建立乙醯胺基苯酚誘導小鼠肝損傷模式並以此模式探討大蒜精油護肝功效

Abstract

根據行政院衛生署九十六年國人十大死因統計資料指出，慢性肝疾病與肝硬化位居十大死因第七位，因此護肝相關議題應受台灣人所關注。四氯化碳誘導大、小鼠肝損傷模式為目前行政院衛生署公告之護肝功效健康食品評估辦法，由於四氯化碳已被列為管制藥品，故本研究目的一為建立乙醯胺基苯酚(acetaminophen; APAP)誘導BALB/c小鼠肝損傷模式。研究目的二為應用此動物模式評估大蒜精油(garlic oil; GO)的護肝功效。在誘導小鼠肝損傷實驗設計上分為急性與慢性試驗，急性期部份，將小鼠分為四組，分別為GAC (正常對照組)、GA1 (APAP 400 mg/kg bw)、GA2 (APAP 600 mg/kg bw)與GA3 (APAP 800 mg/kg bw)。小鼠以腹腔注射不同劑量APAP後三小時進行犧牲，採集血液與肝臟樣本分析肝損傷相關生化指標，包含ALT (alanine aminotransferase)、AST (aspartate aminotransferase)、GSH (glutathione)、GPx (glutathione peroxidase)、GRd (glutathione reductase)、CAT (catalase)、SOD (superoxide dismutase)、TBARS (thiobarbituric acid reactive substances)與肝臟病理組織切片。結果顯示，GA1、GA2與GA3組的肝臟GSH含量及抗氧化酵素GPx與CAT之活性顯著低(p<0.05)於GAC組。GA1、GA2與GA3組之血清ALT與AST含量顯著高(p<0.05)於GAC組，但由於GA3組造成過度嚴重肝損傷，使得血清ALT與AST含量低於GA2組。GA1、GA2與GA3組的小鼠肝臟病理組織切片皆呈現肝損傷現象，損傷程度以GA1組最為輕微而GA3組最為嚴重。綜合上述結果評估出APAP最適合誘導急性期BALB/c小鼠肝損傷的劑量為600 mg/kg bw。慢性期部份，將小鼠分為四組，分別為GCC (正常對照組)、GC1 (APAP 200 mg/kg bw)、GC2 (APAP 400 mg/kg bw)與GC3 (APAP 600 mg/kg bw)。各組小鼠每週以腹腔注射兩次不同劑量APAP並持續八週，每週進行生存率之評估，於第八週結束後進行犧牲，採集血液與肝臟樣本分析肝損傷相關生化指標。結果顯示，GC1、GC2與GC3組的肝臟GSH含量及抗氧化酵素GRd、SOD與CAT之活性顯著低(p<0.05)於GCC組。GC1、GC2與GC3組之血清ALT與AST含量顯著高(p<0.05)於GCC組，且ALT與AST含量隨著給予的APAP劑量增加而增加。GC2與GC3組的小鼠肝臟病理組織切片皆呈現嚴重肝損傷現象，但GC3組在八週期間的小鼠生存率顯著低於GCC組，表示小鼠對於APAP 600 mg/kg bw之慢性誘導下的耐受力較低。綜合上述結果評估出APAP最適合誘導慢性期BALB/c小鼠肝損傷的劑量為400 mg/kg bw。在大蒜精油護肝實驗設計上，將小鼠分為六祖，分別為G1 (正常對照組)、G2 (APAP負對照組)、G3 ﹝N-acetyl-L-cysteine (NAC)正對照組﹞、G4 (GO 2.5 mg/kg bw)、G5 (GO 25 mg/kg bw/day)與G6﹝dially disulfide (DADS) 11 mg/kg bw/day﹞。小鼠在處理APAP前一週開始每天管餵NAC、GO與DADS，維持一週後開始每週以腹腔注射兩次APAP (400 mg/kg bw)並持續四週，四週結束後將小鼠進行犧牲，採集血液與肝臟樣本分析肝損傷相關生化指標與肝臟解毒酵素glutathione S-transferase (GST)與cytochrome P450 2E1 (CYP2E1)之活性。實驗結果顯示，GO (2.5與25 mg/kg bw)處理的組別相較於APAP負對照組具有降低肝功能指標酵素ALT、AST含量與增加肝臟GSH含量及抗氧化酵素GRd、GPx與SOD之活性，另外亦具有增加肝臟解毒酵素GST以及降低CYP2E1之活性。DADS處理的組別相較於APAP負對照組具有降低ALT、AST活性與增加肝臟GSH含量及抗氧化酵素GRd、GPx、SOD與CAT之活性，並且增加肝臟解毒酵素GST以及降低CYP2E1之活性。而由肝臟病理組織切片證實GO與DADS可以保護肝細胞免受到APAP的氧化傷害。综合以上結果，GO及其所含揮發性純物質DADS可藉由提高抗氧化系統與調節肝臟解毒酵素而具有保護因APAP誘導之肝損傷的效果，綜上結果推斷大蒜精油具有發展成為護肝功效健康食品的潛力。

Chronic liver disease and cirrhosis is the seventh leading causes of death in Taiwan in 2007. This indicates hepatoprotection is very important for Taiwanese. Nowadays, the model of carbon tetrachloride (CCl4)-induced hepatotoxicity is an official methodology used to evaluating the hepatoprotective effect of Health Foods in Taiwan. However, CCl4-induced hepatotoxicity is not common to people and CCl4 is recognized as a controlled-drug in Taiwan. Finding another chemical to replace CCl4 is necessary. Acetaminophen (APAP) is a clinically used analgesic and antipyretic drug, but overdose of APAP can cause liver injury in experimental animals and human. The first aim of this study was to establish the applicability of APAP-induced hepatotoxicity model in BALB/c mice for evaluating the hepatoprotective effect of Health Foods. Garlic (Allium sativum), a bulbous root with a strong flavor, is used widely in culinary preparations and as a folk medicine. It is rich in organosulfur compounds with biological activities, such as hepatoprotective, detoxifying and antioxidative activities. Previous studies showed that steam-distilled garlic oil (GO) gave hepatoprotective activity, but the dosage used was much higher than human’s consumption in daily life. Therefore, the second aim of this study was to evaluate the protective effects of lower dosage of GO on APAP-induced hepatotoxicity in BALB/c mice and to check the practicability of this model. The results indicated that the adequate doses of APAP to induce acute and chronic liver injury in BALB/c mice are 600 and 400 mg/kg bw, respectively. The administration of GO at the dose of 2.5 and 25 mg/kg bw could decrease ALT (alanine aminotransferase) and AST (aspartate aminotransferase) values in mice. GO also could modulate enzymes (glutathione S-transferase and cytochrome P450 2E1) of hepatic detoxification system, as well as increase hepatic glutathione contents and antioxidative enzymes (glutathione reductase, glutathione peroxidase and superoxide dismutase) activities. The hepatoprotective effect of GO was also confirmed in histopathological examination of the liver. In conclusion, the model of APAP-induced hepatotoxicity may be useful to evaluate the hepatoprotective effect of Health Foods. GO is a potential candidate as Health Food with liver protective function.