Study On The Effect And Regulatory Mechanism Of Protein 4.1R On Foam Cell Formation

Background:Atherosclerosis?Atherosclerosis,AS?is a disease of lipid metabolism and a chronic sterile inflammatory disease that has a huge impact on human health.Mononuclear cells in the blood migrate to the intima of the arteries and differentiate into macrophages.Macrophages uptake oxidized low-density lipoprotein through scavenger receptors on the membrane surface,load lipids,and eventually become"macrophage-derived foam cells?MDFC?",MDFC plays an important role at all stages of atherosclerotic lesion development,from initial lesions to advanced plaques.MDFC presents an inflammatory phenotype that secretes inflammatory cytokines.These cytokines act on other immune cells and exacerbate inflammatory responses.The migration ability of MDFC decreases,and it resides in the intima,which in turn causes plaque instability.At the same time,MDFC can secrete reactive oxygen species?ROS?.ROS promotes the production of various pro-inflammatory cytokines,exacerbate endothelial cell damage and LDL oxidation.Since MDFC plays an important role in development of AS,it is essential to explore the factors or molecules involved in its formation,which will provide new targets for the prevention and treatment of AS.CD36 is the main scavenger receptor for macrophage recognition and uptake of oxLDL.After CD36 binds to oxLDL,it triggers the activation of the downstream signaling pathway of CD36.These signaling pathways can promote the further uptake of oxLDL,increase the secretion of inflammatory cytokines,and inhibit cell migration,which together promote the formation of MDFC.Polymerized actin?F-actin?also plays an important role in the formation of MDFC.In MDFC,F-actin can inhibit cell migration and promote the internalization of CD36 and oxLDL complex.Protein 4.1R was originally discovered as a red blood cell membrane protein,and it has been shown to be widely expressed in eukaryotic cells.Protein 4.1R acts as an adaptor between the cytoskeleton and membrane proteins in a variety of cells.Protein 4.1R affects the function of the cytoskeleton and participates in F-actin-related cell activities.Protein 4.1R can affect cell function by affecting the expression of membrane receptors,and also by affecting the activation of downstream signaling pathways.Objective:In our study,oxLDL was used to induce wild-type(4.1R^+/+)and 4.1R gene-deficient(4.1R^-/-)macrophages to build an MDFC model.By studying the effect of protein 4.1R on the accumulation of cholesterol,inflammatory phenotype and migration ability in MDFC,we investigated the role of protein 4.1R in the formation of MDFC.Then,we studied the effect of protein 4.1R on the activation of CD36expression Lyn in MDFC,and the effect of protein 4.1R on the polymerization of actin in MDFC,to explore the mechanism of protein 4.1R involved in the formation of MDFC.Methods:1.The content of lipid droplets and cholesterol in cells was detected by oil red O staining and cholesterol content detection kit.2.Quantitative real-time PCR were used to detect 4.1R mRNA expression levels in MDFC.3.T7 Endonuclease I and Western Blot were used to verify the 4.1R gene knockout in macrophages.4.Oil red O staining and cholesterol test kit were used to detect cholesterol content in 4.1R^+/+MDFC and 4.1R^-/-MDFC.5.Quantitative real-time PCR and ELISA were used to detect the expression and secretion of inflammatory cytokines IL-1?,IL-6 and MCP-1 in 4.1R^+/+MDFC and 4.1R^-/-MDFC.6.Flow cytometry was used to detect ROS expression in 4.1R^+/+MDFC and4.1R^-/-MDFC.7.Wound-healing assay and Transwell experiments were used to examine the effect of 4.1R deletion on the ability of MDFC to migrate.8.Quantitative real-time PCR detection of CD36,SR-A mRNA expression levels in 4.1R^+/+MDFC and 4.1R^-/-MDFC.9.The uptake of oxLDL by 4.1R^+/+macrophage and 4.1R^-/-macrophages was detected by fluorescence microscopy and flow cytometry.10.Flow cytometry was used to detect the expression of CD36 on the surface of4.1R^+/+MDFC and 4.1R^-/-MDFC.11.Western Blot detected the activation of key signal molecules Lyn in 4.1R^+/+MDFC and 4.1R^-/-MDFC.12.Flow cytometry and immunofluorescence detection of actin aggregation in4.1R^+/+MDFC and 4.1R^-/-MDFC.Results:1.MDFC model successfully constructed using oxLDL.2.Protein 4.1R expression was increased in MDFC.3.T7 Endonuclease I and Western Blot confirmed the loss of 4.1R expression in4.1R^-/-macrophages at the gene and protein levels,respectively.4.Compared with 4.1R^+/+MDFC,4.1R^-/-MDFC decreased the lipid content and the accumulation of cholesterol in cells.5.The expression of proinflammatory cytokines IL-6,IL-1?,MCP-1 and ROS in 4.1R^-/-MDFC was significantly lower than in 4.1R^+/+MDFC.6.The migration capability of 4.1R^-/-MDFC is greater than 4.1R^+/+MDFC.7.The transcription level of CD36 in 4.1R^-/-MDFC was lower than that of4.1R^+/+MDFC.8.The uptake of oxLDL of 4.1R^-/-macrophages was lower than that of 4.1R^+/+macrophages.9.The expression of CD36 on the surface of 4.1R^-/-MDFC was lower than that of 4.1R^+/+MDFC.10.After oxLDL stimulation,the expression of p-Lyn proteins in 4.1R^-/-MDFC was lower than in 4.1R^+/+MDFC.11.Compared with 4.1R^+/+MDFC,4.1R^-/-MDFC has lower level of actin polymerization.Conclusion:Protein 4.1R plays a positive regulatory role in the process of MDFC formation.In MDFC,the deletion of protein 4.1R can reduce the accumulation of intracellular lipids.Moreover,the absence of protein 4.1R can reduce the inflammatory phenotype and increase the migration ability in MDFC.Furtherly,we found that protein 4.1R may participate in the formation of MDFC by reducing the expression of CD36 and reducing the activation of Lyn,as well as reducing the polymerization of actin.