探討雞肝水解物於酒精誘導肝損傷之護肝功效

Abstract

長期不當酒精攝取將造成細胞內能量平衡破壞、氧化壓力、脂質堆積等並引起發炎反應，進一步發展成纖維化及肝硬化，這一系列的損傷通稱為酒精性肝損傷(alcohol liver disease, ALD)。本實驗室先前研究發現，利用pepsin水解之雞肝蛋白能產製具有抗氧化及調節血脂能力的雞肝水解物(chicken-liver hydrolysates, CLHs)。故本研究的目標為評估CLHs在酒精誘發肝損傷小鼠模式下之保健功效。 CLHs的游離胺基酸組成的結果顯示，CLHs主要成分為Glu、Leu、Lys及Ala；另外，雞肝中牛磺酸(taurine)、雙胜肽-肌肽(carnosine)與甲肌肽(anserine)的含量在水解後亦有提升。在動物實驗部分，48隻8週齡雄性C57BL/6小鼠被隨機分配到六個組別，包括: 1) CON: control liquid diet+0.2 mL ddH2O; 2) Ethanol: Lieber-DeCarli ethanol diet+0.2 mL ddH2O; 3) E_CLHL: Lieber-DeCarli ethanol diet+80 mg CLHs/kg body weight (BW) in 0.2 mL ddH2O; 4) E_CLHM: Lieber-DeCarli ethanol diet+320 mg CLHs/kg BW in 0.2 mL ddH2O; 5) E_CLHH: Lieber-DeCarli ethanol diet+1280 mg CLHs/kg BW in 0.2 mL ddH2O; 6) E_Silymarin: Lieber-DeCarli ethanol diet+150 mg silymarin/kg BW in 0.2 mL ddH2O。水、CLHs及silymarin以每日管餵方式進行處裡並於八週後犧牲採取其血清與肝臟進行分析。結果顯示，酒精餵飼導致小鼠肝臟及脾臟腫大(p<0.05)，其血清AST、ALT及ALKP含量亦較高(p<0.05)；除此之外，H&E染色切片結果顯示肝臟出現免疫細胞浸潤及肝細胞腫脹的情況，而這個結果與提升的肝臟發炎激素(TNF-α, IL-6, 和IL-1β) (p<0.05)互相呼應。另外，肝臟中亦出現三酸甘油酯(triglyceride, TG)含量顯著提升 (p<0.05)及脂質過氧化物(TBARS)產量上升(p<0.05)的情況。上述結果經過CLHs及silymarin的補充後得到改善(p<0.05)。而其效果推斷來自CLHs具提升脂肪酸β-氧化、降低脂肪酸生合成、增加抗氧化酵素活性(SOD、CAT及GPx)、總抗氧化能力(TEAC)及提升酒精代謝酵素的活性(ALDH) (p<0.05)。 綜觀上述研究結果，CLHs可經由加速肝臟酒精代謝、增加細胞抗氧化能力、調節脂質恆定進一步減緩發炎反應來改善酒精所誘發肝損傷，而其保健效果甚至不亞於臨床藥物silymarin的補充。

Chronic alcohol consumption mainly leads to hepatosteatosis and increases oxidative stress, thus inducing an inflammatory response, further causing fibrosis or worse, cirrhosis. The serial progressions are well-known as alcoholic liver disease (ALD). According to studies from our lab, pepsin-digested chicken-liver hydrolysates (CLHs) own antioxidative capacity and lipid lowering effects. Hence, the aim of this study was to investigate the hepatoprotective effect of CLHs against alcohol damage in vivo. Regarding the free amino acid profile, glutamic acid, leucine, lysine, and alanine were majorities in CLHs. The contents of taurine, anserine, and carnosine were elevated after the pepsin hydrolyzation. In in vivo study, 48 eight-week-old male C57BL/6 mice were randomly divided into six groups: 1) Control: control liquid diet+0.2 mL ddH2O; 2) Ethanol: Lieber-DeCarli ethanol diet+0.2 mL ddH2O; 3) E_CLHL: Lieber-DeCarli ethanol diet+80 mg CLHs/kg body weight (BW) in 0.2 mL ddH2O; 4) E_CLHM: Lieber-DeCarli ethanol diet+320 mg CLHs/kg BW in 0.2 mL ddH2O; 5) E_CLHH: Lieber-DeCarli ethanol diet+1280 mg CLHs/kg BW in 0.2 mL ddH2O; 6) E_Silymarin: Lieber-DeCarli ethanol diet+150 mg silymarin/kg BW in 0.2 mL ddH2O for 8 weeks. Distilled water, CLHs and silymarin were provided daily by an oral gavage, respectively. The results indicated that the liver and spleen of alcoholic diet fed mice were enlarged (p<0.05); higher (p<0.05) serum AST, ALT and ALKP levels than control diet fed ones were also observed. Moreover, increased hepatic immunocyte infiltration and swollen hepatocytes were shown on the H&E staining which in correspond to elevated (p<0.05) hepatic pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) levels. An increased triglyceride contents in liver tissues (p<0.05) were observed. In addition, alcoholic diet fed mice also had higher (p<0.05) hepatic/serum TBARS values. All the above damages were ameliorated by CLH or silymarin supplementation (p<0.05) which may result from up-regulated fatty acid β-oxidation and down-regulated fatty acid synthesis, increased antioxidative abilities (SOD, CAT and GPx activities; TEAC levels), as well as elevated alcohol metabolic enzymatic activities (ALDH) (p<0.05). Taken together, our data suggested that pepsin-digested CLHs have the abilities to increase alcohol metabolism and antioxidative capacities, as well as regulate lipid homeostasis, further modulating inflammation and lessening alcohol-induced liver damage. Besides, the protective effects of CLHs were even better than silymarin.