雞肝水解物於硫代乙醯胺誘導肝損傷大鼠模式下之保護功效的探討

Ameliorative effects of pepsin-digested-chicken liver hydrolysates on thioacetamide-induced liver damage

陳柏如(Po-Ju Chen)

指導教授 ： 陳億乘

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10786

摘要

根據世界衛生組織2013年的統計資料，肝癌於癌症中排名第六；在台灣，慢性肝病及肝硬化為2013年第九大死因。有鑑於肝纖維化的可逆性，於肝病發生至纖維化前給予保健食品的補充將有助於延緩肝病的進程。經胃蛋白酶水解製備而成的雞肝水解物(chicken liver hydrolysates, CLHs)中含有牛磺酸、L-肌肽/甲肌肽等機能性雙胜肽，具有改善肝臟纖維化的潛力。本研究主要探討補充CLHs對硫代乙醯胺(thioacetamide, TAA)誘導大鼠肝損傷下是否具有減緩發炎及纖維化的效果，並以L-肌肽為正對照組。結果顯示：TAA會導致體重顯著下降(p < 0.05)，並具有相對較腫脹之肝臟、腎臟、脾臟。雖然CLHs與肌肽對於臟器重量並無明顯減緩腫脹之效果，但有助於降低血清中ALT、AST值(p< 0.05)。病理切片觀察亦顯示，Sirus red染色中發現補充雞肝水解物及L-肌肽的組別，膠原蛋白的堆積明顯降低。同時，TGF-β 路徑上下游基因部分受到CLHs的調控(TGF-β, SMAD4, αSMA, and Col1α) (p< 0.05）。 H E及ED1免疫染色中，巨噬細胞及單核球浸潤的發炎現象在同時投予CLHs時可獲得改善。此外，在補充CLHs的組別發現肝臟中TNF-α、IL6和TGF-β的含量較TAA組低(p < 0.05)，指出具減緩發炎反應的作用，而這樣的功效可能來自於CLHs能夠顯著提升(p < 0.05)肝臟中的抗氧化酵素活性(SOD和CAT)、非酵素型抗氧化能力(TEAC和reduced GSH)及降低氧化指標(TBARS value)有關，進一步減緩TAA所造成的氧化壓力，而改善了肝臟發炎以及纖維化的現象。綜合上述，雞肝水解物(CLHs)的補充確實具有減緩大鼠肝臟發炎及纖維化之功效。然其中主要有效成分及詳細的作用機制，則需進一步研究加以證實。

關鍵字

肝纖維化 ； 肝發炎 ； 雞肝水解物 ； 硫代乙醯胺 ； 氧化壓力

並列摘要

Liver diseases are severe problems globally; meanwhile, liver cancer ranked sixth among cancer categories in the world. In Taiwan, a chronic liver disease is the ninth leading cause of death in 2013. Hence, this study was to evaluate the amelioration of chicken liver hydrolysates (CLHs), which manufactured by a pepsin digestion for 2 h, against TAA-induced liver fibrosis via a rat’s model. After a pepsin digestion for 2 h, brolier by-products, chicken livers, can be functional hydrolysates CLHs where there are plenty amount of some functional amino acids and dipeptide, i.e. taurine, branched amino acids (valine, leucine, and isoleucine), carnosine, and anserine.
Male Wistar rats treated with TAA (100 mg/kg, ip. three times weekly for 10 weeks) were orally supplemented with CLHs (200 and 600 mg/kg BW daily for 10 weeks). Results have shown that the TAA-treated groups have significantly lowered body weight gain and induced swollen liver, spleen, and kidney (p<0.05); meanwhile, higher serum ALT and AST levels, as well as liver TNF-α, IL6, and TGF-β contents (p<0.05) were also detected. A cells infiltration was also shown on H E stained biopsies in livers of TAA-induced groups, and further identified to be monocytes and macrophages by immunohistochemistry (IHC) analysis of ED1. More (p<0.05) collagen accumulation in livers was also observed and measured under TAA treatment, corresponding to histopathological observations by a Sirius red staining. The significantly upregulated mRNA expressions of TGF-β and SMAD4 caused by TAA treatment further induced an enhancement of α-SMA gene expressions. The apparently elevated protein expressions of α-SMA were found in both IHC and western blotting analysis (p<0.05). Those liver inflammatory and fibrotic evidences were ameliorated by CLHs (p<0.05) which are partially related to antioxidant effects including increased antioxidant enzymatic activities (SOD and CAT activities), reduced GSH contents, and TEAC levels, as well as decreased TBARS values in livers (p<0.05).
Hence, it is fully demonstrated that CLH supplementation can alleviate TAA-induced liver fibrosis by both antioxidant and inflammation modulating effects. Based on the current results, functional CLHs not only enhance the added value of chicken livers, but also be a hepatoprotective agent in the niche market. The possible functional components in chicken liver hydrolysates which are responsible for hepatoprotection on TAA-induced liver damage warrant further investigations in the new drug discovery.

並列關鍵字

chicken liver hydrolysates ； liver fibrosis ； thioacetamide ； oxidative stress?

參考文獻

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