桂花、菊花與芝麻素對肝細胞脂質堆積、發炎與倉鼠脂肪肝之影響

The effects of Osmanthus fragrans, Chrysanthemum morifolium and sesamin on steatosis and inflammation in HepG2 cells and fatty liver in hamsters

辛靜玫(Ching-Mei Hsin)

指導教授 ： 吳文惠 博士

國立臺灣師範大學/教育學院/人類發展與家庭學系/碩士(2010年)

摘要

代謝症候群包含腹部肥胖、高三酸甘油酯、低HDL與高血壓，若未經適當控制，則可能轉為第2型糖尿病或心血管疾病。許多研究提出非酒精性脂肪肝是代謝症候群的相關病症，會由單純的脂肪變性進展到肝炎、肝纖維化、肝硬化。有研究指出，芝麻素是PPAR-α天然配子，具促進脂肪酸氧化及抑制脂肪酸合成的作用。菊花桂花萃取物可以保護細胞免受自由基的傷害。故本研究探討這三種材料對非酒精性脂肪肝與代謝症候群的影響。
實驗一：在人類肝細胞 (HepG2) 模式中，以1 mM脂肪酸 (oleic acid, palmitic acid, stearic acid, linoleic acid and arachidonic acid, 1mM) 誘發脂肪堆積，發現芝麻素、菊花與桂花甲醇萃物均可降低肝細胞內三酸甘油酯堆積與oil red O染色，菊花與桂花甲醇萃物可抑制ROS生成量，具有抗氧化潛力。
以real-time PCR分析HepG2脂肪酸代謝相關基因，發現桂花甲醇萃取物抑制sterol regulatory element binding protein-1 (SREBP-1)、fatty acid synthase(FAS)、glycerol 3-phosphate acyltransferase (GPAT)、acyl-CoA oxidase (ACO) mRNA表現，促進acetyl-CoA carboxylase(ACC) mRNA表現，菊花甲醇萃取物抑制SREBP-1c、ACC 、FAS、GPAT、ACO mRNA表現，芝麻素抑制SREBP-1c與GPAT mRNA表現量。另以0.5 g/ml LPS刺激THP-1分泌促發炎激素做為誘發HepG2發炎模式，以real-time PCR分析HepG2 mRNA表現，實驗結果發現，菊花萃取物與芝麻素抑制TNFα, IL-1β, IL-6, IL-8, MCP-1與TGFβ mRNA表現量，桂花萃取物降低TNFα, IL-1β, IL-8 mRNA表現量。
實驗二、在動物模式方面，餵予雄性倉鼠高脂 (35%) 、高膽固醇 (1%) 飼料，同時投予0.3% 芝麻素、菊花甲醇萃取物或桂花甲醇萃取物，14天後犧牲。發現芝麻素對血脂、血中游離脂肪酸與血糖有下降趨勢但未達顯著，菊花甲醇萃取物則反而顯著增加血液三酸甘油酯與膽固醇之濃度。桂花甲醇萃取物顯著降低肝中膽固醇，菊花與桂花甲醇萃取物會顯著降低腎臟之相對重量。但此三種食材對體重增加量與腹睪脂肪量皆無顯著影響。
實驗三、將雄性倉鼠分為三組，對照組餵予AIN76配方飼料，另兩組分別餵予FFC飼料 (Fat 20%, Fructose 34%, Cholesterol 0.2% wt/wt) 與含有0.3% 桂花甲醇萃取物的FFC飼料，為期10週。發現FFC飲食顯著增加脾、副睪脂肪、腎、肝之相對重量，血中TC、TG與HDL-C與肝臟TC皆顯著增加，肝臟SOD活性則顯著下降，肝臟MCP-1含量顯著上升。肝臟切片以Oil Red O染色觀察到脂質堆積，以H&E染色發現有巨噬細胞入侵的發炎情形。桂花甲醇萃物對以上指標則無改善效果。
總結： 在體外實驗，芝麻素、菊花與桂花甲醇萃取物可顯著降低肝細胞脂肪堆積與發炎。以高脂 (35%)、高膽固醇 (1%) 餵予雄性倉鼠2週，僅見桂花甲醇萃物降低肝中膽固醇堆積。以較低脂肪 (20%) 膽固醇 (0.2%) 及高果糖 (34%) 餵予雄性倉鼠10週可誘發腹部肥胖、高血脂、肝臟發炎等代謝症候群相關病症，但桂花甲醇萃物並未改善代謝症候群。動物實驗未觀察到此三種材料在細胞實驗中顯現的效果，可能由於萃物劑量不足，或是萃物經由動物吸收代謝後降低效果所致。

關鍵字

非酒精性脂肪肝 ； 代謝症候群 ； 倉鼠 ； HASH(0x1d862b30) ； 芝麻素 ； 菊花 ； 桂花

並列摘要

Metabolic syndrome includes central obesity, hypertriglyceridemia, low serum level of high density lipoprotein-cholesterol and hypertension. It may turn to type II diabetes mellitus and cardiovascular disease without proper treatment. Many studies reveal that nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome. NAFLD refers to a wide spectrum of liver disease ranging from simple fatty liver, to steatohepatitis, and to cirrhosis. The aim of this study was to investigate the effects of methanolic extracts of Osmanthus fragrans (OF) and Chrysanthemum morifolium (CR) and sesamin (SE) on NAFLD and metabolic syndrome in HepG2 cells and hamsters.

In experiment 1, the HepG2 cells were treated with a 1mM fatty acid (FA) mixture containing oleic, palmitic, stearic, linoleic and arachidonic acids, and co-incubated with or without OF, CR and SE for 24 hrs. We observed that OF, CR and SE significantly suppressed intracellular TG accumulation. Both OF and CR decreased ROS production in HepG2 cells. The expression of mRNA related to fatty acid metabolism in HepG2 cells was determined by quantitative real-time PCR. The results revealed that OF significantly decreased mRNA expression of sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthase(FAS), glycerol 3-phosphate acyltransferase (GPAT), and acyl-CoA oxidase (ACO) and increased mRNA expression of acetyl-CoA carboxylase(ACC). CR significantly decreased mRNA expression of SREBP-1c, ACC, FAS, GPAT, and ACO. SE significantly decreased mRNA expression of SREBP-1c and GPAT.
To assess the anti-inflammatory potential of OF, CR and SE, HepG2 cells were treated with conditioned medium from LPS-stimulated THP-1 macrophages as a source of pro-inflammatory cytokines and co-incubated with or without test samples for 48 hr, and then the mRNA expression of pro-inflammatory cytokines in HepG2 cells was determined by quantitative real-time PCR. The results revealed that CR and SE significantly decreased mRNA expression of TNFα, IL-1β, IL-6, IL-8, MCP-1, and TGFβ. Similar inhibitory effects on mRNA expression of TNFα, IL-1β and IL-8 were observed with OF.
In experiment 2, male hamsters were randomly divided into 4 groups and fed high fat high cholesterol diet (30% fat, 1% cholesterol) supplemented with or without 0.3% OF, CR or SE. After 14 days of feeding, the SE group seemed to have a lower level of serum TC, TG, NEFA and glucose but without statistical significance. The OF group significantly decreased the content of hepatic TC but the CR group significantly increased the level of serum TG and TC. There was no significant difference in body weight gain and the weight of epididymal fat pad among control, OF, CR and SE groups.

In experiment 3, male hamsters were divided into 3 groups (1) LF: low fat diet (5% fat, AIN76 diet)；(2) HF: high fat (HF) diet (20% fat, 34% fructose, 0.2% cholesterol)；(3) OF: HF diet supplemented with 0.3% OF. After 10 weeks feeding, the HF group had higher levels of serum TC, TG, HDL-C, hepatic TC, and MCP-1, and lower activity of hepatic SOD in comparison with the LF group. Liver sections revealed HF diets increased fat deposition from oil-red O stain and macrophage clusters from H&E stain. Therefore, metabolic syndrome was induced in this model. However, OF supplementation did not improve any of the above parameters.

In conclusion, OF, CR and SE prevent lipid accumulation and inflammation in HepG2 cells, but they do not improve metabolic syndrome in hamsters except a decreased level of hepatic TC by OF. The inconsistence may be due to (1) insufficient dosage of test samples, (2) low intestinal absorption or metabolic alteration of the effective components in hamster studies.

並列關鍵字

nonalcoholic fatty liver disease ； metabolic syndrome ； hamster ； HepG2 ； sesamin ； Chrysanthemum morifolium ； Osmanthus fragrans

參考文獻

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