Effect Of Pigment Epithelium-derived Factor On Expression Of CD36 Protein On Surface Of Macrophages

BackgroundAtherosclerosis is one of the most important pathological causes of cardiovascular disease,AS patients are more likely to suffer from myocardial infarction,aortic stenosis,cerebral hemorrhage,peripheral arterial disease,etc.This is a serious threat to people's health.Lipid aggregation,inflammation and the damage of vascular endothelium are the internal factors for AS.One of the most important pathogenesis is the damage of vascular endothelium,it is because that the damage of vascular endothelium would increased endothelial permeability;changes in the balance of endothelial regulation;increased endothelin release;reduce the synthesis and secretion of NO;increased expression of endothelial adhesion molecules,like ICAM-1,VCAM-1,E-selectin and P-selectin;disrupting endothelial antithrombotic function.AS is also a chronic inflammatory disease,the clinical features of vascular smooth muscle cell proliferation and the formation of atherosclerotic plaque after blood lipid deposition,as is often visible millet congee kind of lipid in the artery wall deposition and stenosis of artery wall caused by artery elasticity decreased.Clinical studies have shown that AS is the most important cause of coronary heart disease,the number of deaths due to coronary heart disease every year in China ranks second in the world,and it is the first cause of death in China.According to the results of epidemiology and cause of death statistics,in the next ten years,the death rate of coronary heart disease in our country will continue to rise.Therefore,it is a hot topic that how to detect the pathological changes and effective treatment of AS.The formation of AS is the beginning stage of macrophage migration of lipoprotein into foam cells.In the whole arsenic process,the phagocytosis of macrophage and the inflammatory reaction of macrophage are always accompanied by macrophages.Macrophage Devour is mediated by cd36,and cd36 is the most important receptor.CD36 is a kind of class B scavenger receptor,which is highly expressed in monocytes,macrophages,microvascular endothelial cells,smooth muscle cells,adipocytes and platelets.CD36 gene is located in chromosome 7 of q11.2,its main function in the human body includes the following two aspects,one is promoting the extraction of specific lipid molecules,just like oxidized low density lipoprotein and Long Chain Fatty Acid;the other is combining with biological macromolecules,through the conduction of cell signaling,triggering the related inflammation,phagocytosis and apoptosis and other cell processes.Pigment epithelium derived factor(PEDF)is a kind of serine protease inhibitor,it does not have the function of inhibiting protease,and is located in the short arm of chromosome 17p13.1 region.PEDF has eight exons and seven introns,the charge distribution of three-dimensional structure has obvious asymmetry.PEDF is a multifunctional protein,in addition to the role of nutrition,but also can play a protective role in the body's nerves.Therefore,it can resist tumor,angiogenesis and vascular permeability,and inhibit the formation of thrombosis and inflammation.PEDF can inhibit the expression of-P selectin on platelet surface,it also can inhibit the activation and aggregation of platelets,block the production of ROS by NADPH oxidase and collagen,and inhibit the formation of thrombus and protect blood vessels.Recent study found that PEDF has inhibitory effect on macrophage inflammatory response,and has protective effect on the stability of AS.CD36 is the key receptor that mediates the phagocytosis of lipids by macrophages.The increase in the expression of CD36 receptor can promote the phagocytosis of macrophage,and thus promote the progress of AS.We speculated that PEDF could inhibit the phagocytosis of macrophage by inhibiting the expression of CD36,and thus play the role of resistance to.Objective1.In vivo experiments revealed the effect of overexpression of PEDF on the expression of CD36 in atherosclerotic plaques of apoE-/-mice.2.In vitro experiments revealed the influence of PEDF on the expression of scavenger receptor CD36 in macrophages.MethodIn vivo,20 male ApoE-/-8-week-old mice as research object,the experimental animals were fed at the animal experimental center of Qilu Hospital of Shandong University for 1 weeks,divided into two groups:the control group and PEDF group,there are 10 mice in each group.The feeding method used the high fat diet(high fat diet:mouse basic feed is 94.3%,lard oil is 3%,cholesterol is 2%,sodium cholate is 0.5%,propylthiouracil is 0.2%.After the material in accordance with the proportion of good,high fat feed drying preservation,used in the feeding of experimental mice.)After feeding for 16 weeks,the mice in group PEDF were injected with PEDF,andthe dosage of injection was 1×108 TU/case,mice in control group were given the same dose of negative virus.After 4 weeks,the mice were sacrificed and the tissue of the root of the aorta was taken into 4%paraformaldehyde solution,and the routine paraffin embedded and slice were used.The RAW264.7 cells were cultured on 6 well plates,the use of high glucose(10%DMEM fetal bovine serum,1%penicillin/streptomycin)cell adherent culture 24h were divided into four groups,respectively ox-LDL group,negative ox-LDL+ virus transfection(G)group,ox-LDL+PEDF group and Con group.When the cells were adherent to the 8h,the PEDF surrounded by adenovirus was transfected into ox-LDL+PEDF cells with MIC value of 30.The transfection of 24h cells was observed after transfection..ox-LDL group g/ml,ox-LDL+G group and ox-LDL+PEDF group were given ox-LDL80,respectively,and were not given to group Con.The expression of CD36 protein was detected using immunohistochemical staining and operated in accordance with the procedures of the SP-9001 immunohistochemistry kit.Immunohistochemistry was used as a Rabbit anti mouse CD36 polyclonal antibody,which was diluted according to the proportion of 1:100.The negative control was PBS.The color of DAB solution used fresh good configuration,color time after 3-5min use tap water to fully flush,using hematoxylin stained,again for dehydration,transparent,neutral balata.The mice in each case specimens were placed in 400 times under the vision of the microscope,the 4 visual fields of each section in aortic tissue distribution and uniform staining,using Image pro-Plus 6 image analysis system for the quantitative analysis of the results.The cells with brown reactive granules were positive,and the positive expression of CD36 protein was calculated by the ratio of the positive expression area and the total area under the visual field.The expression of CD36 protein in RAW264.7 cells was detected by Western blot.Each group of RAW264.7 cells were collected after the extraction of the total protein in each group of cells,using Bradford method to determine the concentration of protein,placed in the environment of-40?.The protein extracts using 10%SDS polyacrylamide gel electrophoresis,transmembrane protein,a closed anti CD36 with the ratio of 1:1000 at a constant temperature of 4? under overnight incubation,two anti incubating for 2h,ECL light after incubating for 5min protein in Image pro-Plus 6 image analysis system for image scanning.The gray value of purpose protein and the ratio of gray reference protein as the relative protein expression level.Use SPSS 20.0 software for statistical analysis of experimental data,the measurement data in(x±3)expression is described,comparison between groups using T test.There was statistical difference between P<0.05.Result1.Expression of CD36 protein in mouse macrophagesThe expression of CD36 protein on the surface of macrophages in the aortic root tissue of two groups of mice was measured.The results showed that the AS group was the control group,the A group was group,the B group was PEDF.In PEDF group the expression of CD36 was(29.00±0.58)%,the control group of mice CD36 expression for(14.00±1.16)%,two groups of mice were compared,the difference was statistically significant(P<0.05),shows that compared with the control group,PEDF mice aortic root at the macrophage surface the expression of CD36 protein was significantly lower.2.Expression of CD36 protein in RAW264.7 cellsUsing Western blotting of CD36 RAW264.7 cells in the four groups of the protein expression was detected,the results shown in Figure 3 in group ox-LDL(0.75±0.06),ox-LDL+G group(0.83±0.07),ox-LDL+PEDF group(0.55±0.00),Con group(0.34±0.03).The Con group as the contol group,the expression level of CD36 protein in ox-LDL group and ox-LDL+G group cells were higher than group Con,there are differences(P<0.05),the expression level of ox-LDL+PEDF cells CD36 protein was significantly lower than that of ox-LDL group and ox-LDL+G group(P<0.05).3.Detection of CD36 expression in RAW264.7 cells by RT-PCRThe use of RT-PCR was detected on the expression of RAW264.7 cell CD36 of the four groups,the results shown in Figure 3,the ox-LDL group(6.93±0.14),ox-LDL+G group(8.34±0,16),ox-LDL+PEDF group(1.81 ±0.23),Con group(1.01±0.03).Group Con as control group,compared with ox-LDL group and ox-LDL+G group,the expression of CD36 increased significantly(P<0.05),ox-LDL+ PEDF group compared with ox-LDL group and ox-LDL+G group,the expression of CD36 was significantly decreased(P<0.05).Conclusion1.PEDF can inhibit the expression of CD36 on macrophage surface.2.After ApoE-/-mice were transfected with PEDF virus vector,the expression of CD36 in AS plaques was significantly inhibited.