口服二十二碳六烯酸與離胺酸改善小鼠脂肪肝現象

Abstract

近年來脂肪肝成為已開發國家中常見的代謝失衡型流行病，初期病徵為脂質代謝失常，而於肝臟大量堆積，同時亦會伴隨發炎反應產生，進而造成肝纖維化、硬化，甚至肝衰竭或導致肝癌。二十二碳六烯酸（Docosahexaenoic acid, DHA）為人體必須的n-3多元不飽和脂肪酸，前人研究已證實DHA對人類健康極為有益，可降低脂質生成相關基因的表現，並改善心血管疾病與高血脂症。離胺酸是人體生長所須的必需胺基酸，參與肉鹼生成，並影響脂肪酸代謝。早期研究發現離胺酸可保護大鼠免於半乳糖胺造成之肝毒性，由此推測離胺酸有保護肝臟的效果。因此本研究欲探討DHA與離胺酸兩種營養素對非酒精性脂肪肝病（Non-alcoholic fatty liver disease）的影響。 活體試驗部分利用C57B/6小鼠做為動物模式，餵以高脂飼糧誘導脂肪肝形成。預備試驗中，高脂飼糧餵飼23週後的小鼠體重明顯高於對照組，其肝臟中有大量脂肪油滴堆積。而後正式試驗中，48隻小鼠分成對照組、0.5%（以平均日採食量計算）離胺酸、1 %離胺酸、1 % DHA、1 % DHA + 0.5%離胺酸以及1% DHA + 1%離胺酸六組（每組八隻），以灌食方式餵食各濃度營養素四週。各處理組的體重均較對照組低，此外，DHA處理組血清中三酸甘油酯與丙氨酸轉氨酶濃度皆顯著低於對照組，顯示飲食中添加DHA可減少脂肪堆積以保護肝臟。此外，DHA處理可降低發炎前驅因子mRNA表現，例如腫瘤壞死因子α（TNF-α）、細胞介白素1β（IL-1β）以及單核球趨化蛋白1（MCP-1）。DHA處理也同時降低脂質生成相關基因表現，例如脂肪酸合成酶（FAS）與乙醯輔脢A羧化脢1（ACC1），在肝臟與脂肪組織都有相同的結果。經由肝臟切片染色結果發現DHA與離胺酸處理皆可減少肝臟脂肪油滴堆積。 體外試驗部分利用人類肝癌細胞株SK-HEP-1處理1 mM油酸（OA）24小時做為脂肪肝細胞模式。細胞試驗分為對照組、0.1 mM DHA、4 mM離胺酸以及0.1 mM DHA + 4 mM離胺酸四個組別，分別在有OA存在與否的情況下添加不同濃度之DHA及離胺酸。實驗結果顯示DHA與離胺酸處理皆可減少FAS以及發炎前驅因子IL-6基因表現。 綜上所述，本研究證實餵飼DHA可降低小鼠血清中三酸甘油酯與丙胺酸轉胺酶濃度，並減少肝臟脂肪油滴堆積；此外，DHA處理可減低脂質生成與發炎前驅因子的表現，因而改善脂肪肝現象。而離胺酸只可減少肝臟脂肪堆積，但其調控機制以及離胺酸對脂質的影響還有待進一步釐清。因此，富有DHA與離胺酸的食物提供了改善非酒精性脂肪肝 的高度潛力。

Fatty liver disease (FLD) is a worldwide illness, characterized with significant amount of triglyceride accumulation in the liver cells. Fatty liver progression with inflammation, FLD may further develop into fibrosis, cirrhosis and liver failure, or toward hepatocellular carcinoma. Docosahexaenoic acid (DHA) is one of essential n-3 polyunsaturated fatty acids for human. Treatment of DHA has been shown to decrease the expression of lipogenic genes and prevent some illnesses, such as cardiovascular diseases. In addition to its critical role to animal growth, lysine plays a fundamental role in carnitine synthesis, a nutrient that helps to convert fatty acids into energy. It has been shown that lysine treatment protects rats against hepatotoxic actions of D-galactosamine. Therefore, this study was designed to investigate the effects of DHA and lysine on non-alcoholic FLD. In the in vivo study, 48 C57B/6 mice were fed with high fat diet containing 35.5% fat for 23 weeks. They were then divided into 6 groups, with oral administration of saline, 0.5% (based on daily dietary intake) lysine, 1% lysine, 1% DHA, 1% DHA + 0.5% lysine and 1% DHA + 1 % lysine for 4 weeks. Mice with high fat diet exhibited heavier body weight than those fed with control chow diet. Histological examination showed that significant and numerous fat droplets accumulated in the liver of mice with high fat diet. Compared with the saline group, mice administrated with DHA or lysine or combined with both at any given levels exhibited a decrease of body weight. Furthermore, serum triglyceride levels and alanine transaminase activities were remarkably reduced in the groups fed with DHA, indicating that DHA may reduce fat accumulation to protect liver from steatosis. Indeed, the suggestion was supported by a decrease of the transcript levels of proinflammatory cytokines genes such as tumor necrosis factor-α, interleukin 1β and monocyte chemotactic protein-1 and lipogenic gene expreesions, such as fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1) in the liver of mice with DHA treatment. Both the treatments of DHA and lysine decreased the accumulation of hepatic lipid droplets. Additionally, reduced mRNA expressions of FAS and ACC1 in mice gonadal adipose tissues by DHA treatment were observed. In the in vitro studies, human hepatocellular carcinoma SK-HEP-1 cells were treated with 1 mM oleic acid in order to establish a cell model for fatty liver. After 24 hr culture, the steatotic hepatocytes were treated with 0.1 mM DHA, 4 mM lysine respectively or with 0.1 mM DHA + 4 mM lysine for 24 hr in the presence of or without OA. Results showed that the treatments significantly downregulated FAS and interleukin 6 transcription levels by DHA and lysine. In conclusion, the present study suggests that DHA treatment decreases mice serum TG levels and ALT activities and reduced the hepatic lipid droplet accumulation. Moreover, treatment of DHA decreases lipogenic and inflammatory gene expressions and contributes to the amelioration of fatty liver. Lysine exerted effects only on decreasing lipid accumulation in the liver, and its regulatory mechanism required further studies. Based on the results above, foods abundant with DHA or lysine may have a high therapeutic potential in the amelioration of non-alcoholic fatty liver disease.