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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

Received: 2017-06-02　　Accepted: 2017-11-29

Corresponding author: 秦树存　　E-mail: shucunqin@hotmail.com

DOI: 10.13294/j.aps.2017.0088

Citing This Article：

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).