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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53713

Title: 以吳郭魚作為非酒精性脂肪肝動物模式及 HepG2 細胞評估養殖蜆脂溶性成分護肝之功效
Evaluation of the hepatoprotective ability of lipid-soluble compounds of freshwater clam using tilapia as an in vivo liver disease animal model
Authors: Lin, Jing-Jen
林敬甄
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 吳郭魚;HepG2 細胞;台灣蜆;非酒精性脂肪肝;植物固醇;多元不飽和脂肪酸
tilapia;HepG2 cells;freshwater clam;nonalcoholic fatty liver disease;phytosterols;polyunsaturated fatty acids
Date: 2019
Issue Date: 2020-07-03T08:43:04Z
Abstract: 本研究建立吳郭魚作為非酒精性脂肪肝 (nonalcoholic fatty liver disease, NAFLD) 之動物模式,以此模式及細胞模式評估台灣蜆 (Corbicula fluminea) 熱水萃後之殘肉 (residual meat of freshwater clam, FCR) 及其乙醇萃取物 (ethanol extract of FCR, FCRE) 減緩 NAFLD 之作用,鑑定 FCR 及 FCRE 之脂溶性機能性成分,並探討提升蜆機能性成分之育肥策略。 吳郭魚攝食高脂飼料 (high-fat diet, HFD) 2 週後,其血漿丙胺酸轉胺酶 (alanine aminotransferase, ALT)、天門冬胺酸基轉移酶 (aspartate transaminase, AST)分別較控制組高 3.7 及 3.3 倍、肝體比增 1.6 倍,肝臟三酸甘油酯和總膽固醇分別增加 2.2、2.3 倍,肝臟脂肪酸含量為控制組的 1.9 倍,單元不飽和脂肪酸 (monounsaturated fatty acids, MUFAs) 最高,佔 53%,其次為飽和脂肪酸 (saturated fatty acids, SFAs) 佔 37%;將 SFAs 去飽和為 MUFAs 的 stearoyl-CoA desaturase (SCD, C18:1n9/ C18:0) 由 2.72 升至 5.26;多元不飽和脂肪酸 (polyunsaturated fatty acids, PUFAs) 的 n3/n6 比值由控制組的 2.4 降至 0.5。肝細胞直徑增為控制組的 1.7 倍,為氣球樣變性 (ballooning degeneration),肝臟並有發炎細胞浸潤,即為非酒精性肝炎 (nonalcoholic steatohepatitis, NASH)。 以攝食 HFD 之吳郭魚作為 in vivo 模式,評估 FCR 或 FCRE 護肝功效,攝食二者 2 週後均顯著 (p<0.05) 降低血漿 AST、ALT、肝體比及肝臟之總膽固醇、三酸甘油酯。FCRE 使肝臟之 SFAs、MUFAs 分別減少 50%、66%,SCD (C18:1n9/ C18:0) 下降 38%,PUFAs n3/n6 比值增加 2.1 倍;FCRE 並減緩肝細胞氣球樣變性、肝臟發炎指標 PGE2 和發炎細胞浸潤。血漿中三酸甘油酯、總膽固醇之濃度也顯著 (p<0.05) 低於 HFD 組,分別減少 50% 和 20% 以上。 吳郭魚作為模式以印證蜆之功效與小鼠之結果相同,均可以 HFD 誘發 NAFLD,並為可逆反應,與餵食小鼠 HFD 10 週後,血漿 ALT、肝臟三酸甘油酯和總膽固醇分別增加 1.9、4.1 和 2.0 倍,症狀相似,但吳郭魚誘導時間為小鼠的 1/5,可做為快篩護肝食品之模式動物。 HepG2 細胞之 in vitro 試驗中,FCRE 提升細胞存活率,減緩游離脂肪酸誘發之脂質堆積,顯著 (p<0.05) 降低脂肪酸合成酶活性、使脂肪酸 β-氧化酵素粒線體之 carnitine palmitoyltransferase-1 (CPT-1)、CPT-2,和過氧化體 (peroxisome) acyl-CoA oxidase (ACO) 之活性顯著 (p<0.05) 增加,並有濃度依存性。 在 FCR 和 FCRE 之脂溶性成分中,鑑定出其機能性含植物固醇,有 campesterol、brassicasterol、β-sitosterol、stigmasterol 及 PUFAs,其 n3/n6 比值均 > 2。FCRE 之總植物固醇 (3.18 mg/g) 和 n3 PUFAs (65.1 mg/g) 含量分別高於 FCR 達 7 倍和 4 倍。 蜆之植物固醇來自所濾食之微藻,以蛋白核小球藻、橢圓小球藻、四尾柵藻及二角盤星藻為主,以微藻或輪蟲育肥,蜆之植物固醇含量分別較控制組增加 13% 和 11%,以蜆殘肉水解物育肥增 5%,大豆蛋白水解物育肥植物固醇含量減少 8%。育肥之蜆。四種餌料均未能增加蜆之 n3 PUFAs,但蜆之肥滿度皆較控制組高,以輪蟲、蜆殘肉水解物育肥蜆之肥滿度均 >15%。 本研究確認吳郭魚可作為非酒精性脂肪肝疾病的模式動物,以製蜆精後之殘肉確認其降低肝臟脂肪酸生合成、提高脂肪酸 β-oxidation、調節肝臟脂肪酸去飽和之作用、減少肝臟脂質堆積、延緩發炎的功效,並發展出提升其機能性成分之育肥策略,自蜆肉護肝素材製成膠囊可於常溫貯藏 3.5 個月。
The purpose of this study was to develop an alternative animal model for assessing nonalcoholic fatty liver disease (NAFLD), applicable to evaluate the hepatoprotective effect of functional foods and ingredients. Tilapia was used as a test animal model and the residual meat of freshwater clam (FCR) and its ethanol extract (FCRE) were determined. The functional compounds of the FCR and FCRE were identified and strategy to increase functional compounds. Tilapia were fed high-fat diet (HFD) for 2 weeks induced their alanine aminotransferase (ALT) and aspartate transaminase (AST) up to 3.7 times and 3.3 times, respectively, and hepatosomatic index (HSI) became 1.6 times greater. The triacylglycerol and total cholesterol content in liver increased up to 2.2 times and 2.3 times, respectively, being higher than those of the control group. The fatty acid content in the liver of HFD-fed tilapia increased to 1.9 times of the control group, of which monounsaturated fatty acids (MUFAs) was the highest (53%), followed by saturated fatty acids (SFAs, 37%). The stearoyl-CoA desaturase (SCD, C18:1n9/ C18:0), which modulates SFAs to MUFAs increased from 2.72 to 5.26. The ratio of polyunsaturated fatty acids (PUFAs) n3/n6 in liver decreased from 2.4 to 0.5. The histological examination of liver revealed that tilapia fed HFD induced nonalcoholic steatohepatitis (NASH), of which the hepatocyte was 1.7 times of the control group, and showed ballooning degeneration and inflammatory infiltrates. Using the HFD-fed tilapia as an in vivo model. Intake of FCR or FCRE caused significant amelioration of AST and ALT in plasma, HSI, and total cholesterol and triacylglycerol content in liver. The concentrations of triacylglycerol (TG) and total cholesterol (TC) in tilapia plasma fed HFD supplemented with FCR or FCRE were significantly (p<0.05) lower than those fed only HFD, TG and TC decreased by 50% and 20% respectively. Feeding FCRE to tilapia decreased SFAs and MUFAs in liver by 50% and 60% respectively, SCD (C18:1n9/ C18:0) decreased by 38%, and the n3/n6 PUFAs ratio increased 2.1 times of those fed HFD alone. The ballooning degeneration, inflammatory infiltrates, and prostaglandin E2 (PGE2) in liver were also improved by feeding FCRE. HFD elicited progression of NAFLD in tilapia similar to what drew forth in mice, expect mice took 10 weeks to induce their ALT in plasma became 1.9 times greater, TG and TC content in liver up to 4.1 times and 2.0 times, respectively, of the control group. In HepG2 cells treated with free fatty acid increased lipid accumulation, while addition of FCRE significantly reduced lipid accumulation and cell death via decreased fatty acid synthase (FAS) activity and increased activities of carnitine palmitoyltransferase-1 (CPT-1), CPT-2 in mitochondria, and acyl-CoA oxidase (ACO) in peroxisome in a dose-dependent manner. The lipid-soluble compounds in FCR and FCRE were identified as the functional compounds. They comprised phytosterols including campesterol, brassicasterol, β-sitosterol and stigmasterol, and PUFAs of which the ratio of n3/n6 >2. The total phytosterols (3.18 mg/g) and n3 PUFAs content (65.1 mg/g) in FCRE was 7 and 4 times of those in FCR, respectively. The strategy to increase the phytosterols content in clam was to fertilize the cultured pond with the microalgae or the rotifer. Both techniques caused the phytosterol to increase by 13% and 11%, respectively, significantly higher than those from the control pond. The n3/n6 ratio of PUFAs in clam were not increased after fertilizing the culture pond. However, rotifer or FCR improved the fatness of clam by >15% increase. For the first time, tilapia was established as an animal model for screening fatty-liver preventive effects in functional foods. In the in vivo and in vitro models of NAFLD, the residual clam meat generated as a by-product of clam essence production significantly decreased de novo lipogenesis, increased fatty acid β-oxidation and modulated fatty acid desaturation, thus suppressed lipid accumulation and inflammation. Strategy on enrichment of the functional compounds in the cultured pond was also first reported in this study. The residual clam meat can be utilize as a hepatoprotective ingredient was developed into the soft-gel capsule, which shelf life was 4.5 months stored at room temperature.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0020132003.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53713
Appears in Collections:[食品科學系] 博碩士論文

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