| 摘要: | 由於病毒感染、飲酒與吸煙的不良習性、藥物濫用、美食主義及環境污染加劇等情形，已大幅增加國人罹患肝病的機會，長期的肝功能失調更是未來發展形成肝癌的禍首，目前國人罹患肝癌的比率已高居癌症的第一位，這個事實也凸顯了國人平日肝臟機能保護的重要性，以及研發具明顯保肝活性食品的迫切性，然而目前對具有保肝活性食品的研究並不多。食用山藥最近在本國農政單位大力的推廣栽種下，產量豐富，目前許多的科學性研究也指出：山藥確實有降低血糖及提昇免疫力、抗疲勞、抗骨質疏鬆、抗氧化等多方面的保健活性，最近的報告更指出台農二號山藥水萃物具有保護乙醯氨基苯酚(acetaminophen)在大鼠肝臟及腎臟所造成的細胞毒性，但對其保肝活性的分子作用機制則仍不清楚。本研究室最近的實驗結果發現：台農山藥二號山藥(Dioscorea alata L.)水萃物具強烈的銅離子鍵結活性，並可明顯抑制芬頓氏(銅離子和過氧化氫)在牛胸腺DNA及人類淋巴母細胞核DNA所造成的傷害，這項活性與山藥保肝活性可能有關，因為典型肝臟毒素所造成肝細胞毒性幾乎都與脂質過氧化(lipidperoxidation)有關，而脂質過氧化的過程中過渡金屬(如銅或鐵離子)扮演重要的前進(processing)角色，因此確認臺農二號山藥水萃物的保肝活性及可能的分子作用機制是本計化的主要目的。本計畫利用大鼠初代肝細胞及人類肝癌細胞(HepG2)探討省產台農二號山藥水萃物保護肝細胞之活性及其可能的分子機制。本計畫利用：(1) ehthidium bromidebinding assay及慧星分析(comet assay)測量山藥水萃物對氧化性核酸傷害可能之影響，(2) 膠體酵素活性染色法(in gel enzyme activity assay)測量山藥水萃物對大鼠初代肝細胞及人類肝癌細胞中catalase, superoxide dismutase活性及同功酵素圖譜的影響，(3) 以細胞生長抑制方法(growth inhibition assay)測量山藥水萃物對acetaminophen處理造成人類肝癌細胞毒殺可能之影響，(4) 以RT-PCR方法測量淋巴母細胞中多種抗氧化酵素包括catalase、glutathione reductase、glutathione peroxidase 1, phospholipids peroxidase, CuZn superoxide dismutase及Mn superoxide dismutase)基因表達的影響等三種方式來評估山藥的抗氧化活性及可能的保肝活性機轉。結果發現：(1) 山藥水萃物具明顯抗氧化活性可有效抑制芬頓氏反應所造成之牛胸腺DNA傷害；對二價銅離子在人類淋巴母細胞造成的核DNA傷害也有很強的抑制作用，(2) 山藥水萃物處理人類肝癌細胞HepG2及大鼠初代肝細胞明顯增加Cu-Zn superoxide dismutase酵素活性，但卻略微降低catalase酵素的活性，(3) 山藥水萃物處理人類肝癌細胞HepG2明顯增加細胞內Cu-Zn superoxide dismutase的mRNA表達及catalase mRNA表現少量增加，然而對glutathione peroxidase、phospholipid glutathione peroxidase及glutathione reduictase mRNA表現則無明顯影響，(4) 山藥水萃物與acetaminophen共同處理無法抑制其在HepG2細胞所造成之細胞毒殺性，在高濃度下0.5%、1%甚至會加劇acetaminophen所造成之細胞毒殺性。本研究首次確認山藥水萃物可調節大鼠初代肝細胞及人類肝癌細胞HepG2中Cu-Zn superoxide dismutase及catalase的酵素活性及mRNA表達，然而此調節功能在保護肝臟功能之角色，仍須藉由其他不同肝毒素作進一步之探討。 關鍵詞：台農二號山藥，大鼠初代肝細胞，人類肝癌細胞，抗氧化酵素，乙醯氨基苯酚
Dioscorea plants have been widely used as traditional medicine and food for health benefits. Several therapeutic properties such as anticough, antidiabetic, antidiarrhea, and anticancer have been attributed to these plants. Although the steroidal saponins (such as diosgenin) account for some of Dioscorea？H？Hs activity, there is little information on the effective components. Previously, We demonstrated that aqueous extract of Dioscorea alata L. (YAE) inhibited the H2O2-CuSO4 induced damage of calf thymus DNA, and protected cultured human lymphoblastoid cells from CuSO4-induced DNA damage. A recent report also shows that aqueous extract of yam can protect acetaminophen induced liver injury in rat; however the molecular mechanism is still unknown. The present study, we examine the protective effect of aqueous extract of yam (D. alata L.) on acetaminophen induced hepatoxicity and the possible mechanism(s) underlying this effect in rat primary liver cells and human HepG2 cells. Here, we determine the modulation effect of the aqueous extract on antioxidant enzymes genes (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) genes expression by in gel activity assay and RT-PCR, respectively. The supplementation of aqueous extract of yam led to an significant increase the enzyme activity and mRNA expression in rat primary liver cells and human HepG2 cells, whereas it had no effect on the glutathione peroxidase, glutathione reductase expressions. When aqueous extract of yam was administered to HepG2 cells, followed by hepatoxic dose of AAP (10 mM ) for 48 hours did not reduce acetaminophen-induced killing effect. These results suggest that aqueous extract of yam could modulate the antioxidant enzymes which are important for maintaining cell viability by lowering the level of oxygen radical generated from intracellular metabolism. Keywords: yam (dioscorea alata L.), rat primary liver cells, human HepG2, antioxidant enzymes, acetaminophen |
