去乙酰化酶SIRT1在血管内皮细胞氧化低密度脂蛋白代谢中的作用及机制研究

The accumulation of oxidized lipid in the vascular endothelial cell is one of the key events occurred in the development of atherosclerosis. Up to now, there still have no good method to prevent and reverse this pathological accumulation of oxidized lipid. Lectin-like oxidized low density lipoprotein ( ox-LDL ) receptor-1 (LOX-1) is a kind of ox-LDL receptor and is believed to be the main receptor for ox-LDL. The aggregation of ox-LDL in vaslular endothelial cell is associated with the increase of LOX-1-mediated uptake and decrease of degradation of oxidized LDL by endothelial cell.How to prevent this pathological process is important for the prevent and cure of atherosclerosis.Autophagy is a potential drug target for atherosclerosis plaque stabilization.We firstly found that the autophagy/lysosome pathway is involved in the degradation of ox-LDL, thus playing a protective role in the ox-LDL-induced injury of human umbilical vein endothelial cell.SIRT1 is a NAD+-dependent class Ⅲ histone deacetylase that mediates the effects of caloric restriction on lifespan and metabolic pathways in various organisms. SIRT1 activation is a promising therapeutic approach in atherosclerosis, which can regulate inflammatory processes,suppresses the expression of endothelial tissue factor and hence exerts anti-thrombotic properties. It is reported recently that deacetylation of relA/p65-NF-kB by SIRT1 in macrophages suppresses the expression of LOX-1, thereby preventing macrophage foam cell formation.SIRT1 deacetylase also is an important regulator of autophagy. However, whether SIRT1 also plays a role in the metabolism of oxidized lipid by vascular endothelial cell is still unknown. Thus, we next investigate the role of SIRT1 in the accumulation of ox-LDL in vascular endothelial cell, and to probe the effect of SIRT1 on the autophagy/lysosome pathway and the LOX-1 mediated endocytosis of ox-LDL in this process. Experimental content are as follows: Using many mathods, such as,immunofluorescence, western blot and et al, we examine: 1.the accumulation of ox-LDL in vascular endothelial cell; 2.the LOX-1-mediated endocytosis of ox-LDL by vascular endothelial cell;3.the activation of the autophagy/lysosome pathway,and the colocalization of Dil-labled-ox-LDL with the autophagy/lysosome related proteins.This study is important for a better understand of the exact role of SIRT1 in the development of atherogenesis.

血管内皮细胞（VEC）氧化脂质的异常聚集是动脉粥样硬化进程中的重要环节。LOX-1介导的细胞摄取增多及入胞后的降解障碍是氧化低密度脂蛋白（ox-LDL）在VEC聚集的主要机制。我们首次发现上调自噬能够促进ox-LDL降解。VEC SIRT1是一种关键的抗AS调节因子，最近研究表明SIRT1能下调巨噬细胞LOX-1表达，促进自噬。然SIRT1对VEC氧化脂质代谢的影响尚未阐明。因此本课题将在此基础上，运用荧光免疫、流式细胞术等方法，在细胞和整体动物水平检测：1.VEC ox-LDL含量；2. LOX-1介导的ox-LDL细胞结合、转运；3.自噬/溶酶体标志蛋白的表达及与Dil-ox-LDL的细胞共定位，研究SIRT1对ox-LDL在VEC聚集的影响，探讨该作用是否与其抑制LOX-1介导的入胞，促进ox-LDL入胞后的自噬性降解有关。这对全面评估SIRT1的血管保护作用及新药研发具有重要意义。

血管内皮细胞（VEC）氧化脂质的异常聚集是动脉粥样硬化进程中的重要环节。LOX-1 介导的细胞摄取增多及入胞后的降解障碍是氧化低密度脂蛋白（ox-LDL）在 VEC 聚集的主要机制。我们首次发现上调自噬能够促进 ox-LDL 降解。VEC SIRT1 是一种关键的抗 AS 调节因子，最近研究表明 SIRT1 能下调巨噬细胞 LOX-1 表达，促进自噬。然 SIRT1 对 VEC 氧化脂质代谢的影响尚未阐明。本课题运用荧光免疫、免疫印迹、活细胞工作站、流式细胞术等方法，发现：1.Dil标记ox-LDL (Dil-ox-LDL) 在 HUVECs的聚集能被SIRT1 siRNA和SIRT1抑制剂 nicotinamide（NAM）所增加,被SIRT1诱导剂resveratrol (RSV) 减少。敲减自噬关键基因Atg5或运用氯喹抑制溶酶体降解途径均会抑制白藜芦醇的作用。在ox-LDL暴露下，自噬标志物LC3II表达及LC3点状聚集均会被SIRT1 siRNA或NAM抑制, 被RSV增加。而自噬降解底物p62 对其有相反效应。SIRT1 siRNA或NAM会抑制 Dil-ox-LDL与溶酶体降解途径标志物Lamp1、 Cathepsin D 的共定位，RSV会增加其共定位。表明，SIRT1能减少ox-LDL 在 HUVECs的聚集，其作用与其促进ox-LDL的自噬性降解有关。2.雷帕霉素会抑制HUVECs摄取Dil-ox-LDL，抑制ox-LDL诱导的LOX-1 mRNA和protein表达，抑制ox-LDL诱导的mTOR、p70s6k、IκBα磷酸化表达增加，抑制ox-LDL诱导的p65核转运。mTOR、NF-κB敲减和NF-κB抑制剂均能显著抑制Dil-ox-LDL的聚集。mTOR敲减能抑制ox-LDL诱导的LOX-1蛋白表达及IκBα磷酸化。 NF-κB敲减能ox-LDL诱导的LOX-1 蛋白表达，却不能抑制ox-LDL诱导的mTOR磷酸化。表明，雷帕霉素会通过抑制mTOR 磷酸化，进一步抑制NF-κB活化，进而减少LOX-1表达，抑制HUVECs摄取ox-LDL。其结果对全面评估自噬诱导因子SIRT1、雷帕酶素的血管保护作用具有重要意义。