建立乙醯胺基苯酚誘導小鼠肝損傷模式與探討大棗對於小鼠肝損傷之護肝功效

Abstract

肝癌與肝臟相關疾病為2007 年國人十大死因的主要死因之一，顯示國人肝臟疾病罹患率甚高，護肝儼然成為值得關注的議題，在健康食品開發市場上，護肝功效之健康食品佔有一席之地。目前行政院衛生署公告健康食品之護肝功能評估方法為四氯化碳 (carbon tertrachloride, CCl4) 誘導大白鼠或小白鼠慢性肝損傷之動物模式，但四氯化碳在環境中並不常見，且被列為管制藥品。乙醯胺基苯酚(acetaminophen, APAP) 為日常生活中常用的解熱、鎮痛藥物之一，人們常因為誤用或自殺而服用過量的APAP，導致急性肝毒性進而造成肝臟壞死，APAP 為目前少數可誘發肝毒性動物模式之藥物之一，有鑑於此，本實驗室欲以APAP 作為誘導肝損傷之另一選擇模式。本研究以ICR 小鼠作為誘導肝損傷之動物模式，以腹腔注射方式分別給予小鼠APAP 400、500 與600 mg/kg bw 進行急性實驗 (三 小時) ，依據急性實驗結果，再分別給予小鼠APAP 200、400 與600 mg/kg bw進行慢性實驗 (八週) 。結果發現，無論是急性實驗或慢性實驗，給予APAP 可提升ICR 小鼠血清中丙胺酸轉胺酶 (alanine aminotransferase, ALT) 與天門冬胺酸轉胺酶 (aspartate aminotransferase, AST) 活性、增加肝臟脂質過氧化、降低抗氧化物質麩胱甘肽 (glutathione, GSH) 含量與抗氧化酵素麩胱甘肽過氧化酶(glutathione peroxidase, GPx) 、麩胱甘肽還原酶 (glutathione reductase, GRd) 、超氧化物歧化酶 (superoxide dismutase, SOD) 以及過氧化氫酶 (catalase, CAT) 活性。其中，急性實驗以給予APAP 600 mg/kg bw 劑量最為顯著；而慢性實驗則以給予APAP 400 mg/kg bw 劑量最為顯著。此外，在肝臟病理切片結果發現，慢性實驗之肝損傷情形較急性實驗嚴重，且隨著APAP 給予劑量增加，其肝損傷程度亦隨之增加。本實驗室另一研究則以BALB/c 小鼠作為誘導肝損傷之動物模式，於急性實驗分別給予APAP 400、600 與800 mg/kg bw，而慢性實驗則分別給予 APAP 200、400 與600 mg/kg bw。ICR 與BALB/c 小鼠之實驗結果相較，顯示兩種品系小鼠誘導急性肝損傷之最適劑量皆為APAP 600 mg/kg bw；而誘導慢性肝損傷之最適劑量皆為APAP 400 mg/kg bw。然而，於慢性實驗進行一週後，給予不同劑量APAP 之BALB/c 小鼠生存率皆分別高於ICR 小鼠之生存率，顯示BALB/c 小鼠對於APAP 耐受程度較好，再者，央請台大醫院鄭永銘醫師評估，認為以BALB/c 小鼠APAP 400 mg/kg bw 組別之肝臟病理進展與人體最為相似，故我們選擇以BALB/c 小鼠腹腔注射APAP 400 mg/kg bw 作為誘導慢性肝損傷之動物模式，並以此模式來評估大棗對於小鼠肝損傷之保護功效。大棗 (Zizyphus jujube Mill.) 為常見被運用於促進肝臟健康或改善肝功能的中草藥之一，具有增強中焦與氣、養血、鎮定以及中和百藥的功效，且具有護肝、清除自由基、抗氧化等生理功能。本實驗結果發現，口服給予大棗樣品 (3、15 與30 g/kg bw) 可降低肝損傷 (四週) BALB/c 小鼠血清中ALT與AST活性，而高劑量大棗樣品 (30 g/kg bw) 抑制肝臟脂質過氧化情形最為顯著。此外，大棗樣品亦能顯著提升肝臟中GSH 含量，增加抗氧化酵素GPx、GRd、SOD 與CAT 活性，並且能顯著增加解毒代謝酵素麩胱甘肽硫轉移酶 (glutathione S-transferase, GST) 活性，加速GST 將N-acetyl-p-benzoquinone imine (NAPQI) 有毒物質與GSH結合為無毒代謝物排出體外；同時，大棗樣品能顯著降低細胞色素P450 2E1 (cytochrome P450 2E1,CYP2E1) 活性，以減少CYP2E1 將APAP 代謝為有毒物質NAPQI，降低肝損傷小鼠之APAP 毒性，並能在病理切片中觀察大棗樣品降低肝臟發炎與減少充血情形，對肝損傷具有改善之作用。顯示大棗樣品對於乙醯胺基苯酚誘導BALB/c 小鼠肝損傷有護肝之功效。

Liver cancer is the second cause of death in malignant neoplasms in 2007 in Taiwan. Chronic liver disease and cirrhosis are the seventh of the top ten causes of death. Therefore, hepatoprotection is very important for Taiwanese. Nowadays, carbon tetrachloride (CCl4)-induced hepatotoxicity is used to evaluate the hepatoprotective effect on Health Foods (Department of Health, Executive Yuan, Taiwan, R.O.C., 2007).However, CCl4-induced liver damage is not common to people. In the recent studies, acetaminophen (APAP)-induced hepatotoxicity was used to evaluate some foods which have anti-oxidative or hepatoprotective effect. The first aim of this study was to establish the model of APAP-induced liver damage in mice. The results indicated that the appropriate doses to induce acute and chronic liver damage in ICR mice or BALB/c mice both are APAP 600 and 400 mg/kg bw, respectively. We choose BALB/c mice to be the animal model for inducing liver damage. That’s because the survival rate of BALB/c mice is better than ICR mice. Another aim of my study is to evaluate the hepatoprotective effect of Ziziphus jujuba Mill. (ZJ) using this animal model. It is commonly used for keeping human in good health, rengthening qi (氣), nourishing the blood (養血) and harmonizing the functions of different drugs (中和百藥). The results indicated that the administration of ZJ (3, 15, 30 g/kg bw) decreased APAP-induced increases in aspartate aminotransferase (ALT), alanine aminotransferase (AST) and thiobarbituric acid-reactive substances (TBARS) values. ZJ could increase hepatic glutathione (GSH) contents, anti-oxidative enzymes glutathione peroxidase (GPx), glutathione reductase (GRd), superoxide dismutase (SOD) and catalase (CAT) activities.ZJ also could modulate the enzymes of hepatic detoxification system glutathione S-transferase (GST) and cytochrome P450 2E1 (CYP2E1). The hepatoprotective effect of ZJ was also confirmed in histopathological examination of the liver. In conclusion, the model of APAP-induced liver damage in BALB/c mice may be useful to evaluate the hepatoprotective effect on Health Foods, and ZJ may have the potential with hepatoprotective effect in this animal model.