Adropin Protects Against Atherosclerosis By Regulating Endothelial-mesenchymal Transition And Its Mechanism In ApoE^-/- Mice

Objective:Atherosclerosis?AS?is a serious hazard to human health,of which endothelial injury is the initiating factor.Endothelial-mesenchymal transition?EndMT?is involved in the early pathological process of AS,but the exact mechanism is still unclear.Adropin is a newly discovered secretory membrane-binding protein with protective effect on endothelial cells.The aim of this study was to investigate whether Adropin can alleviate AS and its molecular mechanism by inhibiting EndMT.Methods:Animal experiment:ApoE^-/-?apolipoprotein E knockout?mice aged 8 weeks with C57BL/6J background were fed with high fat for 13 weeks to establish AS model,which were divided into three groups?n=11-14?:?1?Normal dieting group?ND?,given normal diet;?2?High fat dieting group?HFD?,given high fat diet;?3?High fat dieting+Adropin group?HFD+Adropin?,given high fat diet and intraperitoneal injection of Adropin 105?g/kg.d for 13 weeks.The aortic root was used to detect the lipid area in AS plaque by oil red O staining,and the plaque area was measured by HE staining.The whole aorta was used to detect relative mRNA expression of endothelial cell marker proteins,including leucocyte differentiation antigen?CD?31 and vascular endothelial cadherin?VE-cadherin?,as well as mesenchymal cells marker proteins,including alpha-smooth muscle actin??-SMA?,smooth muscle 22alpha?SM22??and fibroblast specific protein?FSP?-1 and Adropin by real-time fluorescence quantitative polymerase chain reaction?RT-PCR?.Localized expression of CD31 and SM22?was detected by immunofluorescence assay.Cell experiment:EndMT model was established by using human umbilical vein endothelial cells?HUVEC?cultured in vitro with 100mol/L hydrogen peroxide?H₂O₂?.They were divided into four groups:control group,with normal culture;H₂O₂ group,with H₂O₂ intervention;Adropin group,incubation with30ng/ml of Adropin 10 minutes before H₂O₂ intervention;Adropin+TGF-?plasmid group,transfection of TGF-?plasmid before Adropin intervention.The number of spindle cells transformed from mesenchymal at day 0,2,4 and 6 after culture was counted.At the end of the experiment?day 6?,the expression of transforming growth factor?TGF?-?1,TGF-?2,CD31,VE-cadherin,?-SMA,SM22?and phosphorylation level of signal protein Smad2/3 were measured by Western-Blot.The expression of?-SMA was measured by immunofluorescence and the relative expression of RNA of TGF-?1,TGF-?2,TGF-?receptor?TGF-?R?,CD31,VE-cadherin,?-SMA,SM22?,FSP-1 were measured by RT-PCR.The level of reactive oxygen species?ROS?was measured by fluorescence.Serum total cholesterol?TC?,low density lipoprotein cholesterol?LDL-C?,high density lipoprotein cholesterol?HDL-C?,and triglyceride?TG?were detected by automatic biochemical analysis.Serum oxidized low density lipoprotein?ox-LDL?was detected by ELISA.Results:Animal experiment:Compared with ND group,high fat diet?HFD group?induced significant AS in ApoE^-/-mice.The lipid area?oil red O staining?and plaque area?HE staining?of aortic root AS plaque in HFD group increased[oil red O staining,?0.06±0.02?%vs.?0.27±0.16?%,P<0.05;HE staining,?0.12±0.04?%vs.?0.30±0.08?%,P<0.05],Adropin?HFD+Adropin group?can alleviate AS caused by high fat feeding[oil red O,HE staining,respectively?0.04±0.03?%and?0.10±0.08?%,all P<0.05].Compared with ND group,HFD group showed significant mesenchymal transformation of vascular endothelium in HFD group,which showed that the expression of RNA of CD31 and VE-cadherin in aorta decreased[CD31,?1.71±0.68?vs.?0.23±0.34?,P<0.05;VE-cadherin,?11.24±5.44?vs.?0.36±0.51?,P<0.05],the expression of?-SMA and FSP-1 increased[?-SMA,?0.03±0.01?vs.?2.34±0.99?,P<0.05,FSP-1,?0.27±0.17?vs.?1.92±0.60?,P<0.05],Compared with HFD group,Adropin reduced vascular endothelial-mesenchymal transition[HFD+Adropin group:CD31,?1.54±0.18?,P<0.05;?-SMA,?0.15±0.24?,P<0.05;FSP-1,?0.29±0.30?,P<0.05].Compared with ND group,the expression of Adropin was increased in HFD group,and the expression of Adropin was decreased by the intervention of exogenous Adropin.Compared with ND group,serum TC,LDL-C,ox-LDL,HDL-C levels in HFD group increased and body weight increased,but No effect to TG,whereas Adropin intervention failed to reduce the blood lipid and body weight.Cell experiment:Compared with Control,H2O2 promoted the morphological change of endothelial cells?especially on the 6th day?,which showed the increase of spindle cells and?-SMA positive cells.At the same time,the expression of mRNA and protein of endothelial cell markers?CD31 and VE-cadherin?decreased,while the expression of mesenchymal cells markers??-SMA,SM22??,signal pathway related factors?TGF-?1 and TGF-?2?,and downstream signal protein P?phosphorylation?-Smad2/3 in TGF-?increased.Adropin reversed the above changes[H₂O₂+Adropin vs H₂O₂,spindle cells,?0.19±0.05?vs?0.57±0.07?%,P<0.05;?-SMA positive cells,?0.10±0.04?vs?0.93±0.01?%,P<0.05][p-Smad2/3,Control,?0.36±0.25?;H2O2,?0.74±0.31?;Adropin?0.34±0.21?,Adropin vs H₂O₂,P<0.05].Transfection of TGF-??Adropin+TGF-?group?inhibited the above-mentioned effects of Adropin on endothelial cell interstitial transformation and inhibited the TGF-?/Smad2/3 signaling pathway.Conclusions:Adropin can alleviate atherosclerosis in mice through non-lipid-regulating pathway,and its mechanism may be related to the inhibition of endothelial-mesenchymal transition.TGF-?/Smad2/3 signaling pathway is involved in the above-mentioned process.