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氧化低密度脂蛋白诱导巨噬细胞内质网应激及CD36的可能作用

姚树桐, 桑慧, 杨娜娜, 康莉, 田华, 张颖, 宋国华, 秦树存

泰山医学院 动脉粥样硬化研究所； 基础医学部，泰安 271000

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100 mg/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 protein 94,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 enzyme 1)和X盒结合蛋白1 (X box binding protein 1, XBP1)蛋白表达水平。结果显示，不同浓度(25、50 和100 mg/L) ox-LDL 处理细胞24 h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100 mg/L ox-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100 mg/L ox-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。

关键词： 内质网应激; 巨噬细胞; 氧化低密度脂蛋白; CD3 6 ; 动脉粥样硬化

分类号：R332; R363.2; R329.2

Oxidized low density lipoprotein induces macrophage endoplasmic reticulum stress via CD36

YAO Shu-Tong, SANG Hui, YANG Na Na, KANG Li, TIAN Hua, ZHANG Ying, SONG Guo-Hua , QIN Shu-Cun

Institute of Atherosclerosis； Institute of Preclinical Medicine, Taishan Medical College, Taian 271000, China

Abstract

The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction ofendoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7macrophages 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. Thecellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymaticcolorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistryassay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol wereincreased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipidaccumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expressionof 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 inhibitedsignificantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERSin 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.

Key words: endoplasmic reticulum stress; Macrophage; oxidized low density lipoprotein; CD36; atherosclerosis

收稿日期：2010-06-17　　录用日期：2010-07-26

通讯作者：秦树存　　E-mail: shucunqin@hotmail.com

引用本文：

姚树桐, 桑慧, 杨娜娜, 康莉, 田华, 张颖, 宋国华, 秦树存. 氧化低密度脂蛋白诱导巨噬细胞内质网应激及CD36的可能作用[J]. 生理学报 2010; 62 (5): 433-440.

YAO Shu-Tong, SANG Hui, YANG Na Na, KANG Li, TIAN Hua, ZHANG Ying, SONG Guo-Hua , QIN Shu-Cun. Oxidized low density lipoprotein induces macrophage endoplasmic reticulum stress via CD36. Acta Physiol Sin 2010; 62 (5): 433-440 (in Chinese with English abstract).