機能性繫帶水解物對於硫代乙醯胺誘導大鼠肝損傷之保護功效

Abstract

根據世界衛生組織於2015年的統計，肝癌在癌症致死人數中排名第二，大量文獻指出，氧化壓力在許多肝臟疾病中皆扮演了重要的角色。繫帶為液蛋產業所產生的副產物之一，根據分析結果發現，繫帶經protease A水解後所產生的疏水性及芳香族胺基酸與甲肌肽在抗氧化方面具有相當的潛力，另外所產生的支鏈胺基酸對於一些肝臟疾病有減緩的功效。因此，本研究目的為探討經protease A水解後之繫帶水解物(protease A-digested crude chalaza hydrolysates, CCH-As)對硫代乙醯胺(thioacetamide, TAA)於體內代謝時產生大量過氧化物造成的大鼠肝損傷下是否具有減緩發炎及纖維化的效果。經研究結果顯示，TAA處理會導致大鼠體重及飼料效率降低(p<0.05)，且提升(p<0.05)血清中的肝臟受損指標如ALT、AST及ALP。此外，以切片結果觀察肝臟組織後可以發現，TAA處理會產生肝臟中免疫細胞浸潤及膠原蛋白堆積的現象，其中與發炎相關(TNF-α及IL-1β)及纖維化相關(TGF-β1)的細胞因子在肝臟中皆顯著上升(p<0.05)。但是在補充CCH-As後，相較於TAA組，大鼠體增重及飼料效率顯著提升(p<0.05)，血清中ALT、AST及ALP，以及肝臟中TNF-α、IL-1β及TGF-β1皆顯著降低(p<0.05)。同時CCH-As也負調控了tgfβ1基因的表現量(p<0.05)，進而抑制了下游基因colα1(I)及蛋白質α-SMA的表現量(p<0.05)，使得肝臟中細胞外基質的堆積現象減緩。此外CCH-As也降低(p<0.05)了MMP-2及MMP-9活性的表現量，進而阻斷非正常細胞外基質的增生。另外，CCH-As的補充顯著提升(p<0.05)了肝臟中的抗氧化酵素活性(SOD及GPx)、還原性GSH的含量，降低(p<0.05)脂質過氧化指數(TBARS value)，因而降低了由TAA誘發所產生之氧化壓力，改善肝臟中發炎及纖維化之反應。此外，CCH-As亦顯著增加(p<0.05)肝臟中cleaved caspase-3及cleaved PARP的蛋白質含量，推測此可能加速損傷肝細胞進行細胞凋亡以進行更替。綜觀上述結果，CCH-As對氧化壓力誘導肝損傷甚至纖維化具有改善的效果。

World Health Organization (2015) reported that liver cancer ranked second among all cancer categories worldwide. In most liver diseases, it is undoubted that an oxidative stress plays an important role on the pathological development. Chalazae produced from the liquid-egg industry are always discarded. After protease A hydrolyzation, free or hydrolytic hydrophobic, aromatic, and branched chain amino acids, as well as the free dipeptide, anserine were sharply enhanced compared with those of raw lyophilized chalazae. In addition, we would like to investigate if CCH-As can ameliorate oxidative-stress induced liver fibrosis by thioacetamide (TAA) treatment. TAA treatment lowered (p<0.05) body weight increases and feed efficiencies of rats. However, higher (p<0.05) serum ALT, AST, and ALP levels in TAA-treated groups were also assayed. The immune cell infiltration and more collagen deposition were illustrated in the livers of TAA-treated group compared to that of no TAA-treated group (control group) via the observation of H&E and Sirius red stainings. Additionally, the contents of inflammatory (TNF-α and IL-1β) and fibrogenic (TGF-β1) cytokines in livers of TAA-treated group were higher (p<0.05) than those of control group. The upregulated (p<0.05) gene expression of tgfβ1 caused by the TAA treatment further led to an enhancements (p<0.05) of colα1(I) gene and α-SMA protein expressions. Moreover, MMP-2 and MMP-9 activities were increased (p<0.05) by TAA treatments which indicated an abnormal degradation on extracellular matrix (ECM) in livers. Those abnormal growth performance, serum liver damage indices, histopathological liver inflammatory and collagen deposition evidences were ameliorated (p<0.05) by our CCH-As. Additionally, the cytokine, gene, and protein expressions associated with inflammation, fibrogenesis, and abnormal ECM degradation were also improved (p<0.05) by CCH-As which is highly related to an increased (p<0.05) SOD and GPx activities, as well as reduced GSH levels, and lower (p<0.05) TBARS values. Besides, CCH-A supplementation accelerated apoptosis process by increasing (p<0.05) cleaved caspase-3 and cleaved PARP protein levels in liver cells of TAA-treated rats. Taken together, the results in this study offered that CCH-As show an ameliorative effect on TAA-induced liver damages.