Activating Transcription Factor 6-C/EBP Homologous Protein Pathway Mediatesadvanced Glycated Albumin-induced Macrophage Apoptosis

The incidence of atherosclerosis(AS)worldwide is increasing year by year.It seriously threatens human health and is closely related to cardiovascular and cerebrovascular diseases.Diabetes mellitus is an important risk factor for atherosclerosis.The long-term hyperglycemic environment makes the structure and function of the circulating albumin modified by glycosylated modification,thus exerting the role of promoting atherosclerosis.Numerous studies have shown that endoplasmic reticulum stress(ERS)plays an important role in the progression of atherosclerosis,and is related to the accelerated atherosclerosis process by glycated albumin.In many kinds of damaging factors' effect,ERS can guide macrophage apoptosis through various ways,among which the better widely studied one is C/EBP homologous protein(CHOP)pathway.A large number of previous studies have shown that ATF6,IRE1 and PERK pathways can induce CHOP transcription and apoptosis.Therefore,it is of important clinical significance to explore its possible mechanism of action in depth.Objective: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.Methods:1 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 2000.2 Cell viability and apoptosis were determined by MTT method and Annexin V-FITC/propidium iodide apoptosis detection kit,respectively.3 The activities of lactate dehydrogenase(LDH)in medium and caspase-3 in cells were measured by corresponding detection kits.4 ATF6 nuclear translocation was detected by immunofluorescence cytochemistry and Western method.5 The expression changes of C/EBP homologous protein(CHOP,a key-signaling component of ERS-induced apoptosis)were detected by Western method.The expression of CHOP mRNA was detected by real-time quantitative polymerase chain reaction.The difference between P < 0.05 was statistically significant.Results:1 The results showed that similar to TM,the expression of CHOP,the key molecule of ERS apoptotic pathway,were significantly up-regulated,at the level of eithor mRNA or protein which is concentration dependent.2 ATF6,as a factor that positively regulates CHOP expression,was activated by AGE-alb in a concentration dependent manner.3 siRNA-mediated knockdown of ATF6 signifcantly alleviated the macrophage injury caused by AGE-alb,as mentioned 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.Conclusion:These results suggest that ATF6 and its downstream molecule CHOP are involved in AGE-alb-induced macrophage apoptosis.