| Background: Aging is a major health problem,and it is also an important incentive in the occurrence of cardiovascular diseases,tumors,autoimmune diseases,and infectious diseases.Discovering aging-related cytokines and molecular changes in processes will provide new treatment options and diagnostic strategies for aging-related diseases.Premature aging of endothelial cells is the main cause of various cardiovascular diseases.The study of vascular endothelial cell aging provides important significance for the diagnosis and treatment of cardiovascular diseases.sFRP3(Curl-Related Protein 3): is a natural secreted Wnt pathway antagonist,mainly expressed in the heart and brain tissues.The Wnt signaling pathway plays an important role in biological behaviors such as embryonic development,tissue homeostasis,and tumorigenesis and development Many factors affect the expression of Wnt signals.The family of secreted frizzled related proteins(SFRPs)is an important Wnt antagonist.They bind Wnt or its receptor Fz extracellularly to inhibit Wnt signaling.Therefore,this study first examined the expression levels of 200 protein factors in theplasma of mice of different childbearing age by protein chip technology,and explored the changes of cytokines and related pathways in the natural aging process of mice.Screening of differential protein sFRP3 from the serum of healthy young mice and healthy elderly mice,expanding the sample size,the sFRP3 protein expression levels were detected in the serum of mice of different childbearing age,the supernatant of primary vascular endothelial cells of mice of different childbearing age,and the plasma of healthy humans at different ages.Changes in the natural aging process of primary vascular endothelial cells and provide new targets for the prevention and treatment of aging-related diseases.Objective: The expressions of related protein factors in serum of mice of different childbearing ages and the changes of related pathways were explored.The changes of sFRP3 in the aging process of mouse primary vascular endothelial cells were analyzed,and the role and significance of sFRP3 in aging of vascular endothelial cells were clarified,And explore its mechanism.Methods:(1)This study first took healthy young mice(4 cases)and healthy elderly mice(4 cases)as research objects,and quantitatively measured 200 mouse cytokines by solid-phase microarray technology for the detection of eight groups of mice The protein in the serum sample is obtained by using the Inno Scan 300 chip scanner to obtain the fluorescence signal levels of various proteins,and then statistical analysis of the chip data,including normalization of raw data,screening of differential proteins,clustering of differential proteins,and differential protein KEGG pathway enrichment analysis and other basic analysis(2)Through enzyme-linked immunosorbent assay(ELISA),the serum of 15 young mice and 15 elderly mice were detected,and the plasma of 30 healthy young people and 30 healthy elderly were detected.The expression level of sFRP3 protein(3)The expression levels of sFRP3 protein in the supernatant of primary vascular endothelial cells of 5 young mice and the supernatant of primary vascular endothelial cells of 5 old mice were measured by enzyme-linked immunosorbent assay(ELISA).Results:(1)In order to detect differentially expressed proteins(DEP)in the serum of mice of different childbearing ages,we performed a t-test(p-value)on the results of protein chip fluorescence signals and the foldchange of each protein between the two groups.There are 32 DEPs.Visualized with volcanic maps,it was found that DEPs were differentially expressed.(2)PCA and heat maps were performed for all DEPs between the two groups.Plotting the first two main components shows the difference between the two groups of young mouse samples and old mouse samples.Hierarchical cluster analysis showed the expression levels of 32 differential proteins and found that compared with young mice,Fractalkine,IGFBP5,IL-17 E,I-TAC,MDC,IL-15,IL-21 were found in the serum of elderly mice.,Leptin,MIG,MIP-1a,GITR L,Lymphotactin,Osteoactivin,OX40 Ligand,PIGF-2,ANG-3,CCL28,Epigen,Galectin-7,Gremlin,IFNg R1,MIP-16,Persephin,sFRP3,Shh-N,SLAM,TECK,TGFb1 and TWEAK increased,while the contents of IGFBP-6,CD6 and DLL4 decreased.(3)Through GO enrichment analysis and KEGG enrichment analysis of differentially expressed genes in mouse serum of different childbearing ages,the role of membrane surface,cytokine activity,leukocyte migration,and cytokine-cytokine receptor interaction pathways in aging was found.(4)Through statistical analysis of the sFRP3 fluorescence signal map in the protein chip data and sFRP3 fluorescence signal value in the protein chip data,it was found that the expression level of sFRP3 in the serum of the elderly mice was significantly higher than that of the young mice(P <0.05)(5)The sFRP3 levels in serum samples of 15 young and 15 elderly mice were detected by ELISA,and the expression level of sFRP3 protein in the serum of elderly mice was significantly increased(P<0.0001).When the expression level of sFRP3 protein in the supernatant of primary vascular endothelial cells and the supernatant of vascular endothelial cells in five elderly mice was found,the expression level of sFRP3 protein in the supernatant of primary vascular endothelial cells in elderly mice was significantly higher than that inyoung mice.Increased(P<0.01),and at the same time when measuring the expression level of sFRP3 protein in the plasma of 30 healthy young people and 30 healthy elderly people,it was found that the expression level of sFRP3 in the plasma of the elderly was significantly increased compared with young people(P <0.0001)Conclusions:(1)In the natural aging process of mice,we found 32 differentially expressed proteins,as described above.(2)Through gene enrichment and pathway analysis,we found changes in cellular composition,molecular functions,biological processes,and cytokine-cytokine receptor interaction pathways in the natural aging process of mice.(3)In the natural aging process of healthy mice,we found that the expression level of sFRP3 protein in serum increased significantly with age.In the natural aging process of healthy people,we found that the expression level of sFRP3 protein in plasma increased significantly with age.(4)Increased expression of sFRP3 is related to aging of vascular endothelial cells in mice... |
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