2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658308摘要：非酒精性脂肪肝病(NAFLD)是全球性最常見的慢性肝疾病之一。NASH 的病程從單純的脂肪肝到非酒精性脂肪肝炎(NASH)、肝纖維化及肝硬化，甚至可能轉變成肝癌。單純的脂肪肝被認為是相對的良性而且是可逆轉的。但是 NASH 是較嚴重的狀況，會有肝細胞損傷、發炎以及肝纖維化，且有較高的機率轉變成末期肝病。但是，從單純的脂肪肝是如何會演變成 NASH 的機制到目前仍不甚清楚。NASH 與動脈硬化心血管疾病有一些共同的特點，它們都有脂肪蓄積、巨噬細胞活化和浸潤以及發炎的現象。最近的幾個動物實驗顯示，氧化低密度脂蛋白(oxLDL)可能與 NASH 的發生有關。而我們初步實驗結果顯示，倉鼠在餵養高脂高膽固醇(HFC)飼料之後，血漿總膽固醇、三酸甘油酯、LDL-C 及帶負電的 LDL(LDL(-)，血漿中的 oxLDL)都明顯增加，且有明顯的有 NASH 的發生。根據上述資料，我們推測倉鼠發生 NASH 可能與巨噬細胞吞噬oxLDL 有關，且倉鼠可能是探討 oxLDL 在 NASH 發展過程所扮演角色的一個動物模式。另外，我們初步的體外實驗結果顯示，oxLDL 會活化巨噬細胞 NF-κB 及 MAPKs，並引起 TNF-α, IL-1β及 IL-6 表現。進一步顯示 oxLDL 可能是引起發炎進而進展成NASH 的發炎因子。 在這個計畫我們擬以動物實驗及體外實驗，來探討 oxLDL 在 NASH 發展過程所扮演角色。在第一年，我們將建立 oxLDL 與 NASH 的關連性，將比較餵養相同 HFC 飼料相同時間的倉鼠及 C57BL/6 小鼠，之後分析血漿肝功能指數、脂蛋白分佈、LDL(-)濃度，肝臟切片以 HE 染色及膠質纖維染色觀察的病理變化，免疫染色偵測 α-SMA、CD68、oxLDL 及 ApoB 等。第二年，我們將以體外實驗探討 LDL(-)引起巨噬細胞的發炎反應。因為倉鼠相對的是小型的動物，不易取得足夠的血液以分離 LDL(-)。紐西蘭白兔將做為取得 LDL(-)的替代來源，白兔可提供較大量的血液，而且在 HFC 飼料飼養下，血漿也又相當高的 LDL(-)濃度。我們也測試過兔子的 LDL(-)與人的 LDL(-)都有類似的作用會引起巨噬細胞的發炎反應。因此在體外實驗我們將以不同濃度 LDL(-)處理來自肝臟或是骨髓的巨噬細胞，探討巨噬細胞 NF-κB 及 MAPKs 活化，及引起 TNF-α, IL-1β及 IL-6 表現的作用機轉及訊息路徑；並探討是那個清道夫受體(scavenger receptors)參與作用。 這個研究的結果將論證 oxLDL 是否直接參與 NASH 的發展，以及驗證倉鼠(比起小鼠)是否適合用於 NASH 的研究及將來治療方針的探討。這個研究的成果將有可能發表2 到 3 篇文章，在 NASH 的研究有所貢獻。<br> Abstract: Non-alcoholic fatty liver disease (NAFLD) is becoming the most common liver disease worldwide. NAFLD comprises a spectrum of disease ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), and may develop to fibrosis, cirrhosis and hepatocellular carcinoma. Simple steatosis is considered a relatively benign and is reversible; however, NASH is a more severe form of NAFLD in which accompanies with hepatocyte injury, inflammation and fibrosis, and may progress to end-stage liver disease. However, the mechanisms that trigger the development of NASH remain unclear. NASH shares many features in common with cardiovascular disease (CVD); both involve lipid accumulation, macrophage activation and infiltration and inflammation. Recent animal studies suggest that oxidized LDL (oxLDL) is a critical factor for the development of NASH. Our preliminary result showing that hamster develops atherogenic lipoprotein profile and has high level of negatively charged LDL (LDL(-), circulating oxLDL) in response to high fat/high cholesterol (HFC) diet feeding; and is readily developing NASH. Base on these, we speculate that, in hamster, the development of NASH may involve uptake of oxLDL by macrophages. And hamster may be a good animal model to study the role of oxLDL in the development of NASH. Our preliminary in vitro study showing that treatment with LDL(-) induces activation of NF-κB and mitogen-activated protein kinases (MAPK), and expression of TNF-α, IL-1β and IL-6. Therefore, it is very likely that LDL(-) is a factor that triggers inflammation and induces development of NASH. In this study, we plan an in vivo and an in vitro study to investigate the role of oxLDL in the pathogenesis of NASH. In the first year, we will establish the association between the levels of oxLDL and the development of NASH by comparing the development of NASH in C57BL/6 and golden Syrian hamster feeding a same HFC diet for 12 weeks. At the end of feeding, we will compare the plasma AST, ALT, LDL(-) and lipoprotein profile, and liver pathology by HE and Masson’s trichrome staining, and immunostaining of α-SMA, CD68, oxLDL and ApoB in liver sections. In the second year, we plan to investigate the effects of LDL(-) induced inflammation in macrophages. Because hamster is small and unable to provide enough blood for LDL(-) isolation. An alternative source of LDL(-) is HFC fed rabbit, which has high level of LDL(-) and can provide enough blood for LDL(-) isolation. Our preliminary study showing that rabbit LDL(-) also induced inflammation in macropahges similar to LDL(-) from other sources. In the in vitro study, we will investigate the expression of inflammatory cytokines and the involvement of NF-κB, MAPK and scavenger receptors in LDL(-) treated macropahges. The result of this study will demonstrate direct involvement of oxLDL in the development of NASH; moreover, will show if hamster model will be a useful model for the study of NASH and for future identification of therapeutic targets. The results of this study have potential to publish at 2 to 3 papers.