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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。
Objective: To investigate the effect of autophagy on the apoptosis of macrophages induced by ox-LDL (ox-LDL), and to explore the possible molecular mechanisms. METHODS: RAW264.7 macrophages were cultured in vitro and were treated with 3-methyladenine (3-MA; 3 mmol / L), rapamycin (Rap; 1μmol / L) The cells were pre-treated with 4-phenylbutyric acid (PBA; 4 mmol / L) for 1 h and then incubated with ox-LDL (100 mg / L) for 12 h. Cell viabilities and apoptosis were detected by MTT assay and Annexin V-FITC double staining respectively. The activities of lactic dehydrogenase (LDH) and intracellular caspase-3 in culture medium were determined by corresponding kit. The changes of the expression of beclin-1, GRP78 and C / EBP homologous protein (CHOP) in endoplasmic reticulum (ER) The change of intracellular microtubule-associated protein 1 light chain 3 (LC3) was observed by laser confocal microscopy. Results: The ox-LDL treatment significantly decreased the viability of macrophages and increased the leakage of LDH, the apoptosis rate and the activity of caspase-3. Ox-LDL treatment induced the above-mentioned damage by the autophagy inhibitor 3-MA Targeted rapamycin antagonist. Ox-LDL induced autophagy in macrophages. The expression of beclin-1 was up-regulated and LC3 was granulated significantly. The induction of autophagy by ox-LDL was enhanced by Rap with 3-MA. In addition, 3-MA promoted the up-regulation of CHOP induced by ox-LDL, while Rap inhibited obviously the induction of CHOP by ox-LDL. PBA significantly inhibited the upregulation of GRP78 induced by ox-LDL, and significantly reduced the autophagy induced by ox-LDL. The expression of beclin-1 was down-regulated and the degree of LC3 granulation was weakened. CONCLUSION: Endoplasmic reticulum stress mediates the induction of autophagy by macrophages by ox-LDL, and a certain degree of autophagy can reduce the apoptosis of macrophages induced by ox-LDL by inhibiting the expression of CHOP.