Study On The Relationship Between Natural Autoantibodies Associated With Inflammatory Factors And Atherosclerosis In Peripheral Blood

Objective:Cardio-cerebrovascular disease(CCD)is one of the leading causes of all disease-related deaths in the world.The Morbidity and mortality of CCD in China are still on a steady rise.Arteriosclerotic CCD(ACCD),such as stroke and coronary heart disease,is the most common type of CCD in China.Atherosclerosis(AS)is the etiology and common pathophysiological basis of all ACCD.AS is a chronic systemic inflammatory disease which mainly affects the intima of large and medium-sized arteries.In general,AS has a long latency and is often involved in damaging multiple vascular beds at the same time.Early diagnosis and treatment can slow down or prevent the deterioration of atherosclerosis.Therefore,it is imperative to develop an effective detection method for early diagnosis of atherosclerotic conditions.With the development of research,the detection of diagnostic markers based on peripheral blood has aroused great interest of researchers.Recent studies have shown that the identification of natural antibodies may provide a new method for the early detection of AS-related diseases.Almost all kinds of cells involved in the pathogenesis of AS express cytokines,which can act on a variety of targets and play a variety of roles.Cytokines such as tumour necrosis factor?,interleukin-1 and IL-6 usually promote AS formation.Other cytokines,such as transforming growth factor-?,IL-10,and IL-35 inhibit AS formation via interacting with T-regulatory cells(Treg).Moreover,vascular endothelial growth factor 1(VEGFR1)and VEGFR2 are important regulators of angiogenesis,and may be involved in the formation of AS.Treg cells,characterized by CD4+CD25+T cells,can inhibit the formation and progression of AS.CD25 antibody can promote the formation of AS plaque by consuming Treg cells.FOXP3,which is specifically expressed by Treg cells,is one of the most reliable molecular markers of Treg cells,as well as a major regulator of Treg cell development and immune suppression.This study aims to detect circulating natural antibodies of these factors related to AS development,explore the mechanism behind the formation of atherosclerotic carotid plaque on the basis of previous studies,in order to illustrate the feasibility of natural antibodies as diagnostic markers of AS and to provide experimental evidence and new ideas for the diagnosis of AS.Methods:1.A total of 208 patients with atherosclerosis and gender and age matched 187healthy individuals were enrolled in this study.All patients were diagnosed as having atherosclerotic carotid plaque at the Department of Neurology in Second Hospital of Jilin University in Changchun from January 2017 to December 2017.Peripheral blood was obtained at the same time with other blood tests,and plasma samples used for antibody testing was stored at-80 ~oC until use.All the information including gender,age,and history of smoking and drinking,and other clinically relevant data were collected.2.The amino acid sequences of IL1,IL1,IL6,IL8,TNF,CD25,FOXP3 and VEGFR1 were obtained from NCBI database.The linear polypeptide antigen was designed through bioinformatics database and online tool(http://www.iedb.org).The peptide antigen was synthesized by Jier biochemical company in Shanghai and the purity of>95%was guaranteed.3.The expression levels of natural autoantibodies in the peripheral blood of the subjects were measured by an optimized ELISA.4.Nonparametric test was used to analyze the expression difference of natural antibody in patients with atherosclerosis and healthy subjects,and the correlation between the expression level of natural antibody and the clinicopathological characteristics of atherosclerosis patients.The possibility of natural antibody used for diagnosis of atherosclerosis was evaluated by the receiver operating characteristic(ROC)curve and area under the ROC curve(AUC).A panel of diagnostic markers were established based on logistic regression.Pearson correlation coefficient was used to evaluate the correlation between the expression levels of each antibody.P<0.05 was considered statistically significant.Results:1.Fourteen linear polypeptide antigens were designed and synthesized based on the amino acid sequences retrieved from NCBI database.The linear polypeptide antigens are as follows:IL1? H-WETHGTKNYFTSVAHPNLFIATKQDYWVC-OHIL1?-1 H-SLNCTLRDSQQKSLVMSGPYELKALHLQG-OHIL1?-2 H-KHAYYSGNEDDLFFEADGPKQMKCH-OHIL6 H-LTKLQAQNQWLQDMTTHLILRSC-OHIL8 H-DCQCIKTYSKPFHPKFIKELRVIESD-OHTNF?-1 H-CQLQWLNRRANALLANGVELRDNQLV-OHTNF?-2 H-KSAIKSPCQRETPEGAEAKPWYEPK-OHCD25-a H-KPGHCREPPPWENEATERIYHFVVGQMVY-OHCD25-b H-IYHFVVGQMVYYQCVQGYRALHRGPAESVE-OHCD25-c H-KHTSQFPGEEKPQASPEGRPESETSCH-OHFOXP3-a H-DMFAFFRNHPATWKNAIRHNLSLHKCD-OHFOXP3-b H-KCTFPNPSAPRKDSTLSAVPQSSYH-OHVEGFR1-a H-DEGVYHCKATNQKGSVESSAYLTVQGTSDK-OHVEGFR1-b H-CQITWFKNNHK IQQEPGIILGPGSSTD-OH2.The expression level of anti-IL1?Ig G in the plasma of AS patients was0.894±0.156.The expression level of anti-IL1?Ig G in the plasma of healthy control subjects was 0.843±0.169.The expression level of anti-IL1?Ig G in the plasma of AS patients was increased,and the difference was statistically significant(P<0.01).The expression level of anti-TNF?-1 Ig G in plasma of AS patients was1.238±0.304.The expression level of anti-TNF?-1 Ig G in the plasma of healthy control subjects was 1.139±0.259.The expression level of anti-TNF?-1 Ig G in the plasma of AS patients was increased,and the difference was statistically significant(P<0.01).The expression level of anti-VEGFR1-b Ig G in the plasma of AS patients was1.498±0.449.The plasma expression level of anti-VEGFR1-b Ig G in the plasma of healthy control subjects was 1.568±0.390.The expression level of anti-VEGFR1-b Ig G was decreased in AS patients,and the difference was statistically significant(P<0.05).3.The AUC was 0.578 for the anti-IL1?Ig G assay,0.592 for the anti-TNF?-1 Ig G assay,and 0.564 for the anti-VEGFR1-b Ig G assay,respectively,while the AUC was0.651 for combination of the three antibodies.The expression of circulating natural antibodies was analyzed according to gender factors.The results showed that compared with healthy female subjects,plasma levels of anti-IL1?Ig G,anti-IL1?-2 Ig G and anti-TNF?-1 Ig G were all increased in female patients with atherosclerosis,and their AUC was 0.618,0.588 and 0.650,respectively.Compared with healthy male subjects,plasma levels of anti-VEGFR1-a Ig G and anti-VEGFR1-b Ig G were decreased in male patients with atherosclerosis,and the AUC was 0.578 and 0.594,respectively.According to the age factors,the expression of circulating natural autoantibodies in young and old age groups was analyzed respectively.The results showed that the expression of anti-IL1?Ig G and anti-IL1?-2 Ig G in peripheral blood of AS patients was increased in the younger group(P<0.05).The expression level of anti-TNF?-1 Ig G in peripheral blood was increased in both young and old AS patients.Pearson correlation coefficient showed that there was a certain correlation between the expression levels of each antibody,among which the expression of natural autoantibodies of polypeptide antigen derived from the same amino acid sequence had the highest correlation.The non-parametric test results showed that there was no significant correlation between the expression level of circulating natural autoantibodies and the clinical characteristics of patients with AS,such as triglyceride level,smoking history,drinking history,AS plaque site,AS size and et al.Conclusions:1.The plasma levels of anti-IL1?,anti-TNF?-1 and anti-VEGFR1-A natural autoantibody(Ig G)in the atherosclerosis patients were significantly higher than those in the healthy control group(P<0.05).The area under ROC curve was 0.578,0.592and 0.564,respectively.These results suggest that plasma anti-IL1,anti-TNF and anti-VEGFR1 natural autoantibodies(Ig G)may be biomarkers of atherosclerosis.2.The combined diagnostic model of anti-IL1?,anti-TNF?and anti-VEGFR1natural autoantibody(Ig G)in the plasma of atherosclerosis patients significantly improved the diagnostic efficacy,with the area under ROC curve up to 0.651(P<0.01),which was helpful to improve the diagnostic ability of atherosclerosis.3.The plasma levels of anti-IL1?,anti-IL1?-2and anti-TNF?-1 natural autoantibodies(Ig G)in the female atherosclerosis patients were significantly higher than those in the healthy control group(P<0.05).The area under ROC curve was 0.618,0.588 and 0.650,respectively.These three kinds of natural autoantibodies are closely related to the pathogenesis of female atherosclerosis.The plasma levels of anti-VEGFR1-a and anti-VEGFR1-b natural autoantibodies(Ig G)in male atherosclerosis patients were significantly lower than those in healthy controls(P<0.05).The area under ROC curve was 0.578 and 0.594,respectively.It is suggested that plasma anti-VEGFR1 natural autoantibody(Ig G)is closely related to the incidence of atherosclerosis in males.The level of TNF?-1 natural autoantibody(Ig G)in peripheral blood of female patients was higher than that of male patients(P<0.05).4.The plasma levels of anti-IL1?,anti-IL1?and anti-TNF?-1 natural autoantibodies(Ig G)in the atherosclerosis patients aged?61 years were significantly higher than those in the healthy control group(P<0.05),suggesting that these three kinds of natural autoantibodies were closely related to the onset of atherosclerosis in patients aged?61 years.The plasma level of anti-TNF?-1 natural autoantibody(Ig G)in the>61 years patients with atherosclerosis was higher than that in the healthy control group(P<0.05),suggesting that the antibody is closely related to the onset of atherosclerosis in the>61 years patients.5.Anti-IL1,anti-IL6,anti-IL8,anti-CD25,and anti-FOXP3 natural autoantibodies(Ig G)showed no statistically significant difference between the atherogenic group and the healthy control group(P>0.05),and were not suitable as biomarkers of atherosclerosis.