| Background:Midkine（MK）is a heparin-binding growth factor,which is significantly increased in patients with atherosclerosis（As）-related cardiovascular diseases.Studies found that MK played an important role in cardiovascular diseases,and associated with the occurrence and development of As,some studies indicated that MK was considered as one of the independent predictors of cardiovascular events,however,the specific mechanism of MK involved in atherosclerosis remains unclear.The ATP binding cassette A1（ABCA1）is an important protein that mediates cholesterol efflux from macrophages,which promotes the process of reverse cholesterol transport and affect the occurrence and development of As.Studies have demonstrated that MK inhibited the expression of ABCA1 and reduced the cholesterol efflux from macrophages,however,the molecular mechanism was widely unknown.The aims of this study are observed the effect of MK on high-fat diet apoE-/-atherosclerosis model mice and the changes of protein expression,explore the roles of MK in the occurrence and development of atherosclerosis and its preliminary molecular mechanismMethods:1)Male apoE-/-mice were used as the research object（randomly group,6 in each group）and fed with high-fat diet for 6 weeks to replicate the animal model of As,and intraperitoneally inject MK,SR11302（AP1 inhibitor）and Wortmannin（PI3K inhibitor）etc.Biochemical analyzer detected blood lipid levels,HE staining observed the changes of structure in mouse arterial tissue and liver tissue,oil red O staining observed lipid accumulation;Immunohistochemistry and Western blotting methods detected the changes of ABCA1,AP1 protein expression in mouse aorta respectively;2)Culture RAW264.7 macrophages in vitro and induced with 50 mg/L oxidized low-density lipoprotein（ox-LDL）,carry out the following experiments:（1）the cells were loaded with MK（0,50,100,200 ng/ml）or16 h or 200 ng/m L MK for different times（0,8,16,24,48 h）,q RT-PCR and Western blotting detected the expression of ABCA1 and AP1;（2）Interfered with AP1 and PI3K signal molecules by inhibitors,Western blotting detected the changes of corresponding protein expression.Results:1)High-fat feeding apoE-/-mice replicated the As model,in the experiment,compared with the control group,the blood lipid levels of apoE-/-mice were increased after treated with MK（P<0.05）,the As lesion area in the arterial sinus were increased（P<0.05）,the lipid accumulation in the lesion and the liver were increased,and the expression of ABCA1 the arterial was down-regulated,and the expression of AP1 was up-regulated;2)After the high-fat apoE-/-mice were intervened by AP1 inhibitor SR11302,compared with the MK treatment group,the blood lipid levels of MK+SR11302 co-treatment group were decreased（P<0.05）,and As lesions in the aortic sinus were reduced（P<0.05）,lipid accumulation and fibrosis in the lesions were reduced;and the expression of ABCA1 in the aortic tissue of the MK+SR11302 co-treatment group was up-regulated.3)After high-fat apoE-/-mice were treated with PI3K inhibitor Wortmannin,compared with mice treated with MK alone,the blood lipid levels of the MK+Wortmannin co-treatment group was reduced（P<0.05）,the As lesions in the arterial sinuses were reduced,and the degree of lipid accumulation was reduced;protein detection found that the expression of AP1 in aortic tissues of the MK+Wortmannin co-treatment group was down-regulated,the expression of ABCA1 was up-regulated.4)In vitro culture of RAW264.7 macrophages,in the experiment,MK down-regulated the expression of ABCA1 in RAW264.7 macrophages;MK also up-regulated AP1 expression in a time-and concentration dependent manner;after intervened AP1 by AP1 inhibitor SR11302,compared with the group treated with MK alone,the expression of ABCA1 in co-treated with MK+SR11302 group was up-regulated,and the intracellular lipid accumulation was reduced.In addition,the phosphorylated protein detected results showed that after treat with MK,the expression of phosphorylated PI3K protein in macrophages was up-regulated.Conclusion:1.MK increased the area of As lesions in high-fat diet apoE-/-mice,increased lipid accumulation,and down-regulated the expression of ABCA1;2.AP1may participated in regulation of MK on the ABCA1 expression and affected the occurrence and development of As;3.The phosphorylation of PI3K signal molecule was one of essential mechanism that mediated MK to regulate AP1 and ABCA1... |
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