The Effect And Mechanism Of Apolipoprotein AI Mimetic Peptide D-4F On Apoptosis Of Macrophage Derived Foam Cells Via Endoplasmic Reticulum Stress

ObjectiveThe purpose of the present study was to investigate the effect ofapolipoprotein AI （Apo AI） mimetic peptide D-4F on the apoptosis ofmacrophage-derived foam cells and endoplasmic reticulum stress （ERS）-C/EBP homologous protein （CHOP） signal pathway, and to elucidate thepossible mechanism underlying the antiatherogenic function of D4F.MethodsMale apoE-/-mice （8weeks of age） were fed with a high-fat diet （15.8%fat and1.25%cholesterol） for8weeks, and given physiological saline （modelgroup,n=8）, scrambled D4F （1mg/kg·d, sD4F group, n=8） or D4F （1mg/kg·d, D4F group, n=8） by intraperitoneal injection during the final6weeks. The same week old male C57BL/6J mice were fed with normal chow dietand injected intraperitoneally with physiological saline as control group （n=8）. All experiments were approved by the laboratory animals’ ethical committeeof Taishan Medical University and followed national guidelines for the care anduse of animals.The serum oxidized low density lipoprotein （ox-LDL） levelswere measured by ELISA method, and the serial aortic root cryosections （6μm）, were prepared to test the lesion of atherosclerosis and plaque lipid contentusing HE staining and oil red O staining, respectively. The plaque collagencontent and the cell apoptosis in aortic root were determined using Massonstaining and TUNEL, respectively. MOMA-2（macrophage marker）, CHOP andglucose regulated protein78（GRP78）（two ERS markers） in atherosclerotic plaque were detected by immunohistochemistry. RAW264.7macrophages werepretreated with different concentrations （0,12.5,25and50mg/L） of D4F or50mg/L of sD4F for1h, and then were incubated with ox-LDL（100mg/L） for24h or were incubated with ERS inducers tunicamycin （TM,5mg/L） for12h. Apoptosis was analyzed with the flow cytometry, and the expression of CHOPprotein and mRNA were analyzed by western blot and real-time quantitativePCR, respectively.ResultsThe levels of each index of the sD4F group had no difference with themodel group. Compared with the control group, the levels of serum ox-LDL ofthe model group were significantly increased. However, the levels of serumox-LDL of the D4F group reduced by25.5%compared with model group（P<0.05）. D4F significantly reduced the area of the aortic root lesion and thelipid positive percentage, and increased the collagen fiber content in the plaque（P<0.05or P<0.01）. Compared with the model group, macrophageaccumulation and the apoptotic rate in the aortic plaque of the D4F groupsignificantly decreased, which were65.6%（P<0.05） and61.2%（P<0.01）of model group, respectively. and the expression of the GRP78and CHOPsignificantly reduced, which were68.1%（P<0.05） and61.1%（P<0.01） ofmodel group, respectively. In vitro experiment, D4F significantly decreased theapoptosis of macrophages induced by ox-LDL, and lowered the expression ofthe CHOP both at the protein and mRNA levels in a dose-dependent manner（P<0.05or P<0.01）. In addition, D4F significantly reduced macrophageapoptosis and inhibited the up-regulation of CHOP at the levels of mRNA andprotein induced by TM （P<0.05）.ConclusionD4F can reduce the apoE-/-mouse atherosclerotic lesions and apoptosis, themechanism of which may be related to inhibiting the macrophage endoplasmicreticulum stress-associated apoptosis pathway mediated by CHOP.