The results showed that pretreatment of quercetin with Clone 9 liver cells reduced the cytotoxicity and LDH leakage mediated by t-BHP. In addition, quercetin recruited t-BHP-induced exhausting of MT was blocked with the pre-treatment of EDTA. In conclusion, the results indicate that quercetin induces the MT expression through increasing the NO level and stimulating JNK/p38 MAPK, PI3K and Nrf2-ARE pathways, and further contributes to the cytoprotective effect against oxidative stress.

類黃酮化合物屬於植物多酚類,其廣泛存在於自然界植物中,可調控多種基因的表現,且擁有許多良好之生理活性,對人類健康具有保護功效。金屬硫蛋白 (metallothionein, MT) 是一種抗氧化酵素,肝臟中表現量最高,其在防止重金屬與氧化壓力傷害下扮演一重要角色。然而目前類黃酮誘導金屬硫蛋白表現之分子機制並未完全明瞭,因此本研究乃選用廣泛存在植物中之八種天然類黃酮化合物,探討其抗氧化力對人類肝癌細胞 (HepG2) 中金屬硫蛋白影響之關聯性,並進行金屬硫蛋白表現之分子機制調控,及對大鼠正常肝細胞Clone 9之細胞保護效應研究。由RT-PCR結果顯示,quercetin、morin、rutin及naringenin等類黃酮化合物可顯著性 (p < 0.05) 提升HepG2細胞中MT-1及MT-2 mRNA表現,在抗氧化力分析上,由TEAC及ORAC結果得知八種類黃酮均具有不錯之抗氧化力,其中以quercetin、morin及rutin抗氧化力最佳,由線性圖分析類黃酮抗氧化力及金屬硫蛋白基因表現之相關性,類黃酮化合物對MT-1 mRNA基因表現與其抗氧化力之相關性分別為 0.7262 (p < 0.05) 及0.8991 (p < 0.01),對於MT-2 mRNA基因表現與其抗氧化力之相關性分別為0.7749 (p < 0.01) 及0.6880 (p < 0.05)。以quercetin 進行調控金屬硫蛋白表現之分子機制探討,由西方墨點法結果發現,quercetin可誘導HepG2細胞中金屬硫蛋白之蛋白質表現,並呈現劑量及時間依存性。在訊息路徑探討方面,quercetin可活化細胞質中Nrf2轉位進入細胞核以增加ARE序列之活性,並促進 MAPK及PI3K等訊息傳遞激酉每之活化,以JNK的抑制劑 (SP600125) 及p38 MAPK的抑制劑 (SB203580) 及PI3K的抑制劑 (LY294002) 處理,可阻斷quercetin對金屬硫蛋白及轉錄因子Nrf2之蛋白質的誘發效果,然而以ERK的抑制劑 (PD98059) 處理卻無顯著效果。以亞硝酸鹽試驗探討誘發這些路徑活化的上游因子,得知quercetin可誘發HepG2細胞中一氧化氮之生成,推測quercetin是透過促進一氧化氮的表現來活化MAPK及PI3K等訊息傳遞激酉每。進行誘發金屬硫蛋白表現對大鼠正常肝細胞Clone 9的保護效應。在t-BHP處理下會誘發細胞之氧化損傷,造成細胞存活率下降及乳酸脫氫酉每滲漏。而以quercetin預處理之Clone 9細胞在t-BHP傷害模式下,具有提升細胞存活率及降低乳酸脫氫酉每滲漏之細胞保護效果。而細胞預培養以 EDTA 會抑制 quercetin 誘導 MT 表現的效果,並且顯著地抑制了 quercetin 之細胞保護效應 (p < 0.05)。綜合上述結果得知quercetin透過刺激一氧化氮產生以活化HepG2細胞中JNK、p38 MAPK及PI3K及轉錄因子Nrf2,以誘發金屬硫蛋白之表現,並對Clone 9細胞產生抵抗氧化傷害之保護效應。
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