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活化转录因子6-C/EBP同源蛋白途径介导晚期糖基化白蛋白诱导的巨噬细胞凋亡

康攀攀^1,2, 姚树桐^2,3, 郭甜甜⁴, 王志超², 田华², 焦鹏², 周健¹, 秦树存^2,*

¹承德医学院附属医院，承德 067000；²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，泰安 271000；³泰山医学院基础医学院，泰安 271000；⁴泰山医学院口腔医学院，泰安 271000

摘要

本文旨在研究内质网应激(endoplasmic reticulum stress, ERS)感受器活化转录因子6 (activating transcription factor 6, ATF6)是否介导晚期糖基化白蛋白(advanced glycated albumin, AGE-alb)所诱导的巨噬细胞凋亡，并阐明其可能的分子机制。体外培养RAW264.7巨噬细胞，给予AGE-alb (2、4和6 g/L)处理24 h，以正常白蛋白和ERS诱导剂衣霉素(tunicamycin, TM)处理24 h的巨噬细胞分别作为阴性和阳性对照组，并采用小干扰RNA (small interfering RNA, siRNA)技术沉默ATF6表达。分别采用MTT法和Annexin V-FITC/碘化丙碇双染法检测细胞活力和凋亡情况；试剂盒测定培养基乳酸脱氢酶(lactate dehydrogenase, LDH)和细胞内caspase-3活性；免疫荧光细胞化学法检测ATF6核转位情况；Western blot法检测ATF6和促凋亡蛋白C/EBP同源蛋白(C/EBP homologous protein, CHOP)蛋白表达变化，实时荧光定量聚合酶链反应法检测CHOP mRNA表达变化。结果显示：与TM相似，AGE-alb在蛋白和mRNA水平均显著上调ERS凋亡途径关键分子CHOP表达，该作用呈浓度依赖性；Western blot和免疫荧光细胞化学法均显示AGE-alb处理细胞后ATF6明显由胞浆向细胞核内转移；采用siRNA技术沉默ATF6则明显减轻AGE-alb所致的细胞活力降低、LDH漏出、凋亡率增加及caspase-3活化，并可抑制AGE-alb所诱导的CHOP表达上调。上述结果表明，ATF6及其下游分子CHOP介导AGE-alb所诱导的巨噬细胞凋亡。

关键词： 晚期糖基化白蛋白; 活化转录因子6 ; C/EBP同源蛋白 ; 巨噬细胞 ; 凋亡

分类号：R285.5;R363.2

Activating transcription factor 6-C/EBP homologous protein pathway mediates advanced glycated albumin-induced macrophage apoptosis

KANG Pan-Pan^1,2, YAO Shu-Tong^2,3, GUO Tian-Tian⁴, WANG Zhi-Chao², TIAN Hua², JIAO Peng², ZHOU Jian¹, QIN Shu-Cun^2,*

¹Affiliated Hospital of Chengde Medical University, Chengde 067000, China；²Institute of Atherosclerosis, Key Laboratory of Atherosclerosis in Universities of Shandong, Taishan Medical University, Taian 271000, China；³College of Basic Medical Sciences, Taishan Medical University, Taian 271000, China；⁴School of Dentistry and Oral Health, Taishan Medical University, Taian 271000, China

Abstract

The purpose of this study was to investigate whether activating transcription factor 6 (ATF6), a sensor to endoplasmic reticulum stress (ERS), would mediate advanced glycated albumin (AGE-alb)-induced macrophage apoptosis and to elucidate the possible molecular mechanisms. RAW264.7 macrophages were cultured in vitro and treated with AGE-alb (2, 4 and 6 g/L), normal control albumin or tunicamycin (TM, 4 mg/L) for 24 h. ATF6 small interfering RNA (siRNA) was transfected to RAW264.7 cells by Lipofectamine 2000. Cell viability and apoptosis were determined by MTT method and Annexin V-FITC/propidium iodide apoptosis detection kit, respectively. The activities of lactate dehydrogenase (LDH) in medium and caspase-3 in cells were measured by corresponding detection kits. ATF6 nuclear translocation was detected by Western blot and immunofluorescence cytochemistry. Protein and mRNA levels of C/EBP homologous protein (CHOP, a key-signaling component of ERS-induced apoptosis) were detected by Western blot and real-time fluorescence quantitative PCR, respectively. The results showed that similar to TM, AGE-alb increased the expression of CHOP at both the protein and mRNA levels in a concentration dependent manner. ATF6, as a factor that positively regulates CHOP expression, was activated by AGE-alb in a concentration dependent manner. siRNA-mediated knockdown of ATF6 significantly inhibited AGE-alb-induced macrophage injury, as indicated by the increased cell viability and the decreased LDH release, apoptosis and caspase-3 activation. Additionally, ATF6 siRNA attenuated AGE-alb-induced CHOP upregulation at both the protein and mRNA levels. These results suggest that ATF6 and its downstream molecule CHOP are involved in AGE-alb-induced macrophage apoptosis.

Key words: advanced glycated albumin; activating transcription factor 6 ; C/EBP homologous protein ; Macrophage ; apoptosis

收稿日期：2017-06-02　　录用日期：2017-11-29

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

DOI: 10.13294/j.aps.2017.0088

引用本文：

康攀攀, 姚树桐, 郭甜甜, 王志超, 田华, 焦鹏, 周健, 秦树存. 活化转录因子6-C/EBP同源蛋白途径介导晚期糖基化白蛋白诱导的巨噬细胞凋亡[J]. 生理学报 2017; 69 (6): 767-774.

KANG Pan-Pan, YAO Shu-Tong, GUO Tian-Tian, WANG Zhi-Chao, TIAN Hua, JIAO Peng, ZHOU Jian, QIN Shu-Cun. Activating transcription factor 6-C/EBP homologous protein pathway mediates advanced glycated albumin-induced macrophage apoptosis. Acta Physiol Sin 2017; 69 (6): 767-774 (in Chinese with English abstract).