| BackgroundAtherosclerotic cardiovascular disease(ASCVD)is the main fatal disease at home and abroad.At present,the primary and secondary prevention strategies of ASCVD are mainly to seek to correct abnormal serum cholesterol levels,but studies have confirmed that low density lipoprotein cholesterol(LDL-C)can be reduced to very low levels even through active intensive lipid-lowering therapy.The incidence of major cardiovascular events(cardiovascular death,non-fatal myocardial infarction,readmission of unstable angina pectoris,coronary revascularization and non-fatal ischemic stroke)decreased by only about 30%.A large number of studies have shown that atherosclerosis(AS)is an inflammatory disease,and the inflammatory reaction is accompanied by the whole process of the occurrence and development of AS.Clinical studies have proved that anti-inflammatory therapy can achieve obvious clinical effects and reduce cardiovascular events.Macrophages are the core site of inflammatory reaction and foam cell formation in the occurrence and development of AS.Therefore,finding new targets that can affect the inflammatory response of macrophages and elucidating its specific mechanism may be a new idea for the prevention and treatment of AS,and can open up a new way for clinical treatment of ASCVD.Proline/Serine-rich coiled-coil proteinl(PSRC1),encoded by cellular PSRC1 gene,is a microtubule binding protein that is essential for the mitotic process of normal eukaryotic cells.In 2007,Samani NJ et al published a genome-wide association study(GWAS)on coronary artery disease(CAD)in NEJM,which proved for the first time that PSRC1 gene polymorphism is associated with coronary heart disease.Subsequently,studies have confirmed that the genetic polymorphism of PSRC1 is associated with the occurrence of AS in people of different races.At the same time,through the analysis of quantitative trait loci of gene expression,the researchers found that the level of PSRC1 gene expression was negatively correlated with the incidence of CAD.These studies have confirmed that PSRC1 gene is related to the occurrence of AS,but the exact mechanism of its action is still unknown.ObjectivesThe purposes of this study are to identify the interaction between ANXA2 and PSRC1 at the cellular level in vitro;to clarify the specific mechanism of the interaction between ANXA2 and PSRC1 to reduce the release of ANXA2 in vitro and in vivo;to explore the mechanism of the interaction between ANXA2 and PSRC1 to inhibit macrophage activation and slow down AS;to clarify how ANXA2 and PSRC1 interact and reduce the release of ANXA2,and how to regulate macrophage activation and inhibit the occurrence and development of AS,so as to providenew targets for antiinflammatory therapy of A S.MethodsCellular level:RAW264.7 cells were divided into Ad-PSRC1+oxLDL group and Ad-GFP+oxLDL group,the proteins bound to PSRC1 stimulated by oxidized low density lipoprotein(ox-LDL)were identified by unlabeled quantitative proteomics,the results of protein spectrum were verified by immunoprecipitation(Co-IP),and the intracellular co-localization of PSRC1 and ANXA2 was detected by immunofluorescence.In addition,in Ctrl group,ox-LDL group,Ad-GFP+oxLDL group and Ad-PSRC1+oxLDL group,the level of ANXA2 in the supernatant of the four groups was detected by ELISA to determine the effect of overexpression of PSRC1 and stimulation of oxLDL on the level of ANXA2 in the culture supernatant,and the mRNA expression of ANXA2 in RAW264.7 cells was detected by transfection of AdGFP or Ad-shANXA2,RT-PCR to identify the knockdown effect of Ad-shANXA2.Animal level:the model of ApoE-/-mice with ANXA2 knockdown was established by intravenous injection of Ad-shANXA2.The mice were divided into four groups:chow diet+Ad-GFP group(n=6),chow diet+ Ad-shANXA2 group(n=6),high fat diet+ Ad-GFP group(n=6)and high fat diet+Ad-shANXA2 group(n=6).The body weight,blood lipids and blood glucose levels of mice were measured.The levels of interleukin-1 β(IL-1β)and interleukin-6(IL-6)in serum of mice were detected by ELISA test.The gross and root sections of mouse aorta were separated and stained with Oil Red O to observe the area of atherosclerotic(AS),the lipid content of AS lipid plaque was observed by HE staining,and the collagen fiber content and plaque stability were observed by Masson staining.ResultsThe binding between PSRC1 and ANXA2 was significantly increased under the stimulation of ox-LDL,and the binding between them was specific.PSRC1 and ANXA2 were co-localized in cells.The level of ANXA2 was significantly decreased after shANXA2 transfection into RAW264.7 cells(P<0.05).After PSRC1 overexpression in RAW264.7 macrophages,the ANXA2 supernatant of cell culture was significantly decreased(P<0.05).After decreasing the expression of ANXA2,the plaque area of aorta and aortic root of mice was significantly decreased(P<0.05),and the secretion of IL-1 β and IL-6 in serum was significantly decreased(P<0.05).Knock down of ANXA2 expression could reduce body weight and improve blood lipid level in mice.At the same time,plaque area,lipid core area and macrophage infiltration in plaque were significantly decreased,fiber content and smooth muscle quantity of aortic plaque were increased,and plaque stability was also increased.ConclusionsOver-expression of PSRC1 in macrophages can attenuate the progression of atherosclerosis,and the mechanism may be related to the increased binding of PSRC1 to ANXA2,and the inhibition of the release of ANXA2 into the extracellular environment,thereby inhibiting the release of inflammatory factors. |
