自噬通过抑制C/EBP同源蛋白表达减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡  

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

作　　者：田华[1,2] 马守原[3] 康攀攀 郝奇[2] 焦鹏[1] 邵夏炎[2] 徐晓燕[5] 秦树存[1] 姚树桐[1,2] 

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]中国人民解放军总医院南楼心血管内科,北京100853 [4]承德医学院附属医院,河北承德067000 [5]泰山医学院药学院,山东泰安271000

出　　处：《中国病理生理杂志》2016年第12期2192-2198,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81570410;No.81370381);国家级大学生创新训练项目(No.201510439126;No.201510439100)

摘　　要：目的:研究自噬对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)所致巨噬细胞凋亡的影响,并探讨可能的分子机制。方法:体外培养RAW264.7巨噬细胞,分别给予3-甲基腺嘌呤(3-methyladenine,3-MA;3 mmol/L)、雷帕霉素(rapamycin,Rap;1μmol/L)或4-苯丁酸(4-phenylbutyric acid,PBA;4 mmol/L)预处理1 h,再加入ox-LDL(100 mg/L)继续培养12 h。分别采用MTT法和Annexin V-FITC双染法检测细胞活力和凋亡情况;相应试剂盒测定培养液乳酸脱氢酶(lactic dehydrogenase,LDH)和细胞内caspase-3活性;采用Western blot法检测自噬标志分子beclin-1和内质网应激标志分子葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)及促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein,CHOP)表达的变化;采用激光共聚焦显微镜观测细胞内微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的变化。结果:ox-LDL处理可显著降低巨噬细胞活力,并增加LDH漏出、凋亡率及caspase-3活性;ox-LDL对细胞的上述损伤作用可被自噬抑制剂3-MA促进而被自噬诱导剂雷帕霉素拮抗。ox-LDL诱导巨噬细胞自噬反应,表现为beclin-1表达上调,LC3颗粒化显著;ox-LDL对自噬的诱导作用可被3-MA抑制而被Rap增强。另外,3-MA可促进ox-LDL所诱导的CHOP进一步上调,而Rap可明显拮抗ox-LDL对CHOP的诱导作用。PBA可显著抑制ox-LDL所诱导的GRP78上调,且明显减轻ox-LDL所诱导的自噬反应,表现为beclin-1表达下调,LC3颗粒化程度减弱。结论:内质网应激介导ox-LDL对巨噬细胞自噬的诱导作用,而一定程度的自噬可通过抑制CHOP表达从而减轻ox-LDL所诱导的巨噬细胞凋亡。AIM：To investigate the protective effect of autophagy on oxidized low density lipoprotein（ oxLDL）-induced macrophage apoptosis and the underlying molecular mechanisms.METHODS：The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine（ 3-MA）,1 μmol/L rapamycin（ Rap） or 4 mmol/L 4-phenylbutyricacid（ PBA） respectively for 1 h and then treated with ox-LDL（ 100 mg/L） for 12 h.The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining,respectively.The activities of lactate dehydrogenase（ LDH） in the medium and caspase-3 in the cells were determined by detection kits.The protein levels of beclin-1（ a molecular marker of autophagy）,glucose-regulated protein 78（ GRP78,an endoplasmic reticulum stress marker）and C/EBP homologous protein（ CHOP,a key-signaling component of endoplasmic reticulum stress-induced apoptosis）were examined by Western blot.Microtubule-associated protein 1 light chain 3（ LC3,another molecular marker of autophagy） was observed under laser scanning confocal microscope.RESULTS：Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability,and dramatic elevation in LDH leakage,cell apoptosis and caspase-3 activity,which were promoted by 3-MA（ an autophagy inhibitor） and inhibited by Rap（ an autophagy inducer）.ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3,which were inhibited by 3-MA and promoted by Rap.Interestingly,3-MA enhanced,while Rap blocked,the CHOP upregulation induced by ox-LDL.Moreover,PBA（ endoplasmic reticulum stress inhibitor） significantly inhibited ox-LDLinduced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3.CONCLUSION：Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages,and moderates activation of autophagy may protect macrophages from ox-LDL

关 键 词：自噬 内质网应激 氧化低密度脂蛋白 巨噬细胞 细胞凋亡 

分 类 号：R363.2[医药卫生—病理学]