RESUMO
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.