基質金屬蛋白分解脢9在非酒精性脂肪肝病中所扮演之角色

The Role of MMP-9 in Non-alcoholic Fatty Liver Disease

黃裕鍇(YU-KAI, HUANG)

指導教授 ： 邱智賢 ； 吳兩新

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10807

摘要

近年來，非酒精性脂肪肝病(Non-alcoholic fatty liver disease, NAFLD)已經成為主要且常見之慢性肝病。常見之非酒精性脂肪肝病包括脂質浸潤(Steatosis)以及脂性肝炎(Non-alcoholic steatohepatitis, NASH)，雖目前對於NAFLD之致病機轉仍未明朗，近年已有文獻顯示，基質金屬蛋白分解脢 9(Matrix metalloproteinase 9, MMP-9可能參與其中。以往，MMP-9蛋白僅被認為具有酵素截切活性而與胞外基質(Extracellular matrix, ECM)之代謝與持恆有關；然而，最近之研究指出，MMP-9 亦可以其獨有之Hemopexin domain與細胞膜上特定受體結合併進而影響細胞行為。為了解MMP-9蛋白於NAFLD病程中所扮演之角色與其影響，同時進行了in vivo與in vitro試驗以釐清二者之關係。
在In vivo試驗中,以高脂飼糧(60%能量來自脂肪)餵飼12周齡之MMP-9基因剔除與C57BL/6野生型小鼠達15周。餵飼結束後，包含肝重比、肝切片以及肝臟三酸甘油脂定量分析之結果皆顯示MMP-9基因剔除會顯著增加肝臟油滴堆積，導致NAFLD病程更為嚴重。此外，與肝臟脂質相關調控機轉，如：脂質攝入 (Cd36, Ldlr, and Lrp1) 與脂質生合成 (Fasn and Dgat1) 皆因MMP-9基因剔除而顯著增加；更進一步地，與脂質合成相關之轉錄因子 (Pparg and Srebp1c) 亦因MMP-9基因剔除而顯著增加。此一結果證實了MMP-9基因剔除藉由改變脂質代謝途徑而使肝臟大量堆積油滴，導致NAFLD病程更為嚴重。
為分辨對於肝臟脂質代謝途徑之調控是藉由MMP-9蛋白分解ECM所間接調控或是MMP-9蛋白與受體結合所調控，共處理兩種不同MMP-9蛋白抑止劑與油酸於FL83B肝細胞株以檢驗不同抑止劑對油滴堆積之影響。結果顯示，僅只有抑制ECM分解之抑止劑I顯著地增加了油酸誘導之油滴堆積現象；與其相反，MMP-9蛋白結合受體之抑止劑II並無類似效果。
在我們的研究中，MMP-9失去酵素截切活性會改變脂質代謝途徑並顯著地增加肝臟脂質堆積導致NAFLD病程更為嚴重，這一結果提供了在治療NAFLD病程中之可能標的，然而，MMP-9蛋白與肝臟脂質代謝途徑之詳細調控機轉仍須更多試驗釐清。

關鍵字

非酒精性脂肪肝病 ； 脂質浸潤 ； 油滴堆積 ； 基質金屬蛋白分解酶9 ； 酵素截切活性

並列摘要

Non-alcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in recent years. NAFLD ranges from hepatic steatosis to non-alcoholic steatohepatitis (NASH), which can further progress into irreversible liver damages. Although the molecular mechanisms of NAFLD progression are still uncertain, recent studies suggest the matrix metalloproteinase 9 (MMP-9) is involved. In past, MMP-9 has been known as a proteinase can only degrade extracellular matrix (ECM), especially gelatin. However, recent studies reveal, MMP-9 protein can be a ligand binding to the specific receptor by its unique PEX domain to alter the cell behaviors. To study the role of MMP-9 in NAFLD, we conducted both of in vivo and in vitro experiments.
In vivo, 12-week-old C57BL/6 wild type and MMP-9 knockout mice (catalytic domain mutated) were fed with control or high fat diet (60% of energy from fat) for 15 weeks. After high fat diet challenge, the results of liver to body weight ratio, histological section and TG assay (p < .05) indicated that MMP-9 knockout would significantly aggravate the NAFLD progression. Besides, mRNA expression of genes related to hepatic lipid metabolism were analyzed by quantitative PCR. Significant upregulation of genes involved in lipid uptake (Cd36, Ldlr, and Lrp1) and lipogenesis (Fasn and Dgat1) was found in MMP-9 knockout mice (p < .05); moreover, the expression of transcription factor related to lipid anabolism (Pparg and Srebp1c) was also increased (p < .05). These data confirmed the involvement of MMP-9 in hepatic lipid metabolism and the progress of NAFLD.
To clarify the effect of MMP-9 in lipid metabolism is regulated by proteolytic domain or by PEX domain, we performed the model of oleic acid (OA) induced lipid accumulation in FL83B hepatocyte cell and co-treated with two different inhibitors in vitro. The results showed the proteolytic domain inhibition would significantly increase the level of OA induced lipid accumulation (p < .05); in contrast, PEX domain inhibition had no similar effect.
Our findings suggest that MMP-9 knockout enhances the expression of genes related to lipid accumulation to aggravate the NAFLD progression. Furthermore, this kind of regulation is proteolytic activity dependent. These results provide us novel insight into development of NAFLD therapy. However, whether MMP-9 participate in this metabolic regulation via ECM remodeling or degradation of other protein targets needs further studies to be elucidated.

並列關鍵字

NAFLD ； Steatosis ； Lipid accumulation ； MMP-9 ； Proteolytic activity

參考文獻

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