氧化低密度脂蛋白通过CD36介导的氧化应激诱导巨噬细胞自噬  

Oxidized low-density lipoprotein induces autophagy in macrophages via CD36-mediated oxidative stress

作　　者：姚树桐[1,2] 李严严[1] 刘庆华[3] 岳峰[4] 田华[1] 桑慧[2] 杨娜娜[1] 秦树存[1] 

机构地区：[1]泰山医学院动脉粥样硬化研究所,山东省高校动脉粥样硬化重点实验室,山东泰安271000 [2]泰山医学院基础医学院,山东泰安271000 [3]泰山医学院附属医院检验科,山东泰安271000 [4]泰山医学院泰安市中心医院内分泌科,山东泰安271000

出　　处：《中国病理生理杂志》2015年第6期1002-1007,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81202949;No.81370381)

摘　　要：目的:研究氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)对巨噬细胞自噬的诱导作用,并探讨可能的分子机制。方法:体外培养RAW264.7巨噬细胞,给予抗CD36单克隆抗体(2 mg/L)、二亚苯基碘鎓(diphenyleneiodonium,DPI;5μmol/L)、3-甲基腺嘌呤(3-methyladenine,3-MA;3 mmol/L)或雷帕霉素(1μmol/L)预处理1 h,再加入ox-LDL(100 mg/L)继续培养12 h。采用MTT法检测细胞活力,采用相应试剂盒测定培养液乳酸脱氢酶(lactic dehydrogenase,LDH)、细胞内烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶、超氧化物歧化酶(superoxide dismutase,SOD)活性以及活性氧簇(reactive oxygen species,ROS)和丙二醛(malondialdehyde,MDA)水平,以评价细胞膜完整性和氧化应激反应。采用免疫印迹技术检测自噬标志分子beclin-1和微管相关蛋白1轻链3-II(microtubule-associated protein 1 light chain 3-II,LC3-II)表达变化。结果:ox-LDL诱导巨噬细胞自噬反应,表现为beclin-1和LC3-II上调;与自噬抑制剂3-MA相似,抗CD36单抗可显著抑制ox-LDL所诱导的LC3-II和beclin-1表达。抗CD36单抗明显抑制ox-LDL所诱导的氧化应激,包括抑制NADPH氧化酶活性和ROS、MDA水平以及升高SOD活性,其作用与NADPH氧化酶抑制剂DPI相似。另外,DPI显著抑制ox-LDL所诱导的beclin-1和LC3-II表达,且ox-LDL所诱导的细胞活力降低和LDH漏出可被3-MA促进并可被自噬诱导剂雷帕霉素拮抗。结论:ox-LDL可诱导巨噬细胞自噬,其机制可能与CD36介导ox-LDL摄取进而触发的氧化应激有关,且一定程度的自噬可减轻ox-LDL所诱导的巨噬细胞损伤。AIM：To investigate the effect of oxidized low-density lipoprotein （ ox-LDL） on autophagy in mac-rophages and the underlying molecular mechanisms.METHODS：RAW264.7 macrophages were pretreated with 2 mg/L anti-CD36 monoclonal antibody （anti-CD36 mAb）, 5 μmol/L diphenyleneiodonium （DPI）, 3 mmol/L 3-methyladenine （3-MA） or 1μmol/L rapamycin for 1 h and then treated with ox-LDL （100 mg/L） for 12 h.The viability of the cells was measured by MTT assay.The activities of lactic dehydrogenase （ LDH） in the medium and nicotinamide adenine dinucleoti-de phosphate （ NADPH） oxidase, superoxide dismutase （ SOD） in the cells as well as the levels of intracellular reactive ox-ygen species （ ROS） and malondialdehyde （ MDA） were determined to characterize the membrane integrity and the oxida-tive stress, respectively.The protein levels of beclin-1 and microtubule-associated protein 1 light chain 3-II （ LC3-II） , 2 important molecular markers of autophagy, were examined by Western blotting.RESULTS：ox-LDL induced autophagy in 〈br〉 RAW264.7 macrophages as assessed by upregulation of beclin-1 and LC3-II.Similar to 3-MA, an autophagy inhibitor, an-ti-CD36 mAb significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II.Anti-CD36 mAb suppressed the ox-LDL-induced oxidative stress as revealed by decreased NADPH oxidase activation, ROS and MDA generation as well as increased SOD activity.Similar results were observed in the cells pretreated with DPI, a NADPH oxidase inhibitor.Mo-reover, DPI significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II.Inaddition, the decrease in the cell viability and increase in LDH release induced by ox-LDL were promoted by 3-MA and blocked by rapamycin （ an auto-phagy inducer）.CONCLUSION： ox-LDL induces autophagy in RAW264.7 macrophages, which may be involved in CD36-mediated ox-LDL uptake and subsequent activation of oxidative stress, and moderate activation of autophagy may pro-tect macrophages from ox-LDL-

关 键 词：自噬 氧化低密度脂蛋白 CD36 氧化应激 巨噬细胞 

分 类 号：R363.2[医药卫生—病理学] R332[医药卫生—基础医学]