| Background:Cardiovascular disease is the leading cause of death and disability globally.Atherosclerosis is the main pathological basis of cardiovascular diseases.Studies have shown that BCL2-associated immortality gene 3 is involved in the process of cardiovascular disease by regulating epithelial mesenchymal transformation.Endothelial mesenchymal transformation is a special form of EMT in which endothelial cells with a specific phenotype are gradually transformed into mesenchymal cells with a spindle elongated morphology.We previously found that the expression of BAG3 was significantly reduced in the atherosclerosis model.This project explored the effect of BAG3 on atherosclerosis and its molecular mechanism.Purpose:The purpose of this study is to discover new regulatory factors of atherosclerosis,clarify the specific mechanism of their effects on the occurrence and development of atherosclerosis,and provide new ideas and strategies for the treatment and prognosis of atherosclerosis,which has very important social significance.Methods:In vivo experiment,in order to explore the effect of BAG3 on atherosclerosis,ApoE-/-mice were fed a high-fat diet for 12 weeks to establish an animal level atherosclerosis model.Mice were divided into（1）blank group,fed with normal diet for 12 weeks（2）model group,fed with high fat diet for 12 weeks（3）BAG3 overexpressed lentivirus group,injected with BAG3 lentivirus through tail vein,and fed with high fat diet for 12 weeks after successful modeling（4）CON335 lentivirus group,injected with tail vein CON335 lentivirus.After successful modeling,high-fat diet was fed for 12 weeks.After 12 weeks of feeding,the changes of serum lipid levels in mice were detected.The aorta of mice was stained with Oil Red O to observe the lipid deposition in aorta,and the expression of plateletendothelial cell adhesion molecule and α smooth muscle actin was observed by immunofluorescence.In vitro experiments,human umbilical vein endothelial cells were stimulated with oxidized low density lipoprotein to induce endothelial injury.cells were divided into（1）NC group,transfected NC（2）ox-LDL +NC group,transfected NC with 100 μg/m L ox-LDL solution,（3）ox-LDL+BAG3 group,transfected pc DNA-BAG3 with 100 μg/m L ox-LDL solution.Immunofluorescence,western blotting and real-time quantitative PCR were used to determine whether overexpression of BAG3 could affect the expression levels of CD31 and α-SMA.Transwell assay was used to detect the effect of BAG3 on cell migration.Studies have found that ZEB1 is the downstream target of BAG3 in thyroid cancer and participates in the regulation of EMT process.We speculate that BAG3 regulates EndMT by targeting ZEB1 in atherosclerosis.HUVECs were divided into four groups:（1）NC group,transfected NC（2）ox-LDL +NC group,100 μg/m L ox-LDL solution was added after transfection NC（3）ox-LDL +si ZEB1 group,100 μg/m L ox-LDL solution was added after ZEB1 si RNA transfection（4）ox-LDL +si ZEB1+si BAG3 group,100 μg/m L ox-LDL solution was added after ZEB1 si RNA and BAG3 si RNA transfection.The fluorescence intensity of CD31 and α-SMA was observed by immunofluorescence method.WB and q RT-PCR were used to detect the changes of CD31,α-SMA,vascular endothelial cadherin and fibroblast specific protein expression levels and the effect on cell migration.Results:The results of blood lipid level detection and aortic Oil Red O staining showed that overexpression of BAG3 significantly improved the blood lipid level of atherosclerotic mice and reduced the lipid deposition in the aorta of mice.In vitro experiments confirmed that ox-LDL stimulation induces EndMT in HUVECs.WB and q RT-PCR results showed that overexpression of BAG3 significantly inhibited the expression levels of α-SMA and FSP1,and increased the expression levels of CD31 and VE-cadherin.Compared with ox-LDL+NC group,overexpression of BAG3 could enhance the protein fluorescence intensity of CD31 and decrease the protein fluorescence intensity of α-SMA.Overexpression of BAG3 can effectively reduce EndMT-induced cell migration.Knockdown of ZEB1 expression in HUVECs significantly reduced endothelial cell injury.However,after transfection with ZEB1 si RNA and BAG3 si RNA,WB and q RT-PCR experiments showed that the knockdown of ZEB1 and BAG3 could inhibit the expression levels of α-SMA and FSP1 in HUVECs,and increase the expression levels of CD31 and VE-cadherin.Compared with oxLDL+si ZEB1 group,knockdown ZEB1 and BAG3 decreased the protein fluorescence intensity of CD31,enhanced the protein fluorescence intensity of α-SMA and enhanced the migration ability of HUVECs.Conclusion:In summary,our study showed that BAG3 significantly improved atherosclerosis in ApoE-/- mice.BAG3 improves EndMT induced by endothelial injury by acting on ZEB1. |
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