The Role And Molecular Mechanism Of[Gly14]-humanin In Atherosclerosis

Objective:Atherosclerosis(AS)is the main pathological basis of ischemic cardiovascular and cerebrovascular disease.Humanin(HN)is a small endogenous peptides derived from mitochondria.Animal experiments have shown that exogenous S14G-HN(HNG)has anti-atherosclerotic effects,and its mechanism is poorly understood.Oxidative low-density lipoprotein(ox-LDL)-induced vascular endothelial cell damage is a key trigger point in the development of AS.Ox-LDL can also be taken up by vascular endothelial cel through a receptor-dependent pathway based on LOX-1,which in turn damages endothelial cells.This study aims to clarify the anti-atherosclerotic effect of HN in a high-fat diet ApoE-/-atherosclerosis model mouse,and to investigate whether this effect of HNG is related to the inhibition of LOX-1 receptor which mediating uptaking ox-LDL of vascular endothelial cells.Methods:Six-week-old male C57BL/6 mice with normal diet served as a control group.Male ApoE-/-mice were randomly divided into high-fat diet group(HFD group),high-fat diet with saline injection group(HFD+saline group),and high-fat diet with HNG injection group(HFD+HNG group),aortic arch embedded sections were taken 16 weeks later and HE stained.human umbilical vein endothelial cells(HUVECs)was treated with HNG at different concentrations for 12h,and the CCK8 kit was used to detect the effect of HNG on cell viability.HUVECs were pretreated with different concentrations of HNG for 1 h,and then HUVECs were co-incubated with ox-LDL for 12 h.Intracellular cholesterol content assay was used to detect cholesterol content in HUVEC.HUVECs was pretreated with different concentrations of HNG for 1 h,and then incubated with Dil-labeled oxidized low density lipoprotein(Dil-ox-LDL)for 12 h.The intracellular lipid accumulation of HUVECs was observed by flow cytometry.Western blot was used to detect the effects of ox-LDL and HNG on the expression of ox-LDL uptake receptor LOX-1and ERK pathway-related proteins in HUVECs.Annexin V-FITC/PI apoptosis kit was used to detect the effect of HNG on apoptosis.Results:1.The HE staining results showed that compared with the control group,the aortic plaque area in the HFD group was significantly increased,there was no difference in plaque area between the HFD+saline group and the HFD group,and the plaque area was significantly reduced in the HFD+HNG group.2.The CCK8 kit results showed that different concentrations of HNG did not damage HUVEC activity.Intracellular cholesterol content assay and flow cytometry results showed that HNG was able to inhibit ox-LDL-induced lipid accumulation in HUVECs in a concentration-dependent manner.3.Ox-LDL increased the expression of LOX-1 in a concentration-dependent manner,and HNG inhibited the increase of LOX-1 expression induced by ox-LDL.4.After knocking down LOX-1,the uptake of ox-LDL was significantly reduced,and HNG did not further reduce the uptake of Ox-LDL.3.The Annexin V-FITC/PI apoptosis kit results showed that ox-LDL concentration-dependently increased HUVECs apoptosis,HNG concentration-dependently decreased HUVECs apoptosis,and LOX-1 inhibitors inhibited ox-LDL-induced HUVEC apoptosis,HNG cannot further reduce apoptosis.Ox-LDL promoted phosphorylation of ERK in a time and concentration-dependent manner,HNG inhibited ox-LDL-induced ERK phosphorylation,and the ERK inhibitor PD98059 inhibited ox-LDL-induced LOX-1 expression increasing and intracellular lipid content increasing.Conclusion:The above results show that HNG exerts an anti-atherosclerotic effect.It reduces the uptake of ox-LDL by inhibiting the ox-LDL uptake receptor LOX-1 in HUVECs,and its regulation of LOX-1 is related to the inhibition of ox-LDL-induced activation of ERK pathway.