The Inductive Effect Of Glycosylated Low Density Lipoptotein On C/EBP Homologous Protein-mediated Apoptotic Pathway In Macrophages

ObjectiveThe purpose of the present study was to investigate the regulative effect of glycosylated low density lipoprotein(G-LDL)on the macrophage-derived foam cell formation and endoplasmic reticulum stress(ERS)-C/EBP homologous protein(CHOP)-mediated macrophage,which would further explore the molecular mechanism of the development of diabetic atherosclerosis,and provide a new theoretical basis for further elucidate the pathogenesis of atherosclerosis(AS).Methods1.The preparation of G-LDL and determination of its glycosylated level: LDL were collected from healthy human plasma by sequential ultracentrifugation,and incubated in the dark for 7 days with 250 mmol/L D-glucose at 37°C under sterile conditions.ELISA was used to detect the carboxymethyl-lysine(CML)in lipoprotein in order to determine the level of glycosylation.2.RAW264.7 macrophages were cultured in vitro and randomly divided into the following groups:(1)normal control group: cells were routinely cultured.(2)LDL group: adding the 100mg/L LDL in the cell culture medium.(3)G-LDL group: adding the different concentration(25,50,100 and 200 mg/L)of G-LDL in cell culture medium.(4)Tunicamycin(TM)group: adding 4 mg/L TM in the cell culture medium.(5)4-phenylbutyric acid(PBA)+G-LDL group: cells were pretreated with 5 mmol/L PBA for 30 min,and then treated with100 mg/L G-LDL.Every group continued to culture for 24 h.The effect of G-LDL on the viability and apoptosis of macrophages was detected by MTT assay and Annexin V-FITC/PI double staining method,respectively.The intracellular lipid accumulation was measured by oil red O and nile red staining.The protein and m RNA levels of CHOP,a pro-apoptotic molecule under ERS,were analyzed by western blot and real-time quantitative PCR,respectively.ResultsCompared with LDL,the CML levels increased significantly in G-LDL(P<0.01).Like TM,an ERS inducer,G-LDL induced macrophage injury as assessed by the decreased viability and increased apoptosis in the dose-dependent manner.G-LDL caused lipid accumulation in macrophages,compared with the normal control group(P<0.01).Western blot and real-time PCR results showed that G-LDL significantly up-regulated the expression of CHOP both at the protein and m RNA levels in a dose-dependent manner(P<0.05 or P<0.01).In addition,compared with G-LDL treated group,the macrophage injury and upregluation of CHOP protein and m RNA levels induced by G-LDL were significantly inhibited by PBA(an ERS inhibitor)pretreatment.ConclusionGlycosylated low density lipoprotein may induce the injury and apoptosis of macrophage-derived foam cells through activating the CHOP-mediated apoptotic pathway.