CREG1 Alleviates Atherosclerosis Via Increasing SRA Degradation Through Lysosome

BackgroundCardiovascular diseases are the main cause of death worldwide,and atherosclerosis is the dominant cause of cardiovascular diseases.Therefore,further research on the biological mechanism of the occurrence and development of atherosclerosis is of great significance for the prevention and treatment of atherosclerosis.Cellular repressor of E1A stimulated genes 1（CREG1）can regulate cell proliferation,differentiation,and senescence,and plays an important role in the biological process of vascular associated disease.Previous studies have found that CREG1 expression is decreased during the process of atherosclerosis,and exogenously added recombinant CREG1 has an anti-atherosclerosis effect.Macrophages phagocytose oxidized low-density lipoprotein（OX-LDL）and transform into foam cells is crucial for the development of atherosclerosis.The phagocytosis and metabolism of OX-LDL by macrophages are closely related to scavenger receptor class A member 1（SRA）and lysosome.Therefore,this study conducted an in-depth study on the anti-atherosclerosis effect of CREG1 and took the regulation of SRA and lysosome function of CREG1 as the research focus.With this research focus,we explored the regulatory mechanism of the biological process of macrophages transforming into foam cells by CREG1,to provide a new intervention target for the prevention and treatment of atherosclerosis.Objectives1.To determine CREG1 levels in macrophages has a regulatory effect on the occurrence and development of atherosclerosis.2.To explore the mechanism of CREG1 affects the transformation of macrophages into foam cells in the atherosclerotic microenvironment.3.To clarify the mechanism of CREG1 regulates SRA,which is a key molecule in the transformation of macrophages into foam cells.Methods1.The atherosclerosis model has been constructed by feeding apolipoprotein E-deficient(Apo E^-/-)mice with Western diet（WD）.The levels of CREG1 in various tissues were detected by western blot（WB）and quantitative real-time polymerase chain reaction（q RT-PCR）2.Apo E^-/-background CREG1 macrophage-specific knock out mice(Apo E^-/-CREG1^fl/flLyz2^cre)were fed with WD to establish an atherosclerosis model.Oil red O staining was used to detect the degree of atherosclerosis in mouse aorta and aortic root.Immunofluorescence staining was used to detect the level of SRA in aortic root atherosclerotic plaques.The expression levels of related proteins in atherosclerotic plaques were detected by WB and q RT-PCR.3.Apo E^-/-background mouse bone-marrow-derived macrophage（BMDM）was extracted from CREG1 macrophage-specific knock out mice and treated with OX-LDL.Oil red O staining was used to detect the formation of foam cells.The level of SRA mediated phagocytosis was detected by fluorescence labeling of acetylated low-density lipoprotein.The expression levels of related proteins were detected by WB and q RT-PCR.4.Apo E^-/-background mice were injected with adeno-associated virus-F4/80-CREG1（AAV-F4/80-CREG1）and then fed with WD to establish an atherosclerosis model.Oil red O staining was used to detect the degree of atherosclerosis in mouse aorta and aortic root.Immunofluorescence staining was used to detect the level of SRA in aortic root atherosclerotic plaques.The expression levels of related proteins in atherosclerotic plaques were detected by WB and q RT-PCR.5.BMDM was extracted from Apo E^-/-background CREG1 macrophage specific overexpression mouse（AAV-F4/80-CREG1）and stimulated with OX-LDL.Oil red O staining was used to detect the formation of foam cells.The level of SRA mediated phagocytosis was detected by fluorescence labeling of acetylated low-density lipoprotein.The expression levels of related proteins were detected by WB and q RT-PCR.6.Mouse macrophage cell lines were transfected with si RNA targeting CREG1and si RNA targeting SRA to achieve CREG1 and SRA knockdown,then treated with OX-LDL.Oil red O staining was used to detect the formation of foam cells.7.Mouse macrophage cell lines were transfected with CREG1 plasmid and SRA plasmid to achieve overexpression of CREG1 and SRA in macrophages,then treated with OX-LDL.Oil red O staining was used to detect the formation of foam cells.8.Mouse macrophage cell lines were transfected with CREG1 plasmid,and protein synthesis inhibitors,proteasome degradation inhibitors,and lysosomal degradation inhibitors were respectively given under OX-LDL stimulation.The expression levels of related proteins were detected by WB.9.Mouse macrophage cell lines were transfected with CREG1 plasmid.After OX-LDL stimulation,lysosome labeling dye was used to detect lysosome acidification.Results1.In vivo results showed that CREG1 expression was decreased in atherosclerotic plaques,the degree of atherosclerosis was significantly increased in Apo E^-/-background CREG1 macrophage-specific knock out mice,and the level of SRA was increased in atherosclerotic plaques.In vitro results show that macrophage CREG1deficiency leads to increased SRA levels,increased foam cell formation,and increased SRA mediated phagocytosis in the atherosclerotic microenvironment.2.In vivo studies showed that the degree of atherosclerosis in Apo E^-/-background CREG1 macrophage-specific overexpression mice was significantly reduced,and the level of SRA in atherosclerotic plaques was reduced.In vitro studies have shown that CREG1 overexpression in macrophages leads to decreased SRA levels,decreased foam cell formation,and decreased SRA mediated phagocytosis in the atherosclerotic microenvironment.3.In vitro studies showed that SRA knockdown inhibited the increase in foam cell formation caused by CREG1 deficiency in macrophages,and SRA overexpression inhibited the decrease in foam cell formation caused by CREG1 overexpression in macrophages.4.In vitro studies showed that CREG1 did not affect the m RNA level and protein synthesis process of SRA,but could increase the degradation of SRA,mainly through lysosome degradation pathway,and could promote lysosome function.ConclusionsThe level of CREG1 in macrophages is closely related to the occurrence and development of atherosclerosis.In the atherosclerotic microenvironment,macrophage CREG1 can increase the degradation of SRA through lysosomes,reduce the phagocytosis of OX-LDL by macrophages,inhibit the formation of foam cells,and alleviate atherosclerosis.