動脈粥狀硬化始於血管內皮細胞的失調，誘發單核球進入血管壁並分化成巨噬細胞，巨噬細胞吞噬氧化態低密度脂蛋白，促使發炎反應的產生，進而造成動脈硬化斑塊生成。MicroRNAs (miRs) 會與標的mRMA結合，使mRNA降解或阻斷蛋白質轉譯，影響內皮細胞、平滑肌細胞及巨噬細胞的功能。近年miRs在動脈粥狀硬化的發炎反應扮演的角色逐漸被研究， miR-146a會降低巨噬細胞內由氧化低密度脂蛋白所誘發的脂質累積和在不同細胞中抑制發炎反應。由於動脈粥狀硬化症的發展過程由多種細胞參與，推測miR-146a在不同細胞中，藉由不同標的mRNA調控動脈粥狀硬化的發展。因此，本實驗以miR-146a+/+ ApoE-/-及miR-146a-/- ApoE-/-動脈硬化小鼠模式探討miR-146a缺失對小鼠動脈粥狀硬化發展的影響。經餵食高膽固醇飼料十周後，miR-146a+/+ ApoE-/-與miR-146a-/- ApoE-/-小鼠相比，體重與血漿中三酸甘油脂顯著地增加，高密度脂蛋白的含量卻減少。血管內動脈硬化斑塊的數目與大小顯著地增加，且斑塊中巨噬細胞的組成也顯著地增多。為了釐清miR-146a在巨噬細胞內的角色，分離miR-146a+/+ ApoE-/-及miR-146a-/- ApoE-/-小鼠巨噬細胞，以氧化態低密度脂蛋白刺激， miR-146a-/- ApoE-/-小鼠的巨噬細胞，細胞內累積的脂質顯著比miR-146a+/+ ApoE-/-小鼠的巨噬細胞多。測量巨噬細胞表面感測脂蛋白的接受器，以及與膽固醇流出相關蛋白的表現量，發現miR-146a會提高巨噬細胞表面蛋白- ATP-binding cassette transporter A1 (ABCA1) 及G1(ABCG1)的表現量，使細胞內膽固醇流出的能力提升，進而使巨噬細胞內累積的膽固醇下降。我們也發現miR-146a是透過其標的基因Toll-like receptor 4(TLR4)影響轉錄因子Liver X receptors (LXRs)所調控的ABCA1及ABCG1的表現量。實驗結果顯示，小鼠中miR-146a的缺失會影響血清中的生化值，並且會促進動脈粥狀硬化進程。在巨噬細胞內，大量表現miR-146a會抑制TLR4表現量，進而上調控ABCA1及ABCG1的表現量，使細胞內累積的脂質下降，以及增進膽固醇流出細胞的能力，進而減緩動脈粥狀硬化進程。

Atherosclerosis starts with endothelial cells dysfunction. Various adhesion molecules recruit monocytes migrate into the vessel wall and differentiate into macrophages. Subsequently, macrophages uptake oxLDL and drive inflammatory response in atherosclerotic plaques. MicroRNAs (miRs) control the function of endothelial cells, smooth muscle cells and macrophage by targeting mRNA degradation or translation. Recent years, the roles of miRs in inflammatory response during atherosclerosis have been discovered. MiR-146a has been demonstrated to reduce the cholesterol levels, and oxidized low density lipoprotein-induced inflammatory response in macrophage. MiR-146a may have different functions in vascular cells by targeting mRNA to reduce the progression of atherosclerotic. In the present study, we assessed the impact of the genetic loss of miR-146a in a mouse model of atherosclerosis and elucidated the mechanism of miR-146a in regulation of atherosclerosis. We studied the mechanism of miR-146a in ApoE-/- mice which is an experimental model of atherosclerosis. In comparison with miR-146a+/+ ApoE-/- mice fed with high cholesterol diet for 10 weeks , miR-146a-/- ApoE-/- mice showed an increase number and area of atherosclerotic plaque, lipid content and abundant macrophages accumulated in the plaque. In addition, miR-146a-/- ApoE-/- mice also showed an increase in circulating triglyceride and decrease in HDL levels compared with miR-146a+/+ ApoE-/- mice. To elucidate the roles of miR-146a in macrophages, bone marrow derived-macrophages were isolated from miR-146a-/- ApoE-/- and miR-146a+/+ ApoE-/- mice. In comparison with macrophage of miR-146a+/+ ApoE-/-, macrophages of miR-146a-/- ApoE-/- showed a significant oxidized LDL uptake and reduction in cholesterol efflux capacity The expression of cholesterol transporters, including the ATP-binding cassette sub-family A1 (ABCA1) and sub-family G1 (ABCG1) were decreased in miR-146a-/- ApoE-/- macrophages. We demonstrated that toll like receptor 4 (TLR4) is directly targeted by miR-146a. Increase and activation of TLR4 by LPS blocked the liver X receptors target ABCA1 and ABCG1 expression. Overexpression of miR-146a and silence of TLR4 increased the cholesterol efflux capacity and reduced the oxidized LDL uptake. These data demonstrated that TLR4 is involved in miR-146a-mediated regulation of ABCA1 and ABCG1 expression, and miR-146a reduces the progression of atherosclerotic plaque by increasing ABCA1 and ABCG1 protein.

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