以 Lieber-DeCarli 之動物模式探討慢性酒精毒性對於抗氧化狀態及肝臟形態變化之影響

本研究利用 Lieber-DeCarli流質飲食模式，探討不同性別於酒精性肝臟疾病方面之表現。此外，本研究也針對酒精與四氯化碳所造成之肝傷害進行探討並作系統性之比較。

首先，在第一部份，不同性別對於長期攝取酒精所造成肝損傷之影響的研究中，使用年齡相同之雄性與雌性Wistar品系大白鼠，根據 Lieber-DeCarli模式，分別餵食對照或含有酒精的液體飼料，為期 12週。將 40隻大白鼠依照血漿中肝功能指數天門冬胺酸轉胺 ?(aspartate aminotransferase, AST)與丙胺酸轉胺 ?(alanine aminotransferase, ALT)之活性分成 4組：雄性對照組 (MC)、雄性酒精組 (ME)、雌性對照組 (FC)以及雌性酒精組 (FE)，每組 10隻。餵食酒精 12週之後，結果顯示，長期餵食酒精會造成顯著的影響包括：增加相對肝重 (%)，提高血漿中AST與 ALT之活性，增加血漿中總膽固醇 (total cholesterol, TC)、高密度脂蛋白膽固醇 (high density lipoprotein cholesterol, HDL-C)、乳酸 (lactate)、游離脂肪酸 (non esterified fatty acid, NEFA)、羥基丁酸 (-hydroxy- butyrate)與腫瘤壞死因子 (tumor necrosis factor-, TNF-)之濃度，增加肝臟中三酸甘油酯 (triglyceride, TG)與 TC之含量，誘導肝臟中細胞色素 P450 2E1 (CYP2E1)蛋白質之表現，提高肝臟中黃嘌呤氧化 ?(xanthine oxidase, XO)與骨髓過氧化 ?(myeloperoxidase, MPO)之活性，增加血漿與肝臟中脂質過氧化產物即硫巴比妥酸反應物質 (thiobarbituric acid reactive substances, TBARS)之含量，減少肝臟中還原型麩胱甘 ?(glutathione, GSH)之含量並降低還原型麩胱甘 ?/氧化型麩胱甘 ?(oxidized glutathione, GSSG)之比值，降低肝臟中抗氧化酵素麩胱甘 ?過氧化 ?(glutathione peroxidase, GPX)、過氧化氫 ?(catalase, CAT)與超氧化物歧化 ?(superoxide dismutase, SOD)之活性，提高空腸中脂解 ?(lipase)之活性，降低空腸中雙醣 ?(sucrase、maltase與 lactase)之活性，而且病理學上明顯有脂肪肝形成之情形。此外，相較於雄性組，雌性組大白鼠血漿中AST活性明顯升高，血漿中 NEFA與 -hydroxybutyrate濃度顯著增加，肝臟中 TG與 TC含量明顯增加，肝臟中 TBARS含量顯著增加，肝臟中 GSH含量明顯減少、 GSH/GSSG比值顯著下降，肝臟中抗氧化酵素 GPX與 CAT之活性均顯著降低，而且脂肪肝程度較嚴重。由此可知，不論雄性或雌性，長期攝取酒精都會造成空腸中消化酵素活性明顯受到影響。另外，長期攝取酒精也會導致肝功能指標上升、脂質代謝異常、氧化壓力上升、脂質過氧化產物增加、抗氧化物質含量減少、抗氧化酵素活性降低以及病理上造成肝損傷；其中又以雌性組所造成的影響更嚴重。

在第二部份，比較酒精與四氯化碳造成肝傷害之實驗中，以雄性Wistar大白鼠 50隻作為實驗動物，依肝功能指標 AST與 ALT分成 5組：對照組 (C)、酒精組 (E)、酒精治療組 (ES)、四氯化碳組 (CCL)以及四氯化碳治療組 (CCLS)，每組 10隻，實驗期為 12週。結果顯示，長期餵食酒精或注射四氯化碳都會造成的影響包括：肝重與相對肝重 (%)明顯增加，血漿中 AST與 ALT活性於實驗期第 2、 4、 6、 8、 10、 12週皆明顯上升，血漿中 TG濃度於實驗期第 10、 12週顯著減少，肝臟中 TG與 TC含量均顯著增加，肝臟中 CYP2E1蛋白質表現量明顯受到誘導，肝臟中MPO活性顯著上升，血漿中 TBARS濃度於實驗期第 2、 4、 6、 8、 10、 12週皆明顯增加，肝臟中 TBARS含量顯著增加，肝臟中 GSH含量顯著減少、 GSH/GSSG比值明顯下降，肝臟中抗氧化酵素 GPX與 SOD活性皆顯著降低，而且病理學上明顯有脂肪肝與脂肪變性形成之情形。此外，於實驗結束後，相較於酒精組，四氯化碳組大白鼠肝重與相對肝重 (%)均明顯增加，血漿中 AST與 ALT之活性均明顯上升，血漿中 TG、 TC與 HDL-C濃度顯著減少，肝臟中 TG與 TC之含量均顯著增加，肝臟中 XO與MPO活性均顯著升高，血漿中 TBARS濃度明顯增加，肝臟中 GSH含量顯著減少、 GSSG含量顯著增加、 GSH/GSSG比值明顯下降，肝臟中抗氧化酵素麩胱甘 ?還原 ?(glutathione reductase, GRD)與 CAT活性皆顯著降低，而且脂肪肝與脂肪變性之情形更嚴重。由此可知，酒精與四氯化碳都會造成肝功能指標上升、脂質代謝異常、氧化壓力上升、脂質過氧化產物增加、抗氧化物質含量減少、抗氧化酵素活性降低以及病理上造成肝損傷；其中又以四氯化碳所造成的影響更嚴重。

Effects of Chronic Alcoholic Toxicity on Antioxidative Status and Hepatic Morphologic Changes by Lieber-DeCarli Animal Model

This dissertation had two major parts; one was to evaluate the correlation between gender differences and chronic alcoholic liver disease. In addition, effects of gender differences on jejunal lipase and disaccharidase activities, the liver function tests, metabolic disorders and oxidative damage were also examined. Another major part was to examine oxidative stress of alcoholic injury and carbon tetrachloride- induced damage in relation to risk factors for liver disease.

The first part of this study was to investigate the effects of gender differences on alcoholic liver disease in rats with chronic ethanol consumption. Age-matched male and female Wistar rats were fed control or ethanol-containing liquid diets for 12 weeks following the Lieber-DeCarli model. According to both the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, 40 rats were divided into four groups as follows: male control group (MC), male ethanol group (ME), female control group (FC), and female ethanol group (FE). After ethanol feeding for 12 weeks, the findings indicated significant main effects of ethanol consumption on increased relative liver weight (%); elevated plasma AST and ALT activities; raised plasma total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), lactate, non-esterfied fatty acid (NEFA), -hydroxybutyrate, and tumor necrosis factor-alpha (TNF-) concentrations; increased hepatic triglyceride (TG) and TC contents; induction of hepatic microsomal cytochrome P450 2E1 (CYP2E1); elevated hepatic xanthine oxidase (XO) and myeloperoxidase (MPO) activities; increased both plasma and hepatic thiobarbituric acid reactive substances (TBARS) levels; reduced hepatic glutathione (GSH) content and the ratio of GSH/oxidized GSH (GSSG); decreased hepatic antioxidant enzymes, glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) activities; elevated jejunal lipase activity; lowered all jejunal disaccharidase, sucrase, maltase and lactase, activities; and the formation of fatty liver; when compared to control group. Furthermore, the results also showed significant main effects of gender differences on elevated plasma AST activity; increased both plasma NEFA and -hydroxybutyrate concentrations; augmented hepatic TG and TC contents; raised hepatic TBARS level; reduced hepatic GSH content and the ratio of GSH/GSSG; decreased hepatic antioxidant enzymes, GPX and CAT activities; and degree of fatty liver; when compared to male group. In conclusion, our results suggest that long-term ethanol consumption significantly increased jejunal lipase and decreased jejunal disaccharidase (sucrase, maltase, and lactase) activities in both male and female rats. Our results also show that chronic ethanol administration induced a greater susceptibility to liver damage in female rats than in male rats.

In the second part, we examined the effects of ethanol- or carbon tetrachloride-induced liver injury in male Wistar rats following the Lieber-DeCarli liquid diet. According to both the plasma AST and ALT activities, 50 rats were assigned to five groups: C (control feeding), E (ethanol feeding), ES (ethanol feeding combined with the supplementation of silymarin, 200 mg/kg BW/day), CCL (CCl4 injection and control feeding) and CCLS (CCl4 injection and control feeding combined with the supplementation of silymarin, 200 mg/kg BW/day). Rats in groups CCL and CCLS were intraperitoneally injected with 0.75 mL/kg BW of 40% CCl4 dissolved in olive oil once a week, while rats in groups C, E and ES were intraperitoneally injected with 0.75 mL/kg BW of olive oil only. Our dada indicated significant main effects of both ethanol feeding and carbon tetrachloride injection on increased relative liver weight (%); elevated plasma AST and ALT activities at weeks 2, 4, 6, 8, 10 and 12; lowered plasma TG concentrations at weeks 10 and 12; increased hepatic TG and TC contents; induction of hepatic microsomal CYP2E1; elevated hepatic MPO activity; increased plasma TBARS concentrations at weeks 2, 4, 6, 8, 10 and 12; increased hepatic TBARS level; reduced hepatic GSH content and the ratio of GSH/GSSG; decreased hepatic antioxidant enzymes, GPX and SOD, activities; and pathologically changed liver; when compared to control feeding. Moreover, after 12 weeks, the results also showed significant main effects of carbon tetrachloride injection on increased relative liver weight (%); elevated plasma AST and ALT activities; reduced all the plasma TG, TC and HDL-C concentrations; augmented hepatic TG and TC contents; elevated hepatic XO and MPO activities; increased plasma TBARS concentration; reduced hepatic GSH content and the ratio of GSH/GSSG, whereas increased GSSG level; decreased hepatic antioxidant enzymes, GRD and CAT activities; and severe fatty change in livers; when compared to ethanol feeding. In conclusion, our results suggest that both long-term ethanol feeding and carbon tetrachloride injection significantly increased oxidative stress, lipid peroxidation, and decreases in GSH concentrations and the ratio of GSH/GSSG in rats. Our results also show that carbon tetrachloride injection induced a greater susceptibility to liver damage than ethanol feeding.

Key words: ethanol, gender differences, carbon tetrachloride, lipase, disaccharidase, oxidative stress, lipid peroxidation, CYP2E1, xanthine oxidase, myeloperoxidase, fatty liver, fatty degeneration