The Regulating Effect Of Naringenin On Endoplasmic Reticulum Stress-ATF6 Activity And Cellular Cholesterol Efflux In Macrophages

Objective:Naringenin improves lipoprotein profile and protects against cardiovascular disease.ATF6 is an endoplasmic reticulum(ER)stress sensor with the same activation processes with sterol regulator SREBPs.Clinical data revealed the ATF6 level was associated with plasma cholesterol levels.Here,we investigated whether naringenin was involved in the regulation of cholesterol efflux and tested the role of ER stress-ATF6 in the naringenin function.Methods:1.Mouse macrophages(RAW264.7)and monocytes(THP-1)were cultured in vitro and induced by 80 jag/ml oxLDL or fluorescence labeled cholesterol.RAW264.7 and THP-1 cells were treated with naringin at different concentrations(10,25,50 ?M).Cholesterol efflux was detected by fluorescence quantitative enzyme labeling apparatus.The expression of related proteins was detected by fluorescence quantitative reverse transcription PCR(qRT-PCR)and Western blot.Effects of naringin on RAW264.7 and THP-1 cells treated with endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(PBA),inducer TM or ATF6-specific siRNA,Western blot was used to detect the expression of related proteins.Effects of naringin on RAW264.7 cells treated with PI3K inhibitor LY294002,Western blot was used to detect the expression of related proteins.2.eight-week-old ApoE-/-mice were randomly divided into four groups and treated as follows:normal diet group(ND),high-fat model group(HFD),naringenin group(HFD+naringenin 100 mg/kg/day),TM Group(HFD+naringenin 100 mg/kg/day+TM,5 mg/kg/day).naringenin was administered by intragastric administration,TM were injected intraperitoneally.After treatment,the mRNA and protein contents of related proteins were detected by qRT-PCR and Western blot,respectively.Finally,the mouse aorta was pathologically analyzed.Results:1.Results showed that naringenin increased cholesterol efflux to both apoA-I and HDL and gene expressions in ABCA1,ABCG1 and LXRa in RAW264.7 macrophages.Naringenin inhibited the cleaved ATF6 nuclear translocation and its targeted genes GRP78 and XBP-1 expressions.Naringenin functions were mediated through inhibiting ER stress-ATF6 pathway.Naringenin promoted Akt phosphorylation;PI3K inhibitor LY294002 treatment increased nuclear ATF6 and reduced naringenin-enhenced cholesterol efflux and ABCA1 expressions.2.Next,we found HFD-ApoE'A mice supplemented with naringenin increased by more than 1.5-fold in cholesterol efflux capacity in primary peritoneal macrophage than that from only-HFD treated mice.The increasing was significantly reduced by tunicamycin treatment.Naringenin decreased nuclear ATF6,GRP78 and XBP-1 levels at aorta and peritoneal macrophage and reduced atherosclerotic lesion at aortic root,but reversed by tunicamycin.These confirmed participation of ER stress-ATF6 in naringenin efficacy.Conelusion:Naringenin as a regulator for cholesterol efflux,and the regulation was mediated by ATF6 branches of ER stress and P13K/Akt pathway.