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槲皮素预处理对衣霉素所致巨噬细胞凋亡的抑制作用及机制

姚树桐, 苗成, 刘庆华, 李妍妍, 田华, 王芸芸, 马变颖, 方永奇^*, 秦树存^*

泰山医学院动脉粥样硬化研究所，山东省高校重点实验室；基础医学院；附属医院检验科； 2008级生物技术本科，泰安 271000

摘要

本文旨在研究槲皮素(quercetin, QUE)预处理对内质网应激诱导剂衣霉素(tunicamycin, TM)所致RAW264.7巨噬细胞凋亡的抑制作用，并探讨可能的分子机制。体外培养RAW264.7巨噬细胞，给予20、40和80 μmol/L QUE预处理30 min，再加入5 mg/L TM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；免疫荧光细胞化学法和免疫印迹法检测转录激活因子6 (activating transcription factor 6, ATF6)核转位情况；分别采用免疫印迹法和实时定量聚合酶链反应(real-time PCR)技术检测促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein, CHOP)和抗凋亡蛋白Bcl-2蛋白及mRNA表达变化。结果显示，QUE (40和80 μmol/L)预处理显著抑制TM所诱导的细胞活力降低和凋亡。与TM处理组比较，QUE预处理组内质网应激感受器ATF6由胞浆向核内转移明显减弱。TM在蛋白和转录水平均明显上调CHOP表达，并下调Bcl-2表达，而QUE则明显抑制上述变化，且呈浓度依赖性。以上结果表明，QUE可减轻TM所诱导的RAW264.7 巨噬细胞凋亡，可能是部分通过抑制ATF6-CHOP信号途径实现的。

关键词： 槲皮素; 内质网应激; C/EBP同源蛋白; 衣霉素; 巨噬细胞; 凋亡

分类号：R331.3；R363.2；R332

[Inhibitory effect of quercetin preconditioning on tunicamycin-induced apoptosis in macrophages and its mechanism.] [Article in Chinese]

YAO Shu-Tong, MIAO Cheng , LIU Qing-Hua, LI Yan-Yan, TIAN Hua, WANG Yun-Yun , MA Bian-Ying, FANG Yong-Qi ^*, QIN Shu-Cun^*

Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong； College of Basic Medical Sciences； Department of Clinical Laboratory, Affiliated Hospital of Taishan Medical University； Grade 2008 of Biotechnology, Taishan Medical University, Taian 271000, China

Abstract

The purposes of the present study were to investigate the inhibitory effect of quercetin (QUE) preconditioning on endoplasmic reticulum stress (ERS) inducer tunicamycin (TM)-induced apoptosis in RAW264.7 macrophages and the underlying molecular mechanisms. RAW264.7 cells were pretreated with different concentrations (20, 40, and 80 μmol/L) of QUE for 30 min and then treated with TM (5 mg/L) for 12 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The nuclear translocation of activating transcription factor 6 (ATF6) in cells was detected by immunofluorescence analysis and Western blot. Protein and mRNA expressions of C/EBP homologous protein (CHOP) and Bcl-2 were examined by Western blot and real-time PCR, respectively. The results showed that TM reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of TM were significantly inhibited by QUE pretreatment at the concentrations of 40 and 80 μmol/L. Interestingly, we found that QUE also significantly suppressed the TM-induced translocation of ATF6, an ERS sensor, from the cytoplasm to the nucleus. In addition, exposure of RAW264.7 macrophages to TM resulted in a significant increase of the expression of CHOP, a transcription factor regulated by ATF6 under conditions of ERS, as well as a decrease of Bcl-2 at transcript and protein levels. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from TM-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ATF6-CHOP signaling pathway.

Key words: quercetin; endoplasmic reticulum stress; CHOP; tunicamycin; Macrophage; apoptosis

收稿日期：2012-05-04　　录用日期：2012-06-19

通讯作者：方永奇,秦树存　　E-mail: yqfang@tsmc.edu.cn,shucunqin@hotmail.com

引用本文：

姚树桐, 苗成, 刘庆华, 李妍妍, 田华, 王芸芸, 马变颖, 方永奇, 秦树存. 槲皮素预处理对衣霉素所致巨噬细胞凋亡的抑制作用及机制[J]. 生理学报 2013; 65 (1): 47-54.

YAO Shu-Tong, MIAO Cheng , LIU Qing-Hua, LI Yan-Yan, TIAN Hua, WANG Yun-Yun , MA Bian-Ying, FANG Yong-Qi , QIN Shu-Cun. [Inhibitory effect of quercetin preconditioning on tunicamycin-induced apoptosis in macrophages and its mechanism.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (1): 47-54 (in Chinese with English abstract).