摘要
本文旨在研究在鼠源巨噬细胞泡沫化过程中氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)的诱导作用及其机制。体外培养RAW264.7巨噬细胞,分别给予ox-LDL(25、50和100mg/L)、抗CD36抗体+ox-LDL和衣霉素(tunicamycin,TM)等不同处理。采用油红O染色观察细胞内脂质蓄积情况,酶比色法测定细胞内总胆固醇含量,免疫细胞化学法检测ERS标志分子糖调节蛋白94(glucose-regulated protein94,GRP94)表达,免疫印迹法检测GRP94及未折叠蛋白反应关键分子p-IRE1(phosphorylated inositol-requiring enzyme1)和X盒结合蛋白1(X box binding protein1,XBP1)蛋白表达水平。结果显示,不同浓度(25、50和100mg/L)ox-LDL处理细胞24h后,胞浆内可见大量油红O染色阳性脂质颗粒,细胞内总胆固醇含量明显增加,分别为空白对照组的2.1倍、2.8倍和3.1倍;使用抗CD36抗体阻断ox-LDL的摄入,可显著减少100mg/Lox-LDL所致的细胞内胆固醇蓄积。不同浓度ox-LDL和ERS诱导剂TM均可显著增加GRP94及其上游信号分子p-IRE1和XBP1蛋白表达,且表达强度随着ox-LDL诱导浓度的增加而增强;抗CD36抗体显著抑制100mg/Lox-LDL所致的上述3种蛋白表达上调。上述结果提示,ox-LDL可呈剂量依赖性诱导RAW264.7巨噬细胞产生ERS,激活未折叠蛋白反应信号通路;该过程可能由清道夫受体CD36所介导。
The purpose of the present study is to explore the effect of oxidized low density lipoprotein（ox-LDL） on the induction of endoplasmic reticulum stress（ERS） and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process.RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum,and then treated with ox-LDL（25,50 and 100 mg/L）,anti-CD36 monoclonal antibody＋ox-LDL and tunicamycin（TM）,respectively.After incubation for 24 h,the cells were collected.The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry.The expression of glucose-regulated protein 94（GRP94）,a molecular marker of ERS,was determined by immunocytochemistry assay.The levels of GRP94 protein,phosphorylated inositol-requiring enzyme 1（p-IRE1） and X box binding protein 1（XBP1） in RAW264.7 cells were detected by Western blotting.The results indicated that after incubation with ox-LDL（25,50 and 100 mg/L） for 24 h,a large amount of lipid droplets were found in the cytoplasm,and the contents of cellular total cholesterol were increased by 2.1,2.8 and 3.1 folds compared with the control,respectively.Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L.Both ox-LDL and TM,a specific ERS inducer,could up-regulate the protein expression of GRP94 in a dose-dependent manner.Furthermore,p-IRE1 and XBP1,two key components of the unfolded protein response,were also significantly induced by the treatment with ox-LDL.The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody.These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages,which is potentially mediated by scavenger receptor CD36.
出处
《生理学报》
CAS
CSCD
北大核心
2010年第5期433-440,共8页
Acta Physiologica Sinica
基金
supported by the"Taishan Scholars"Project of Shandong Province
China(No.zd056
zd057)
the Special Program of Taishan Medical University
China(No.1065)
