非酒精性脂肪肝雞隻模式與生物指標之建立與應用於篩選改善非酒精性脂肪肝之功能性營養成分

Abstract

隨著隨著肥胖盛行率的增加，非酒精性脂肪肝病 （non-alcoholic fatty liver disease, NAFLD） 已是世界上最常見的肝臟疾病。雖然非酒精性脂肪肝並無明顯病癥且具有可逆轉性，但隨後衍生之非酒精脂肪肝炎 (non-alcoholic steatohepatitis , NASH) 已被指出約有10-29%的機率會在十年內發展為不可逆轉性地肝纖維化與硬化 （liver fibrosis and cirrhosis）， 導致肝功能喪失，甚至發展成肝癌 （hepatocellular carcinoma, HCC）。目前除了侵入性之肝臟切片檢查能作為非酒精性脂肪肝之確切診斷依據，仍未有可作為診斷非酒精性脂肪肝病之準確血液生物標記。 因此，發展適用之動物模式與可應用之簡易診斷方法當前所迫需。 由於雞隻與人類同樣使用肝臟作為脂質生合成的主要器官，但雞隻並未有相應的脂肪肝或肝臟損傷之生物性指標。因此本研究利用雞隻作為非酒精性脂肪肝病之動物模式，並應用轉錄體定序 (Transcriptome sequencing) 與蛋白質體學（Proteomics）尋找能有效應用之非酒精性脂肪肝生物性指標。研究結果顯示，雞隻成功發展出非誘導式的非酒精性脂肪肝病，且運用這些雞隻成功篩選出四個具有成為非酒精性脂肪肝病指標潛力之血漿蛋白質。包含acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL) 以及glutathione S-transferase (GST)。並且藉由添加具有保護肝臟肝臟功能之甜菜鹼 (Betaine) 或二十二碳六烯酸 （Docosahexaenoic Acid, DHA） 於雞隻飼養或初代雞隻肝臟細胞培養試驗中，發現生物性指標之表現量與雞隻或肝臟細胞脂肪堆積量成正比，也從盲測的檢驗中驗證所篩選出的脂肪肝指標確實具有成為雞隻非酒精性脂肪肝病模式生物標記之特性。 接著使用四氯化碳誘導雞隻肝纖維化，並在雞隻飲用水中添加甜菜鹼以逆轉誘導之肝纖維化，藉此探討此四個生物標記是否可用來測定肝纖維化。實驗結果顯示，血漿中的DPP4與GST濃度伴隨肝纖維化而提升，此外，甜菜鹼可顯著抑制四氯化碳誘發之肝纖維化，血漿中的DPP4與GST的濃度也隨肝纖維化的抑制而減低。縱合上述兩個實驗，AACS, DPP4, GLUL以及GST除可作為雞隻非酒精性脂肪肝病之生物標記，DPP4與GST還可被應用於測定肝臟纖維化的血液指標；因此，我們認為合併這四個蛋白質的測定，應可幫助診斷時分辨非酒精性脂肪肝病與肝臟纖維化。 由於肝臟具有極高的再生能力，患有肝硬化與肝癌之患者常以切除部分肝臟作為治療的手段。在本研究中另外探討ADP-ribosylation factor 6 (Arf6) 以及Phosphatidylinositol 4-phosphate 5-kinase A (PIP5K1A)在肝臟再生中扮演的角色。前人研究顯示剔除Arf6影響小鼠胚胎肝臟發育並導致小鼠胚胎死亡，另外曾抑制肝臟生長因子(Hepatocyte growth factor, HGF) 誘導之胚胎肝臟細胞網狀組織的形成（Hepatic cord formation），此結果顯示Arf6在HGF的訊息傳遞中扮演著重要角色。藉由剔除HepG2細胞中之Arf6，Arf6在HGF誘導之HepG2細胞增生，以及傳遞細胞增生訊息之AKT的磷酸化扮演正向調控的角色；另外， Arf6能透過recruitment of PIP5K1A (PIP2 producing enzyme) 至HGF受體c-Met，進而促進HGF誘導之AKT磷酸化。由於HGF是肝臟再生時主要被肝臟分泌的生長激素，因此我們應用PIP5K1A剔除小鼠與部分肝切除手術（2/3 Partial heaptectomy）以探討PIP5K1A在活體小鼠肝臟中所扮演的功能。結果顯示PIP5K1A剔除小鼠其肝臟再生能力與肝臟細胞增殖能力受到抑制。綜合以上，在HGF 誘發訊息傳遞中Arf6-PIP5K1A-PIP2-AKT在肝臟細胞增殖與肝臟再生中扮演重要角色。

With the high prevalence of obesity, non-alcoholic fatty liver disease (NAFLD) becomes one of the most common liver diseases in the world. Although, the NAFLD is a non-viral and reversible disease, it tends to progress from simple steatosis to nonalcoholic steatohepatitis (NASH), then to irreversible fibrosis and cirrhosis, eventually even to hepatocellular carcinoma (HCC). Up to date, there is no reliable biomarkers used to identify NAFLD, and further discern NAFLD, NASH, and liver fibrosis progression. Invasive and costly liver biopsy remains a standard procedure to diagnose and stage NAFLD. Since the liver account for most pf lipogenesis in both human and chicken, chicken is suggested as a suitable model for studying human NAFLD. However, there is no appropriate NAFLD biomarker fin the chickens. Therefore, the aim of this study is to identify the potential non-invasive plasma biomarkers is the chicken NAFLD model. In the study, laying hens progressed into NAFLD spontaneously with age. By using transcriptomic and proteomic analysis, four plasma proteins were identified as potential biomarkers including, acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL) and glutathione S-transferase (GST). The potential of these proteins as biomarkers for NAFLD were confirmed further by feeding chickens with a diet containing hepato-protective nutrients, betaine or DHA, the diet and by supplementing the nutrients into the medium of chicken primary hepatocytes. Results showed that both betaine and DHA supplementations suppressed the development of fatty liver, and both transcript and protein levels of the selected biomarkers showed positive correlation with the degrees of hepatic lipid accumulation. Thus, these results verify the potential of these proteins as NAFLD biomarkers. To examine whether these biomarkers as suitable for diagnosing liver fibrosis, chickens were induced for liver fibrosis by carbon tetrachloride (CCl4) to induce liver fibrosis. Betaine supplementation in drinking water was used to exam its amelioration on CCl4-induced liver fibrosis. Results suggested of hepatic mRNA and plasma DPP4 and GST level were elevated in liver fibrosis group, and betaine supplementation inhibited the development of liver fibrosis with lower plasma DPP4 and GST concentrations. Accordingly, these four biomarkers were concluded as effective biomarker to diagnose NAFLD in chickens. Among the organs liver has a high regenerative capacity. A surgical removal of a portion of the liver is one of treatment strategies for patients with liver cirrhosis or HCC. The study also aimed to investigate the role of ADP-ribosylation factor 6 (Arf6) and phosphatidylinositol 4-phosphate 5-kinase A (PIP5K1A) in liver regeneration. Previous studies have showed that knockout of Arf6 gene cause embryonic lethality due to defect liver development in mice, and Arf6 is required for hepatocyte growth factor (HGF)-dependent formation of hepatic cord formation, which indicates the essential role of Arf6 to mediate HGF signaling. HGF is known as a critical and major growth factor to induce liver regeneration. Using Arf6 knockdown approach, we showed that Arf6 mediates HGF-stimulated hepatocyte proliferation and AKT phosphorylation by recruiting of PIP2-producng enzyme, PIP5K1A to HGF receptor (c-Met) in HepG2 cells. To validate the observation in in vivo models, Pip5k1a knockout mice with 2/3 partial hepatectomy were used to investigate the role of PIP5K1A in liver regeneration. A significant inhibition of liver regeneration and hepatocyte proliferation in Pip5k1a knockout mice were observed. These results suggest the essential role of Arf6-PIP5K1A-AKT to mediate HGF signaling in hepatocyte proliferation to liver regeneration process. In conclusion, transcriptomic and proteomic studies, AACS, DPP4, GST and GLUL were concluded as potential biomarkers for NAFLD and DPP4 and GST for liver fibrosis in the chicken models. Both betaine and DHA supplementation in chicken diet effectively ameliorated NAFLD and liver fibrosis induced by CCl4. In a separate study, Arf6-PIP5K1A-AKT cascade was conclude to mediate HGF signaling transduction in liver regeneration. These findings may shed new light to reveal the molecular mechanism of liver regeneration, and provide a potential diagnostic markers and therapeutic strategies for treating liver diseases.