- 學位論文
不同劑量β-胡蘿蔔素對於大白鼠酒精性肝臟疾病之保護效果及作用機轉之探討
Study on the protective effects and mechanisms of the different β-carotene dosage in rats with alcoholic liver disease
摘要
本研究利用體內及體外試驗，探討β-胡蘿蔔素是否可利用其抗氧化特性，降低酒精之氧化性傷害，並進一步釐清β-胡蘿蔔素預防大白鼠酒精性肝臟疾病的機制。體外試驗部份，利用大白鼠初代肝細胞，以無添加或有添加酒精 (100 mM) 以及β-胡蘿蔔素 (1 μM與5 μM) 的培養基進行培養24及48小時後，評估肝細胞的抗氧化狀態的改變以及細胞凋亡的現象。結果發現，肝細胞以含酒精之培養基培養，肝細胞存活率顯著降低，GSH/GSSG比值顯著降低及脂質過氧化物 (thiobarbituric acid reactive substance, TBARS) 的堆積，並可觀察到CYP2E1、caspase-3與caspase-9的表現量顯著增加，顯示酒精造成肝細胞中氧化壓力增加，進一步使細胞凋亡現象增加。添加1 μ M β-胡蘿蔔素並進行48小時培養，則抑制因酒精所造成的GSH/GSSG比值降低、脂質過氧化物的堆積與CYP2E1的表現，以減少其氧化壓力，而且藉由降低caspase-9的表現量而減少肝細胞進行細胞凋亡，進而降低肝細胞的死亡，顯示β-胡蘿蔔素在此實驗模式中具有抗氧化及抗細胞凋亡之保護作用。體內試驗部分，將Wistar大白鼠依照aspartate aminotransferase (AST) 及alanine aminotransferase (ALT) 分為攝取正常液體飼料組 (C)、補充低劑量β-胡蘿蔔素組 (CLB)、補充高劑量β-胡蘿蔔素組 (CHB)、佔總熱量36%之酒精液體飼料組 (E)、酒精液體飼料並補充低劑量β-胡蘿蔔素組 (ELB) 及酒精液體飼料並補充高劑量β-胡蘿蔔素組 (EHB) 共6組，每組10隻。低劑量β-胡蘿蔔素之補充為0.52 mg/kg BW/day，高劑量β-胡蘿蔔素之補充為2.6 mg/kg BW/day，飼養期共12週，並分析體內抗氧化狀態及肝臟細胞凋亡現象。由結果可知，大白鼠攝取酒精導致血漿中AST與ALT增加，紅血球GSH/GSSG 比值降低。另外，肝臟中TNF-α濃度顯著增加，CYP2E1蛋白質表現量及脂質過氧化物TBARS濃度顯著增加，表示大白鼠體內氧化壓力提高。另外，肝臟中Fas ligand、caspase-8、cytochrome c、caspase-9與caspase-3之表現量顯著增加，顯示酒精引起肝臟中細胞凋亡產生。補充低劑量β-胡蘿蔔素後，可降低攝取酒精之大白鼠血漿中AST與ALT，並可降低CYP2E1表現量及TBARS濃度，亦可增加Bcl-xL蛋白質的表現量，降低caspase-9及caspase-3蛋白質表現量。而補充高劑量之β-胡蘿蔔素可降低攝取酒精之大白鼠血漿中AST並降低肝臟中TNF-α及TABRS濃度，可增加Bcl-2蛋白質表現量，降低cytochrome c蛋白質表現量。由此推測，大白鼠長期攝取酒精會降低其體內抗氧化狀態，而補充低劑量β-胡蘿蔔素可降低酒精造成之氧化傷害，並進一步降低肝臟細胞凋亡發生。綜合以上結果可知，酒精會引起氧化壓力增加，進一步誘發細胞凋亡發生，對肝細胞或肝臟造成傷害。而β-胡蘿蔔素則可能因具有抗氧化特性，藉由降低氧化壓力而減少細胞凋亡產生，進而保護肝細胞或肝臟以預防酒精帶來的傷害，但是β-胡蘿蔔素的投與量與其抗氧化效果並沒有劑量效應。
並列摘要
The purpose of this study is to realize whether β-carotene could present the antioxidant property and to clarify the mechanism of β-carotene on the prevention of alcoholic liver diseases in rats. In the in vitro study, the isolated rat hepatocytes were incubated for 24 and 48 hr in a medium with or without alcohol (100 mM) and β-carotene (1 μM and 5 μM). The ethanol-treated hepatocytes showed lower cell viability, reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and lipid peroxide (thiobarbituric acid reactive substance, TBARS) accumulation in rat hepatocytes; meanwhile, CYP2E1, caspase-3, and caspase-9 expressions increased. In contrast, GSH/GSSG ratio increased while TBARS concentration and expressions of CYP2E1, and caspase-9 decreased in the hepatocytes with medium containing ethanol and 1μM β-carotene after incubation for 48 hr. The results suggest that ethanol treatment decreases cell viability in rat hepatocytes via induced oxidative stress. 1μM β-carotene decreased oxidative stress and prevented ethanol-induced cell apoptosis. In the in vivo study, according to the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT), rats were divided into 6 groups: group C (control liquid diet), group CLB (control liquid diet with β-carotene supplementation, 0.52 mg/kg BW/day), group CHB (control liquid diet with β-carotene supplementation, 2.6 mg/kg BW/day), group E (ethanol liquid diet), group ELB (ethanol liquid diet with β-carotene supplementation, 0.52 mg/kg BW/day), and group EHB (ethanol liquid diet with β-carotene supplementation, 2.6 mg/kg BW/day) (n=10/group). The antioxidative status and hepatic apopotosis were analyzed. After 12 weeks, plasma AST and ALT, TBARS concentration, hepatic CYP2E1 expression were increased and erythrocytic GSH/GSSG ratio was decreased in the E geoup. Hepatic Fas ligand, caspase-8, cytochrome c, caspase-9, and caspase-3 expressions had significantly increased in the E group. The results suggested that ethanol-induced oxidative stress and hepatic apoptosis in ethanol-feeding rats. However, plasma AST and ALT, TBARS concentration and CYP2E1, caspase-9 and caspase-3 expression were significantly lower and Bcl-xL expression was higher in the ELB group than those in the E group. And the AST activity, hepatic TNF-α level, TBARS concentration, and cytochrome c expression were lower and Bcl-2 expression was higher in the EHB group than those in the E group. The results suggested that β-carotene supplementation (0.52 mg/Kg BW/day) could prevent ethanol-induced liver damage by means of providing antioxidant property, decreasing ethanol-induced oxidative stress and inhibiting apoptosis in liver. However, there is no dose-response relationship in the present study. Taken together, ethanol treatment causes oxidative stress increased which could promote apoptosis in liver and then leads to liver injury. β-carotene which acts as an antioxidant could decrease oxidative stress and prevent ethanol-induced liver damage by inhibiting apoptosis in liver.