The Effect And Mechanism Of PSRC1 Overexpression On Lipid Metabolism And Inflammatory Response In Macrophages

BackgroundCurrently,with ASCVD getting more and more attention,which is the most important component of CVD,scholars are more and more profound about the theroy of AS."Lipid infiltration theory","damage-response theory" and "inflammation theory" have become the mainstream theories of AS pathogenesis.Atherosclerosis is a chronic inflammatory disease arising from an imbalance in lipid metabolism and a maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall.Among them,the formation of macrophage-derived foam cells is a major feature of the development of AS.The main manifestation of AS is that vascular endothelial cells are damaged by the continuous chronic damage of modified LDL.Inflammatory cells such as monocytes and lymphocytes adhere and accumulate in the damaged endothelium,and monocytes differentiate into macrophage uptake Lipids and lipoproteins form foam cells by scavenger receptors.Inflammatory cells accumulate a large number of inflammatory mediators such as tumor necrosis factor alpha,interleukin 6,interferon gamma etc.,and then promote the inflammatory response and accelerate the progress of AS.Therefore,looking for new targets of anti-AS and CVD from the perspective of lipid metabolism and inflammation has great scientific significance and clinical value.PSRC1,a proline/serine-rich coiled-coil protein 1,also known as DDA3,is a coding protein.Earlier studies with PSRC1 focused on tumors,cell proliferation,cell division,Adjustment and other fields.After the publication of the human genetic map in the 21st century,Genome-Wild Association Studies(GWAS)published by Samani NJ et al.in the New England Journal of Medicine for the first time in 2007 confirmed that the expression level of PSRC1 gene is closely related to the total cholesterol concentration in the blood.Subsequently,some data showed that there was a potential new multi-effect association between two related SNPs and CRP levels in CELSR2/PSRC 1/SORT1 locus,which may provide a new understanding of inflammatory pathways.Thus,PSRC1 was closely associated with inflammatory response of lipid metabolism and coronary heart disease.In addition,our previous study found that serum amyloid P stimulated mouse macrophages,the cholesterol transfer rate was significantly increased,decreased intracellular cholesterol,cell foaming decreased.At the same time,gene sequencing suggested that PSRC1 gene expression was increased in SAP-stimulated group compared with control group.Silencing PSRC1 gene could reverse the biological effect of SAP on anti-macrophage foam formation.In summary,PSRC1 and lipid metabolism,inflammation and coronary heart disease are closely linked.However,PSRC1 specifically depends on what signaling pathway to play a role in anti-AS data are reported less.Therefore,.In this study,the recombinant adenovirus was transfected into RAW264.7 macrophage model to investigate the effect and mechanism of PSRC1 overexpression on lipid metabolism and inflammatory response in macrophages.ObjectivesTo investigate the effect and mechanism of PSRC1 overexpression on lipid metabolism and inflammatory response by recombinant adenovirus transfection in RAW264.7 macrophages.Methods1.Construction of recombinant adenovirusThe mouse PSRC1 gene sequence was obtained from Genebank.The construction of Ad-PSRC1 and Ad-GFP performed by Obio Technology(China).2.Cell culture and Group of ExperimentsRAW264.7 macrophages in DMEM containing 10%fetal bovine serum and maintained at 37? in a humidified atmosphere of 5%C02.After 12h,the cells were transfected with Ad-GFP and Ad-PSRC1 for 48h.After the starvation treatment of the macrophages for 24h,the macrophages were incubated with 100 ?g/ml oxidized low-density lipoprotein,and the samples were collected for follow-up detection.The experimental was divided into two groups:Ad-GFP and Ad-PSRC 1.3.Detection of lipid content in macrophagesOil red O staining to detect the accumulation of lipid of macrophages,the quantitative values by oil red O positive area percentage of total cellular area(as per the average data of 6 photomicrograph).4.Detection of IL-6 and TNF-a by ELISAAfter the treatment of macrophage,the cell supernatant were extracted by 4?centrifugal machine with 1000g/min in 10min.The detection of IL-6 and TNF-?concentration in accordance with the instructions of the ELISA kit.5?Detection of the mRNA expression by RT-qPCR in macrophagesTotal mRNA was extracted from cultured cells treated with 100 ?g/ml ox-LDL using TRIzol reagent in accordance with the manufacturer's instructions.The mRNA was reverse transcribed using a PrimeScriptTM RT Reagent Kit.Real-time quantitative PCR(RT-qPCR)was performed on a LightCycler 480 II with a SYBR Premix Ex Taq kit.The level of ?-actin mRNA expression was used as the internal control.Quantitative measurements were determined using the 2-??ct method.6?Detection of the protein expression by Western Blot in macrophagesProteins were extracted from macrophages treated with 100 ?g/ml of ox-LDL by homogenization in lysis buffer according to the product information manual.The protein expression were detected by western blot.7?Statistical analysesTotal Data are presented as the mean ±SD.The results were analyzed by Student's t-test followed by the Student-Newman-Keuls test using SPSS 22.0.One-way ANOVA was used to analyze means among three groups.Welch calibration test was used for variance heterogeneity.Post-hoc comparisons were made using least significant difference(LSD)test.Dunnett's T3 test was used when the variances are heterogeneities.P<0.05(two-tailed)was considered statistically significant.Results1?Recombinant adenovirus Ad-PSRC1 and Ad-GFP transfected with the optimal MOI of the macrophagesTo evaluate the efficacy of PSRC1 gene transfection,murine RAW264.7 macrophages were transfected with different multiplicities of infection(MOI=10,100,200 and 400)of Ad-GFP.Concentration-dependent GFP protein expression(green fluorescence)was detected using a Fluorescence Inversion Microscope System after 48 h treatment.The result showed that with the increase of MOI value,the fluorescence intensity increased gradually,indicating that the transfection efficiency of the virus increased correspondingly,and there was no obvious effect on the cell state.2?The mRNA and protein expression of PSRC1 after transfectionThe process of recombinant adenovirus was successful transfected into RAW264.7 macrophage.The experiment using PBS,Ad-GFP and Ad-PSRC1 transfection macrophages,PBS group as Ad-GFP blank control group,48 hours later,the PBS group and Ad-GFP PSRC1 amount of mRNA and protein expression had no significant difference,compared to the PBS group and the Ad-GFP control group,the Ad-PSRC1 group increased the expression of mRNAand protein of PSRC1 in macrophages.3?Effect of PSRC1 overexpression on lipid content in macrophagesThe macrophages were converted into foam cells after ox-LDL treatment of 100?g/ml ox-LDL in 48h.Compared with cells transfected with Ad-GFP(7.67 ± 0.35)%,RAW264.7 cells transfected with Ad-PSRC1(5.03 ± 0.14)%had 34.4%less cellular lipid content.4?Effect of PSRC1 overexpression on the expression of SR-A I and LDLR in macrophagesThe macrophages were converted into foam cells after ox-LDL treatment of 100?g/ml ox-LDL in 48h.Compared with the Ad-GFP group,the Ad-PSRC1 group decreased the expression of mRNA and protein of SR-A I in macrophages,and the Ad-PSRC1 group decreased the expression of mRNA and protein of LDLR in macrophages.5?Effect of PSRC1 overexpression on IL-6 and TNF-a in macrophagesThe macrophages were converted into foam cells after ox-LDL treatment of 100?g/ml ox-LDL in 48h,then the cell supernatant were collected.Compared with the Ad-GFP group,the Ad-PSRC1 group decreased the expression of mRNA and secretion levels of IL-6 and TNF-a in macrophages.6?Effect of PSRC1 overexpression on the ?-catenin-NF-?B signal pathway in macrophagesCompared with Ad-GFP group,the protein expression of P-?-catenin and P-NF-?B in macrophages of Ad-PSRC1 group was significantly decreased,and the expression of ?-catenin was significantly increased;Ad-PSRC1 The expression of?-catenin protein in the nucleus of the macrophages increased significantly,while the expression of NF-?B protein in the nucleus decreased significantly.Conclusion1?PSRC1 overexpression decreased the content of lipid in macrophages.2?PSRC1 overexpression decreased the expression of mRNA and protein of SR-A I and LDLR in macrophages.3?PSRC1 overexpression decreased the expression of mRNA and secretion levels of IL-6 and TNF-? in macrophages.4?PSRC1 overexpression can up-regulated the protein expression of ?-catenin and down-regulated the protein expression of NF-?B in macrophages.PSRC1 overexpression modulates lipid metabolism and reduces inflammatory response,thereby inhibiting the formation of macrophage derived foam cells.The underlying mechanism is likely to downregulate the intake of ox-LDL receptor and native LDL receptor to improve lipid metabolism in macrophages,and downregulate the expression of inflammatory cytokines IL-6 and TNF-? to inhibit the inflammatory response in macrophages,which is partially contributed to increased ?-catenin expression and decreased NF-?B expression.Besides,the study provides a new idea for the diagnosis and treatment of atherosclerosis and coronary heart disease.Meanwhile,further experiments are needed to explore the direct effect and specific mechanism between PSRC1 and atherosclerosis.