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 105μg/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（α-SMA）,smooth muscle 22alpha（SM22α）and fibroblast specific protein（FSP）-1 and Adropin by real-time fluorescence quantitative polymerase chain reaction（RT-PCR）.Localized expression of CD31 and SM22αwas 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-βplasmid group,transfection of TGF-βplasmid 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）-β1,TGF-β2,CD31,VE-cadherin,α-SMA,SM22αand phosphorylation level of signal protein Smad2/3 were measured by Western-Blot.The expression ofα-SMA was measured by immunofluorescence and the relative expression of RNA of TGF-β1,TGF-β2,TGF-βreceptor（TGF-βR）,CD31,VE-cadherin,α-SMA,SM22α,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 ofα-SMA and FSP-1 increased[α-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;α-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 andα-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（α-SMA,SM22α）,signal pathway related factors（TGF-β1 and TGF-β2）,and downstream signal protein P（phosphorylation）-Smad2/3 in TGF-βincreased.Adropin reversed the above changes[H₂O₂+Adropin vs H₂O₂,spindle cells,（0.19±0.05）vs（0.57±0.07）%,P<0.05;α-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-β（Adropin+TGF-βgroup）inhibited the above-mentioned effects of Adropin on endothelial cell interstitial transformation and inhibited the TGF-β/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-β/Smad2/3 signaling pathway is involved in the above-mentioned process.