苦瓜預防脂肪肝功效之研究

Abstract

非酒精性脂肪肝被認為是由非酗酒過量造成肝臟脂質累積超過肝重5%，產生的肝臟代謝性疾病。國內成人人口脂肪肝盛行率高達26~34%，許多臨床研究顯示，非酒精性脂肪肝與肥胖、糖尿病及代謝症候群相關疾病有高度正相關。當脂肪累積過多時會促使肝臟產生發炎，已知非酒精性脂肪肝透過促使肝臟發炎形成非酒精性脂肪肝炎，有機會進一步造成肝硬化、肝纖維化甚至肝功能喪失以及肝癌。目前為止，非酒精性脂肪肝的治療仍無有效的藥物，因此，透過保健食品預防非酒精性脂肪肝的形成，有相當大的開發空間。在許多國家包含亞洲、南美以及東非，苦瓜已有被用來治療糖尿病及相關的代謝疾病，因此苦瓜是否具有預防脂肪肝的功效是相當有趣並且值得深入研究的課題。 首先，在動物實驗的預實驗部份，我們以八週大C57BL/6雄性小鼠分為五組，分別餵與低脂控制飼料（每克飼料熱量約4大卡，12% 熱量來自脂質）、高脂高膽固醇飼料（每克飼料熱量約5大卡，54%熱量來自脂質）、高脂高膽固醇飼料+5%花蓮一號山苦瓜乾燥粉末、 高脂高膽固醇飼料+5%花蓮二號山苦瓜乾燥粉末、 高脂高膽固醇飼料+5%花蓮四號山苦瓜乾燥粉末；高脂高膽固醇飼料由脂質提供之熱量佔飼料總熱量的54%，碳水化合物佔飼料總熱量25%。餵食四週後犧牲，發現花蓮一號及花蓮四號山苦瓜組在體重、副睪脂肪、血糖、肝臟總膽固醇都有顯著的下降，在組織切片染色我們也觀察到肝臟脂肪顆粒的堆積顯著的減少了。此外，以RT-qPCR分析肝臟中與脂質合成相關基因的表現量，結果顯示，相較於高脂高膽固醇組，餵食花蓮一號及四號山苦瓜乾燥粉末組肝臟中SREBP-1c、FAS及ACC等的mRNA表現量明顯的較少，特別是花蓮四號山苦瓜組改善最為明顯。相較之下，花蓮二號山苦瓜組體重、附睪脂肪、肝脂累積、血糖值等則是沒有顯著改善，這也顯示了並非所有品系的山苦瓜皆具有預防脂肪肝的功效。但由於飼料中添加苦瓜粉末使攝食量明顯降低，因此不能排除攝食量減少的影響。 由於在第一次動物實驗中觀察到餵食苦瓜乾燥粉末能有效減少肝臟中膽固醇的累積，為了探討膽固醇在非酒精性脂肪肝的形成是否扮演重要的角色，並控制攝食量。在第二次動物實驗的部份，我們將九週大之C57BL/6雄性小鼠分為七組，以高 IV 脂飼料作為控制組，其他組別分別為高脂高膽固醇組、高脂高膽固醇+2.5%花蓮一號山苦瓜乾燥粉末組、 高脂高膽固醇+5%花蓮一號山苦瓜乾燥粉末組、 高脂高膽固醇+2.5%花蓮四號山苦瓜乾燥粉末組、高脂高膽固醇+5%花蓮四號山苦瓜乾燥粉末組 、高脂高膽固醇+Ezetimibe組，以上七組脂質提供之熱量皆佔飼料總熱量約54%。經過兩週適應期，使其適應配方飼料後，再以實驗飼料以pair-feeding的方式餵養四週。餵食山苦瓜乾燥粉末可顯著降低血糖值及肝臟總膽固醇的含量。從肝臟組織切片結果也可發現脂肪顆粒累積情形有明顯地改善，並且有明顯的劑量關係。在腸道膽固醇運輸部份，我們可以發現餵食花蓮一號及四號山苦瓜，有效地降低了與膽固醇運輸相關的通道蛋白NPC1L1、ABCG5、ABCG8以及與脂肪酸運輸相關的通道蛋白CD36的mRNA表現量。推測花蓮一號及四號降低脂肪肝的功效有可能是透過抑制腸道膽固醇及部份脂肪酸的吸收，其中又以花蓮四號的效果最為顯著。 在兩次動物實驗的結果皆顯示了在餵食含有花蓮一號及花蓮四號苦瓜乾燥粉末飼料的情況下，從組織切片或是生化分析來觀察，都可顯著的預防脂肪肝的形成，然而苦瓜改善脂肪肝的確切機制仍未清楚。 從高脂飼料與高脂高膽固醇飼料餵食組的比較，可知肝臟膽固醇的累積在脂肪肝的形成扮演相當重要的角色，在腸道抑制膽固醇的吸收可能為苦瓜改善脂肪肝的重要機制之一。但苦瓜預防脂肪肝的發生可能是透過多重的作用（例如血糖值的改善），在肝臟是否具有抑制脂肪肝形成隨後引起的發炎反應也是日後研究的重點。此外，在細胞實驗的部分，我們發現花蓮四號山苦瓜的We-EA萃取物具有活化PPARδ的能力，並且We-EA萃取物並無明顯的細胞毒性，PPARδ的活化已被發現與腸道膽固醇吸收有關。因此，給予花蓮一號及花蓮四號山苦瓜具有降低小鼠肝臟膽固醇的累積，是否透過PPARδ的活化所致也是之後研究的重點之一。

Nonalcoholic fatty liver disease (NAFLD) is regarded as a liver metabolic syndrome that the fat accumulation over 5% of the total liver weight. The prevalence of NAFLD in Taiwan is high to 26~34%. Many researches have demonstrated that NAFLD is highly associated with obesity, diabetes and metabolic disorders. Notably, NAFLD has potential to progress through the inflammatory phase of nonalcoholic steatohepatitis (NASH) to fibrosis, cirrhosis (20%), and in some cases to liver failure (9%) or hepatocellular carcinoma (HCC) (1%). Nevertheless, there is no drug for treating NAFLD effectively. Therefore, dietary treatment becomes more important for prevention of NAFLD. Bitter melon (BM) is a popular vegetable that has been used to treat diabetes and metabolic disorder in Asia, South America and East Africa. Since NAFLD is part of the metabolic disorder, in this study we investigated if bitter melon can prevent the development of NAFLD. In animal experiments, we used male C57BL/6 mice as animal model. Mice were fed with low fat (LF), high fat/high cholesterol (HFC), or HFC diet supplemented with 5% Hualien No.1 (H1), No.2 (H2) or No.4 (H4) BM powder (HFC+H1, HFC+H2 or HFC+H4). After 4 weeks feeding, body weight, epididymal fat, plasma glucose level and liver cholesterol were significantly decreased in mice fed with HFC+H1 or HFC+H4 diet. The beneficial effect of BM was also observed in histology analyses by decrease accumulation of lipid droplets in the liver. Hepatic expression levels of SREBP-1c, FAS and ACC mRNA were also decreased in mice fed with diet containing BM powders, especially those fed diet containing H4. However, H2 did not improve body weight, epididymal fat weight or plasma glucose level. VI To exclude that the beneficial effects of BM is due to lower food intake; in experiment 2, HFC+2.5%H1, HFC+5%H1, HFC+2.5%H4 or HFC+5%H4 diets were given by pair feeding. In order to test the role of dietary cholesterol in the development of NAFLD, a group of control mice were fed with HFC diet supplemented with 10 μg ezetimibe/g diet. Similar to the results of experiment 1, diet containing H1 or H4 BM lowered plasma glucose and liver cholesterol. Histological analyses also showed that BM feeding resulted in less lipid accumulation in the liver. Moreover, intestinal NPC1L1 mRNA was decreased in mice fed with H1 or H4 BM. These results suggest that BM ameliorated NAFLD may due to decrease intestinal cholesterol absorption. In addition, intestinal CD36 mRNA was decreased in mice fed with diet containing BM powder. It is possible that fatty acid absorption was also decreased in mice fed diet containing BM powder. In conclusion, our results show that cholesterol plays a pivotal role in the development of NAFLD. And H1 and H4 BM decrease hepatic expression of lipogenic enzymes, cholesterol accumulation and thus ameliorate NAFLD; possibly through decrease intestinal cholesterol absorption. However, the mechanisms underlie require further investigation.