脂联素通过调节巨噬细胞自噬水平发挥抗动脉粥样硬化作用的机制研究  

Adiponectin inhibits atherosclerosis by regulating the level of autophagy in macrophages

作　　者：任新宇[1] 孙岩[1] 周玉杰[1] 

机构地区：[1]首都医科大学附属北京安贞医院-北京市心肺血管疾病研究所十二病房,100029

出　　处：《心肺血管病杂志》2017年第4期314-318,共5页Journal of Cardiovascular and Pulmonary Diseases

基　　金：北京市医院管理局临床医学发展专项经费资助(ZYLX201303);北京市医院管理局"登峰"计划专项经费资助(DFL20150601);国家临床重点专科建设项目经费资助(2013-2014年度)

摘　　要：目的:本研究拟通过探究脂联素(APN)对巨噬细胞自噬水平的影响揭示其抗动脉粥样硬化(AS)的新机制,为临床AS的治疗提供新靶点。方法:采用在体外培养人THP-1细胞系诱导的巨噬细胞,分为对照组,氧化低密度脂蛋白(ox-LDL)组,ox-LDL+APN组三组,给予细胞ox-LDL处理模拟斑块内环境后,采用Cell Counting Kit(CCK-8)方法检测ox-LDL对细胞的毒性,并使用蛋白质印迹方法(Western blot)检测细胞内相关蛋白表达。在给予细胞APN及ox-LDL处理后,使用Western blot方法检测细胞内自噬相关蛋白表达情况,并应用透射电子显微镜(TEM)观察巨噬细胞的超微结构。使用Annexin V/PI染色方法检测细胞凋亡发生情况。结果:CCK-8结果表明巨噬细胞存活率随ox-LDL浓度升高明显下降(P<0.05),Western blot结果示给予ox-LDL处理后,细胞内自噬标记物LC3 II的表达水平明显升高,呈浓度及时间依赖性,且在ox-LDL的浓度为30 mg/L和孵育时间为6h时自噬水平最高(P<0.05)。ox-LDL+APN组的巨噬细胞中LC3 II表达水平较单独给予ox-LDL处理组明显降低,同时电镜结果也表明ox-LDL+APN组的巨噬细胞内自噬小体的形成较ox-LDL组有明显减少(P<0.05)。流式细胞术的Annexin V/PI染色方法检测结果显示APN可抑制ox-LDL诱导的巨噬细胞早期凋亡,且APN对凋亡的抑制作用不依赖于自噬。结论:ox-LDL可使巨噬细胞内自噬水平增加。APN可能通过降低巨噬细胞内自噬水平发挥抗AS的作用。Objective： The aim of this study is to explore the effect of adiponectin on the macrophage autophagy and to reveal its new mechanism of anti-atherosclerosis, providing a new target for the treatment of AS. Methods： The human THP-1 cell lines were cultured in vitro, divided into three groups, control group, oxidized low density lipoprotein （ox-LDL） group and ox-LDL ＋ APN group. The cytotoxicity of ox-LDL was measured by CCK-8. The expression of cellular proteins before and after APN was detected by western blot. The uhrastructure of macrophages was observed by transmission electron microscopy. And we used AnnexinV/PI staining to evaluate tile apoptosis of macrophages. Results： CCK-8 results showed that the survival rate of mac- rophages decreased significantly with ox-LDL concentration increasing（P 〈 0. 05）. Western blot results showed that the expression of intracellular autophagy marker LC3 II was significantly increased after ox-LDL treatment, which is showing a concentration-and time-dependent manner. The highest level of autophagy was observed when ox-LDL concentration was 30 mg / L and incubation time was 6 h（P 〈 0. 05）. The expression of LC3 II in macrophages of ox-LDL ＋ APN group was significantly lower than that of ox-LDL-treated group, transmissionelectron microscopy results also showed that the formation of autophagosomes in macrophages was significantly reduced in ox-LDL ＋ APN group compared with ox-LDL group（P 〈 0.05 ）. The results of AnnexinV/PI staining showed that APN could inhibit the early apoptosis of macrophages induced by ox-LDL, and the inhibitory effect of APN on apoptosis was independent of autophagy. Conchmion： Ox-LDL could increase the level of autophagy in macrophages. APN may play the role of anti-AS by reducing autophagy level in macrophages.

关 键 词：动脉粥样硬化 脂联素 自噬 巨噬细胞 

分 类 号：R54[医药卫生—心血管疾病]