[Gly14]-humanin Repairs Cathepsin D Function By FPRL1 And Promotes Autophagic Degradation Of Ox-LDL In HUVECs

Study Objectives:Abnormal aggregation of oxidized low density lipoprotein(ox-LDL)in vascular endothelial cells is one of the major pathological changes in atherosclerotic lesions.Humanin(HN)is an small molecule polypeptide in vivo.Our previous studies have shown that exogenous administration of S14G-HN(HNG)can promote efflux and reduce ox-LDL accumulation in vascular endothelial cells by inhibiting ox-LDL entry.The purpose of this study is to discuss the effect of HNG on autophagic degradation of ox-LDL in HUVEC and whether its action is achieved by the cell membrane receptor FPRL1.Materials and methods:HUVECs were treated with different concentrations of Dil-ox-LDL(0,30 μg/ml,60 μg/ml,90 μg/ml,120 μg/ml)and different concentrations of HNG(0,100 nM,1 μM,10 μM)for 12 h.The flow cytometry was used to observe the aggregation of Dil-ox-LDL in HUVECs and the effect of HNG on Dil-Ox-LDL aggregation in HUVECs.The different concentrations of ox-LDL and HNG treated HUVECs for 12 h,CCK8 was used to detect the activity of HUVECs.The effect of ox-LDL on the expression of endogenous HN and cathepsin B D K L mRNA was detected by QPCR;P62,LC3-II and cathepsin D protein expression in HUVECs were detected by western blot;Cathepsin D activity kit was used to detect lysozyme enzyme cathepsin D activity;Cholesterol kit was used to detect cholesterol expression;Immunofluorescence was used to detect HN expression in HUVECs.Results:1.Ox-LDL(0,30 μg/ml,60 μg/ml,90μg/ml,120 μg/ml)concentration-dependent aggregated in HUVECs.CCK8 results showed that when ox-LDL concentration reached 90μg/ml,120 μg/ml significantly reduced cell viability and increased cell death.Ox-LDL concentration-dependently increased p62 and LC3-Ⅱ protein expression in HUVECs.2.Immunofluorescence results showed that endogenous HN was expressed in HUVECs.Ox-LDL could change endogenous HN mRNA expression in HUVECs in a time-dependent and concentration-dependent manner.CCK8 results showed different concentrations of HNG(0,100 nM,1μM,10 μM)had no effect on cells vitality.HNG concentration-dependent decreased p62 protein and lipid aggregation,and the expression of LC3-II protein was increased.3.Ox-LDL concentration-dependently increased cathepsin B,cathepsin K,cathepsin L mRNA levels in HUVECs,decreased cathepsin D mRNA levels.Ox-LDL concentration-dependently decreased cathepsin D protein and enzyme activity.HNG concentration-dependent repaired of intracellular ox-LDL-induced decrease in cathepsin D protein expression and activity.HNG did not reduce lipid accumulation in HUVECs after BAF-A1 blocking autophagic flow.4.Ox-LDL had no effect on FPRL1 protein expression.FPRLI siRNA transfected HUVECs,HNG could not reduce lipid aggregation in HUVECs.FPRL1 siRNA or FPEL1 specific antagonist Boc-MLF treated HUVECs blocked FPEL1 pathway,HNG repaired the autophagic flow and the function of cathepsin D disappeared.Conclusion:Humanin can increase the protein level and the activity of cathepsin D by FPRL1 to repair the lysosomal function of ox-LDL-inducted injury,thereby increasing the autophagic flow of ox-LDL damage in HUVECs and further promoting ox-LDL degraded by autophagolysosomal pathways in HUVECs.