建立乙醯胺基苯酚誘導BALB/c小鼠肝損傷動物模式及探討較佳正對照組之藥品

Abstract

慢性肝病及肝硬化在台灣一直是很嚴重的問題，在衛生署2009年統計資料中，肝癌為2008年國人癌症十大死亡率的第二名，其他相關肝臟疾病也為十大死因之一，因此保護肝臟及抑制肝癌的議題就特別受到社會大眾的重視。近年來護肝保健食品發展快速，故建立一套標準的護肝功效之保健食品評估方法就特別重要。衛生署頒訂「以四氯化碳誘發大(小)白鼠慢性肝損傷實驗模式」為目前健康食品護肝功效作用之動物模式評估方法，然而四氯化碳為管制化學物質，一般生活中也不易接觸。乙醯胺基苯酚 (acetaminophen, APAP) 為一般常見解熱鎮痛劑的主要成分，當食用過量易產生肝腎損傷，因此本實驗欲建立APAP誘導肝損傷之護肝效果評估之動物模式，將有助於健康食品之開發。本實驗以BALB/c小鼠作為實驗品系，探討不同性別對APAP誘導肝損傷之影響，並評估具有抗氧化能力的半胱胺酸醋酸酯 (N-acetylcysteine, NAC) 及乳薊 (Silybum marianum) 萃取物–水飛薊素 (Silymarin) 作為正對照組之可行性。Silymarin在過去臨床上常用來作為抑制肝損傷之藥物，在四氯化碳誘發大鼠或小鼠慢性肝損傷實驗模式中更被視為正對照組的使用藥物，而NAC在臨床上則常被用為APAP急性中毒的解毒藥物。在急性實驗中 (給予一次APAP誘導) 發現，母鼠對於APAP的誘導所造成血液中AST及ALT值較公鼠高出20倍，由此可知母鼠對於APAP耐受性不如公鼠，因此後續均使用公鼠來進行實驗。在APAP誘導急性肝損傷模式中，BALB/c公鼠先給予NAC或Silymarine 連續七日，再以600 mg APAP/kg bw進行急性肝損傷誘導，藉此觀察NAC及Silymarin對抑制急性肝損傷的效果。病理組織切片觀察及血液生化值 (AST、ALT) 分析結果顯示，相較負對照組APAP誘導，NAC處理組 (600、1200 mg/kg bw) 明顯改善APAP誘導所造成之肝損傷，但是Silymarin (50~280 mg/kg bw) 處理組的保護效果皆不及NAC，因此後續為期八週之慢性實驗便不再使用Silymarin進行評估。由NAC處理組之慢性實驗結果發現，血液生化指標 (ALT及AST)、肝臟脂質過氧化、病理組織切片、肝臟抗氧化物質 (GSH、GST) 及抗氧化酵素結果顯示，與僅以APAP誘導肝損傷的負對照組相比較下，給予NAC (600、1200 mg /kg bw) 之處理組可明顯改善因APAP誘導之肝損傷現象，但因兩組NAC處理組間則無顯著差異，因此600 mg NAC/kg bw可作為APAP誘導肝損傷試驗之較佳正對照組劑量。綜合上述實驗結果，以BALB/c公小鼠進行APAP誘導慢性肝損傷，並以600 mg NAC/kg bw為正對照組，以進行保健食品之護肝功效評估，應可作為評估健康食品之護肝保健功效之另一動物模式的選擇。

According to the report of Department of Health of Taiwan in 2009, chronic liver disease and cirrhosis are the eighth and the tenth leading causs of death, respectively, and liver cancer is the second leading cancer-caused deaths. Therefore, how to mentain liver health is a very important topic in Taiwan. Nowadays, the model of carbon tetrachloride (CCl4)-induced hepatotoxicity is used to evaluate the hepatoprotective effect of on health food. However, CCl4 is recognized as a damge materia and controlled seriously by the government in Taiwan. Acetaminophen (APAP) is a clinically analgesic and antipyretic drug. Previous researches demoustrated that APAP causes liver injury in experimental animals and human being. The aim of this study was to establish an appropriate model of APAP-induced liver injury, and to evaluate the liver protective effects of N-acetylcysteine (NAC) and Silymarin in BALB/c mice. In the acute experiment, the results showed that APAP increased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (>15000 IU/L) in female mice much more than those (500-1000 IU/L) in male mice after APAP intraperitoneal injection. The death rate in female mice (83%) is higher than that (17%) in male ones. It suggested that female mice were not suitable to be used for the experiments. Additionally, male BALB/c mice with APAP- induced liver injury took NAC and Silymarin once daily for 7 days. The results showed that NAC at 600 and 1200 mg/kg bw decreased ALT and AST values in BALB/c mice with APAP-induced hepatotoxicity. The hepatoprotective effect of NAC was also confirmed using histopathological examination of the liver. The date showed that hepatoprotective effect of NAC was better than that of Silymarin. Furthermore, male BALB/c mice were given with NAC once daily on APAP- induced the chronic experiment. It suggested that NAC at 600 and 1200 mg/kg bw effectively improved the APAP-induced liver injury in BALB/c mice. Therefore, these results showed that NAC (600 mg/kg bw) is better to be positive control in both acute and chronic APAP-induced liver injury in BALB/c mice for evaluation of the heptoprotective effect of health food.