摘要:非酒精性脂肪肝疾病(NAFLD)盛行率日漸增加, 而且不再被認為是單純的良性疾病,而是會發展成肝纖維化及肝硬化的非酒精性脂肪肝炎(NASH)。我們正在執行的計畫成果已發表,我們提出證據顯示陰電性低密度脂蛋白(LDL(-))與NASH 的發生息息相關,體外實驗結果也顯示LDL(-)會引起肝巨噬细胞(Kupffer cell)分泌腫瘤壞死因子(TNF-α),抑制NF-κB 及阻斷LOX-1(LDL(-)受體)都會降低TNF-α 的產生。LDL(-)引起NASH 的機轉是重要,值得繼續深入研究,因此提出以下幾個研究主題。(一)我們的結果顯示LDL(-)引起肝巨噬细胞NF-κB 活化及發炎激素分泌,因此我們推論LDL(-)可能會影響肝巨噬细胞的型態分布,增加刺激免疫反應的M1 肝巨噬细胞及減少抑制發炎的M2 肝巨噬细胞。(二)我們發現以LDL(-)單獨Kupffercell 只引起少量的第一型介白素(IL-1β)分泌,單獨以油酸(Oleic Acid)處理Kupffer cell 幾乎沒有作用,但是當兩者一起處理Kupffer cell 則引起大量IL-1β 分泌。由於IL-1β 分泌需要發炎複合體(inflammasome)活化caspase-1,因此我們將探討LDL(-)與脂肪酸是如何引起caspase-1及IL-1β 分泌。(三)我們在高脂高膽固醇飼料餵食的倉鼠肝臟觀察到血管週邊有免疫細胞入侵的情形,推測肝臟血管內皮細胞可能會表現黏附分子(adhesion molecules, ICAM, VCAM)增加血液中免疫細胞的黏附及入侵。LDL(-)會增加動脈內皮細胞黏附分子的表現,因此我們推測LDL(-)也可能會增加肝內皮細胞表現黏附分子。(四)有一些植物化學成分被認為有抑制抗發炎及減輕NAFLD 的作用但不清楚是否有抑制LDL(-)引起的發炎反應,因此我們將探討一些植物化學成分對上述幾個LDL(-)引起的發炎反應的影響。(五)這些植物化學成分若有好的抑制發炎反應的作用,我們將選取其二以高脂高膽固醇飼料餵食的倉鼠NASH 評估其是否在活體內(in vivo)具有預防NASH 的效果。LDL(-)對Kupffer cell我們提出三年的研究計畫來探討以上的幾個主題,由於我們已建立高脂高膽固醇飼料餵食的倉鼠NASH 模式,且LDL(-)引起Kupffer cell 的發炎反應是最新的發現。因此我們認為這個計畫所提出的研究主題是嶄新具有研究價值的,而且我們具有執行完成這些研究的能力。
Abstract: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and itsworldwide prevalence continues to increase. Simple steatosis is usually a benign and reversiblecondition, but some may progress to non-alcoholic steatohepatitis (NASH). However, thepathogenesis of NASH is mostly unknown. Our current study demonstrated that electronegative LDL(LDL(-)) is highly associated with development of NASH in a high fat/cholesterol (HFC) diet fedhamsters model. Our in vitro study showed that LDL(-) induced production of TNF-α in Kupffer cells,and the effect can be blocked by inhibition of NF-κB or blocking LOX-1. These results are novel anddeserve further investigation. We intend to investigate the effects of LDL(-) on Kupffer cells andendothelial cells in the next 3 years. (1) We aim to investigate if LDL(-) can increasepro-inflammatory M1 and decrease anti-inflammatory M2 Kupffer cells. Because our results haveshown that LDL(-) can activate NF-κB and increases secretion of proinflammatory cytokines inKupffer cells and bone marrow derived macrophages, and thus speculate that LDL(-) may have aneffect on macrophage polarization. (2) We aim to investigate if LDL(-) and free fatty acid, both arerisk factors of NASH, may act synergistically to activate two signals and lead to secretion of IL-1β inKupffer cells. Because, in a preliminary study, we found that LDL(-) or oleic acid alone only slightlyinduced production of IL-1β in Kupffer cells; however, treatment with combination of both resultedin a remarkable production of IL-1β. Macrophages need two signals in order to produce IL-1β, onesignal is to induce transcription and the second signal is to induce caspase-1 for the processing ofIL-1β. Our data support the two-signal hypothesis for IL-1β production. (3) We aim to investigate ifLDL(-) may induce expression of adhesion molecules (such as, ICAM, VCAM) in liver sinusoidalendothelial cells. Because we observed massive immune cells infiltration in the liver portal andsinusoidal of HFC-diet fed hamsters. Since infiltration of these cells may involve expression ofadhesion molecules in endothelial cells, which has been demonstrated in the development ofatherosclerosis and can be happened in the development of NASH. (4) We aim to investigate if somephytochemicals able to inhibit LDL(-)-induced inflammation in liver Kupffer cells and sinusoidalendothelial cells. Some phytochemicals, such as quercetin, rutin, anthocyanin, curcumin, resveratrol,capsaicin, and catechins, exhibit anti-inflammatory effects; but it is not known if thesephytochemicals have anti-NASH effects. (5) Two phytochemicals with potent anti-inflammatoryeffects will be tested for their in vivo effects using the established HFC diet-induced NASH hamstermodel.The effects of LDL(-) on Kupffer cells and sinusoidal endothelial cells may contribute to thedevelopment of NASH, but has not been studied. This 3 y research proposal is based on ourestablished in vivo and in vitro systems, and is well planned and practicable. We believe we will beable to accomplish the proposed experiments.