桑椹及荷葉抽出物抑制酒精誘導小鼠脂肪性肝炎病變和調節體脂肪之作用

Morus alba L. and Nelumbo Nucifera leaf extract prevents alcoholic steatohepatitis and body fat formation via anti-inflammation and inhibition of lipogenesis in C57BL/6J Mice

湯成杰(Chang-Chieh Tang)

指導教授 ： 王朝鐘

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2014年)

摘要

桑椹(MWE)及荷葉萃取物(NLE)對於抑制酒精性肝損傷和體脂肪生成之作用目前尚未釐清。本研究分別利用MWE 或NLE (0.5、1.0和2.0 %) 與Lieber-DeCarli流質酒精飼料合併餵食C57BL/6J小鼠六週建立預防脂肪性肝炎和降低體脂肪生成的動物實驗模式。研究結果證明MWE 和NLE能降低肝功能指數 (AST/ALT/ ALP)及血清和肝臟中三酸甘油酯和膽固醇含量，且抑制三酸甘油酯 (TG) 和膽固醇(TC)合成相關蛋白表現，如固醇類調節結合蛋白(SREBPs)、glycerol-3-phosphate acyltransferase (GPAT)、 HMG-CoA reductase 、脂肪酸合成酶 (FAS) 和 acetyl-CoA (ACC)。此外，顯示MWE 和NLE可經由增加peroxisome proliferator activated receptor-α (PPAR-α)表現，活化palmitoyltransferase-1 (CPT-1) 促進脂肪酸運輸，活化microsomal triglyceride transfer protein (MTP) 促進脂肪酸氧化作用，進而對抗酒精所誘發的脂質生成作用。並能經由活化GSH (麩胱甘肽)、GSH-Px (麩胱甘肽過氧化酶)、SOD (超氧化物歧化酶)和Catalase (過氧化氫酶)等抗氧化性酵素，抑制酒精誘導之氧化效應。從免疫組織染色和 western blot 分析炎性反應介質(proinflammatory mediators) 包含inducible nitric oxide synthase (iNOS)、cyclooxygenase2 (COX2) 、transcription factor nuclear factor-κB (NF-κB)、Interleukin-6 (IL-6) 、tumor necrosis factor- alpha (TNF-α)和酒精性肝炎指標gamma-glutamyl transpeptidase (γ-GT)，結果證明MWE 和NLE也能抑制酒精所誘發之炎症反應。從週邊脂肪組織測量副睪脂體(epididymal fat pat)、副腎脂體 (perirenal fat pat) 和腸間脂體(mesenteric fat pat) 等組織，亦證實MWE 和NLE可以抑制體脂肪形成。最後，我們的研究結果顯示MWE 和NLE能抑制三酸甘油酯和膽固醇在肝臟中累積、促進脂肪酸運輸、降低小鼠體重、調控脂質在肝臟中累積並降低血清中脂質含量、活化抗氧化酵素避免酒精誘導的氧化效應、透過活化 AMPK和PPAR-α路徑抑制脂肪酸合成、增加脂肪酸運輸和刺激脂肪酸氧化作用。因此，桑椹及荷葉萃取物具有潛力發展為健康食品，預防酒精引發之脂肪性肝炎和降低體脂肪的形成。

關鍵字

桑椹萃取物 ； 荷葉萃取物 ； 抗氧化 ； 抗發炎 ； 酒精性脂肪性肝炎

並列摘要

Many plant extracts and their bioactive substances are well recognized for their potential to exert as chemoprotective agents against common alcoholic liver injury. The roles Morus alba L. (MWE) and Nelumbo nucifera leaf water extract (NLE) play in preventing alcoholic liver disease and body fat formation remain unknown. In this study, we were undertaken to evaluate the hepatoprotective effect mechanisms of MWE and NLE in experimental alcoholic steatohepatitis animal models. Six groups of C57BL/6J mice were fed on Lieber-DeCarli regular diet with or without ethanol of 36% energy for 6 weeks. Results found the MWE contain many nutrients and bioactive substances, including fifteen types of polyphenols and anthocyanin compounds. NLE contained polyphenols (phenolic acids and flavonoids) more than 70% maybe the main functional components in NLE that potentially provided benefits for alcoholic liver disease. The parameters of histopathology, immunohistochemistry, antioxidant defense, proinflammatory mediator and lipid synthesis-related proteins demonstrated the inhibitory effect of MWE and NLE on alcoholic steatohepatitis. Plasma and hepatic content analysis showed that MWE and NLE inhibited lipid accumulation by altering the levels of triglycerides (TG) and cholesterol (TC). Treatment with MWE or NLE increased the expression of the p-AMPK/AMPK ratio and PPAR-α. Furthermore, fatty acid oxidation and transport via carnitine palmitoyltransferase-1 (CPT1) and microsomal triglyceride transfer protein (MTP) were through the activation of the AMPK and PPAR-α signal. These results revealed that MWE and NLE prevent alcoholic steatohepatitis by multiple pathways, including reduced lipid synthesis, enhanced fatty acid oxidation and transport responses, inhibited oxidative stress and facilitated anti-inflammation. Suggesting that MWE and NLE might be regarded as beneficial foods, and have potentiality in developing the natural agent against for preventing alcoholic steatohepatitis.

並列關鍵字

Mulberry extract ； Nelumbo nucifera leaves extract ； Antioxidant ； Ati-inflammation ； Alcoholic steatohepatitis

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