陰電性低密度脂蛋白在高油高膽固醇飼料引起之倉鼠非酒精性脂肪肝炎之可能角色

Abstract

非酒精性脂肪肝是非酗酒過量或病毒感染或特定醫療行為引起之肝臟累積過量脂肪之疾病，而非酒精性脂肪肝有機會進一步進展成較惡化的階段，稱之為非酒精性脂肪肝炎。非酒精性脂肪肝炎的特徵除了肝臟脂肪堆積之外，還有肝發炎、肝損傷與肝纖維化等症狀。動脈粥狀硬化亦可發現類似的現象，包含脂質沈積於血管上、巨噬細胞入侵引起發炎以及平滑肌細胞產生纖維化。目前對於非酒精性脂肪肝如何進展到非酒精性脂肪肝炎的機制並無定論，較被廣為接受的理論為「雙衝擊假說」，然而詳細機制仍不清楚。近年來許多研究指出氧化低密度脂蛋白可能是進展至非酒精性脂肪肝炎的危險因子之一，然而這些研究並無直接偵測到氧化低密度脂蛋白含量。陰電性低密度脂蛋白為一群帶有負電荷的輕微修飾之低密度脂蛋白，研究指出心血管疾病、代謝症候群與高膽固醇血症患者血中具有高量的陰電性低密度脂蛋白，然而仍未有研究指出陰電性低密度脂蛋白是否參與非酒精性脂肪肝炎的病程進展。因此本研究論文旨在利用高油高膽固醇飲食誘發之非酒精性脂肪肝炎動物模式探討陰電性低密度脂蛋白在非酒精性脂肪肝炎進展中所扮演的角色。將敘利亞倉鼠與C57BL/6小鼠餵食相同的高油高膽固醇飼料12週後發現此兩品系動物皆累積相當的脂肪於肝臟中。然而病理分析顯示倉鼠發展出較惡化的非酒精性脂肪肝炎，其中包含小泡狀脂肪堆積、肝細胞氣球狀變異、免疫細胞浸潤與橋狀纖維化；而小鼠僅發展出輕微的肝發炎反應與混合型脂肪堆積。此外，高油高膽固醇飲食餵食下，倉鼠顯著增加血漿中低密度脂蛋白膽固醇及陰電性低密度脂蛋白含量，而小鼠僅些微提高，倉鼠陰電性低密度脂蛋白含量更是比小鼠高出16倍。除此之外，我們亦發現高油高膽固醇餵食之倉鼠肝臟中含有大量具ApoB之脂蛋白滯留於肝竇狀隙與肝門靜脈周邊，此區域聚集了多數巨大且含高量游離膽固醇之柯佛氏細胞。進一步研究發現這些含ApoB之脂蛋白主要為陰電性低密度脂蛋白，至此顯示倉鼠肝臟病理變化類似於動脈粥狀硬化初始階段，而此現象在小鼠中並無觀察到。此外，體外實驗顯示自倉鼠或兔子分離之陰電性低密度脂蛋白可分別誘使大鼠原代柯佛氏細胞與小鼠骨髓細胞分化而成的巨噬細胞產生腫瘤壞死因子-α。進一步預處理類凝集素氧化低密度脂蛋白受器-1 （LOX-1）封阻抗體TS92或同時處理IκBα抑制劑BAY-11-7082則顯著降低腫瘤壞死因子-α於大鼠原代柯佛氏細胞的表現。體外實驗的結果顯示陰電性低密度脂蛋白透過與LOX-1並活化NF-κB而促使大鼠柯佛氏細胞產生發炎反應進而造成非酒精性脂肪肝炎。本論文研究結果提供一個直接證據顯示陰電性低密度脂蛋白為造成肝發炎的危險因子之一且在非酒精性脂肪肝炎發展中扮演重要角色。

Nonalcoholic fatty liver disease (NAFLD) is defined as the presence of fat storage in the liver without abuse alcohol, virus infection or certain medications. NAFLD can further progress into the progressed stage called nonalcoholic steatohepatitis (NASH). NASH is considered as the presence of hepatic steatosis and inflammation with hepatocyte injury, hepatic fibrosis is also present in advanced stage of NASH, which is like that of atherosclerosis. The transition from NAFLD to NASH can be explained by the two hit hypothesis, but the mechanisms remain unclear. Recent studies suggest that oxidized low density lipoprotein (oxLDL) is a potential risk that triggers progression from simple steatosis to NASH. However, oxLDL has not been directly measured or detected in these studies. Electronegative low density lipoprotein (LDL(-)) is a pool of minimal modified LDL found in patients with cardiovascular disease, metabolic syndrome and hypercholesterolemia, but its relationship with NASH has not been established. The aim of this study was to examine the role(s) of LDL(-) in the development of NASH in a high fat high cholesterol (HFC) diet induced animal models. Golden Syrian hamsters and C57BL/6 mice were fed with the same HFC diet for 12 weeks. Both species accumulated considerably amount of lipid in liver, but the pathology was much worsen in hamsters. Hamsters developed advanced NASH features including microvesicular steatosis, hepatocyte ballooning degeneration, lobular inflammation and bridging fibrosis, whereas mice developed mixed type of steatosis with mild inflammatory cell infiltration. HFC diet also increased high level of plasma LDL cholesterol in hamsters, but only a slight increase in mice. In LDL fraction, LDL(-) was 16-fold higher in hamsters than in mice. Moreover, massive amount of ApoB containing lipoproteins were retained in sinusoid and portal area in the liver of HFC diet-fed hamsters accompanied with enlarged Kupffer cells accumulated with unesterified cholesterol. Further analysis revealed that the majority of retained ApoB containing lipoproteins are LDL(-), this observation was similar to the initiation stage of atherosclerosis. On the other hand, this phenomenon was not found in mice fed with HFC diet. In addition, in vitro studies showed that LDL(-), from HFC diet-fed hamsters and rabbits, induced TNF-α production in primary rat Kupffer cells and bone marrow derived macrophages. Furthermore, pretreatment of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) blocking antibody, TS92, or co-treatment of BAY-11-7082, an inhibitor of IκBα kinase, significantly decreased TNF-α secretion in rat Kupffer cells, which suggest that LDL(-) trigger Kupffer cell activation during NASH development through NF-κB dependent pathway by binding to LOX-1. These results provide a direct evidence to show that LDL(-) is one of risk factor in hepatic inflammation and play a critical role in the development of NASH