基質金屬蛋白分解酶 9 與 Sirt1 在肝臟脂質代謝中所扮演之角色

Abstract

近年來，非酒精性脂肪肝 (non-alcoholic fatty liver disease, NAFLD) 已成為主要的慢性肝病之一。常見的非酒精性脂肪肝包含，脂質浸潤 (simple steatosis) 以及脂性肝炎 (non-alcoholic steatohepatitis, NASH)，且如果病程持續劣化，將有機會演變成肝硬化 (cirrhosis) 甚至肝癌 (hepatocellular carcinoma)。已知肝臟中失衡的脂質代謝是 NAFLD 的主要病因之一，因此透過了解肝臟中脂質代謝的調控，將有提供發展預防或治療 NAFLD 之方向。基質金屬蛋白分解酶9 (matrix metalloproteinase 9, MMP-9) 為截切胞外的基質之酵素，近年來被發現可能參與在肝臟脂質代謝之調控中，研究指出持續給予小鼠 MMPs 抑制劑 tissue metalloproteinase inhibitor 1 (TIMP-1)，其肝臟會有較多脂質堆積，除此之外，在MMP-9 基因缺失小鼠肝臟中，也被發現具有異常的脂質代謝相關基因表現，因此我們推論，MMP-9 可能在 NAFLD 的病程演變之中扮演重要角色。 為了證實此假說，本試驗利用 MMP-9 基因缺失 (Mmp-9-/-) 小鼠與肝癌細胞株 Hepa1-6 進行動物及細胞實驗。在動物實驗中，即便野生型 (wild type, WT) 與 Mmp-9-/- 小鼠在體重上並沒有顯著差異，qPCR 與 western bolt 實驗結果顯示，Mmp-9-/- 小鼠肝臟中具有較低的 Sirt1 表現量，與較高的 Cd36 表現。Cd36 為肝細胞膜上負責攝入脂肪酸之運輸蛋白，而其表現受到去乙醯基酶 (deacetylase) Sirt1 的抑制，因此我們推論 MMP-9 缺失所造成的肝臟脂質堆積可能是藉由減少肝臟中 Sirt1 的蛋白質含量並間接增加 Cd36 的表現所導致。而透過細胞實驗得知，單只減少肝癌細胞株 Hepa1-6 內的 MMP-9 表現量，便可以看到脂質堆積與 Sirt1 的減少、Cd36 的增加的現象。 綜上所述，本試驗證實 MMP-9 確實參與在肝臟脂質代謝的調控過程，且其可能透過 Sirt1/Cd36 路徑來影響肝臟脂質代謝，然而，MMP-9 是如何影響 Sirt1 與 MMP-9/Sirt1/Cd36 路徑在 NAFLD 病程中所扮演的角色，仍需更多實驗來釐清。

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in recent years. NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis (NASH), which can further develop into cirrhosis and hepatocellular carcinoma. Because the dysregulation of hepatic lipid metabolism is currently thought to be the major cause of NAFLD, it is important to clarify how hepatic lipid metabolism was regulated during NAFLD. Matrix metalloproteinase 9 (MMP-9), an extracellular matrix degrading enzyme, was recently found to be involved in regulation of hepatic lipid metabolism. It was found that tissue inhibitor of metalloproteinase 1 (TIMP-1) treatment increases mouse liver TG content. Besides, altered expression pattern of lipid metabolism-related genes was found in MMP-9 deficient mice. Based on these findings, we hypothesized that MMP-9 may play an important role during NAFLD development. To confirm our hypothesis, we conducted both in vivo and in vitro experiments using MMP-9 knockout mice and Hepa1-6 cell line respectively. Our in vivo results showed that although the body weight was not changed, MMP-9 knockout increased the plasma and hepatic triglyceride level in mice. Importantly, our Western blots and qPCR data showed that MMP-9 knockout significantly decreased the protein level of Sirt1 and increased Cd36 mRNA expression in liver. Sirt1 was previously found to inhibit Cd36, which is a long chain fatty acids uptake protein, mRNA expression as a NAD-dependent deacetylase. These findings suggest that MMP-9 knockout may cause hepatic steatosis by reducing Sirt1 protein expression and indirectly increasing Cd36 expression. Similarly, we found that siRNA mediated MMP-9 knockdown increased lipid accumulation and decreased Sirt1 protein level in Hepa1-6 cells. Taken together, our study has confirmed the regulatory function of MMP-9 in hepatic lipid metabolism. In addition, our data also indicated Sirt1 as a possible downstream target of MMP-9, which may be important in MMP-9 regulated hepatic lipid metabolism. However, the role of MMP-9/Sirt1/Cd36 in NAFLD development still needs more experiments to be clarified.