| Part I Effects of high glucose and high lipid on the IL-18 level in macrophage and endothelial dysfunctionObjective:The major long-term complication of diabetes is vascular disease,which is the leading cause of morbidity and mortality in patients with diabetes mellitus(DM).Diabetes accelerates the pathogenesis of atherosclerosis and is multifactorial,involving the distribution of interactions between apolipoproteins and lipoprotein particle abnormalities,hyperinsulinemia,changes in growth factor expression,and cytokine production.In addition,a large body of evidence suggests that the inflammatory process plays an important role in atherosclerotic lesions,destruction and thrombosis.IL-18,originally described as an IFN-y-inducing factor,is a member of the IL-1 cytokine family and is known to contribute to the pathogenesis of chronic immune inflammatory processes.IL-18 is produced constitutively in many different cells.Types include macrophages,vascular smooth muscle cells and fat cells.However,the mechanism by which IL-18 participates in the specific development of diabetic vascular disease is still unclear.The aim of present part of study was to evaluate the effects of IL-18 level change in macrophages upon the stimulation of high glucose and high lipid and to determine the IL-18 binding protein level and change of adhesion ability.Methods:mRNA by real time quantitative PCR,protein expression by western-blotting,and secretion of IL-18 by ELISA in mouse macrophage lines(RAW 267.4)treated with NG(5.6 + 19.4 mmol/L mannitol)or HG(25 mmol/L)levels in the presence of palmitate(300 mmol/L)for 24 h.The endothelial cells were exposure to the same incubation condition,and the mRNA and protein level of IL-18 BP was determined by real time PCR and western-blotting.To observe the expression of Vascular cell adhesion molecules(VCAM-1)and intercellular cell adhesion molecule-1(ICAM-1)on endothelial cell surface adhesion molecules treated with high glucose and free fatty acid.Effects and adhesion of endothelial cells to THP-1 cell lines.At the same time,the IL-18 neutralizing antibody was used to observe the effect of IL-18 neutralization on the expression of the above adhesion molecules on the surface of endothelial cells and the adhesion of endothelial cells on the THP-1 cell line.All statistical analysis was processed by SPSS 13.0 software package.All data were represented by X±S.Student t test was used to compare the differences between the two mean values,and one-way ANOVA(variance analysis)was used to compare the differences between multiple parameters.The difference was judged with or without statistical significance based on whether p value was less than 0.05.Results:Significantly increased IL-18 mRNA(Relative levels of IL-18 mRNA in the four groups were 1.0,0.1,2.1,0.28,0.94,0.23,1.8,0.2 respectively.)and protein(Relative levels of IL-18 in the four groups were 1.01.52 0.08 0.79 0.22 1.59 0.15,),while increased level of IL-18 secrection(IL-18 protein levels in the four groups were 2.0±0.41?1.2±0.17?2.59±0.52?5.34±1.21.)were found in macrophages exposed to HG and palmitate.(compared with NG group,P<0.05).Increased level.of IL-18 BP was found on endothelial cells exposed to HG and palmitate.Increased level of cell surface adhesion molecules such as ICAM-1(The levels of ICAM-1 protein in the four groups werel.0±0.05?4.1±0.83?3.56±0.36?4.78±1.00.)and VCAM-1(The levels of VCAM-1 protein in the four groups were 1.0±0.05?10.56±2.66?18.22±1.94?11.85±1.65.)were found in endothelial cells exposed to HG and palmitate.In addition,IL-18 could significantly increase the adhesion of THP-1 cells,which could be abolished by IL-18 antibody neutralization(The number of adherent cells in the four groups was 58±11?100±10?139±12.78±6).(compared with NG group,P<0.05).Conclusions:Our study unrevealed a new mechanism of EC dysfunction caused by the de-regulation of the IL-18/IL-18BP pathway,leading to increasedVCAM-1 expression,monocyte/macrophage adhesion,and accelerated atherosclerotic plaque formation in diabetes.Part ? The role and mechanism of IL-18 on smooth muscle cells functionObjective:Atherosclerosis is considered to be a chronic inflammatory disease characterized by enhanced expression of pro-inflammatory cytokines,chemokines and adhesion molecules.Crosstalk between cytokines,chemokines,and infiltrating immune cells amplifies the inflammatory cascade in the vessel wall,leading to the formation of atherosclerosis.Interleukin-18(IL-18)is a pro-inflammatory and atherogenic cytokine that induces the expression of other pro-inflammatory cytokines and adhesion molecules.IL-18 has been localized to human atherosclerotic lesions,and increased circulating IL-18 in acute coronary syndrome has been shown to predict future cardiovascular events.A positive correlation between serum IL-18 levels and carotid intima-media thickness has been demonstrated.Administration of IL-18 aggravates atherosclerosis in mice.In addition,atherosclerosis was reduced in IL-18-deficient apoE knockout mice,suggesting that IL-18 has a causal role in the development and progression of atherosclerosis.Recently,studies have shown that IL-18 induces proliferation of human aortic smooth muscle cells(SMCs).However,it is unclear whether IL-18 induces SMC migration.The aim of present part of study was to evaluate the role of IL-18 on the migration of smooth muscle cells(SMCs),explore related mechanisms and provide certain therapeutic basis for diabetes AS.Methods:In vitro SMCs migration transwell system was established and IL-18 was added for cell migration evaluation.To observe the mechanisms of IL-18 mediated SMCs migration by employment the metal matrix proteinases(MMPs)inhibitors.To observe the mechanisms of IL-18 mediated SMCs migration by evaluate MAPK,PI3K and NF-?B pathways.Effector molecules involved in IL-18-mediated vascular smooth muscle migration were observed by using metal matrix protein MMP3 or MMP8 inhibitors,MMP2,MMP9 siRNA and related inhibitors.At the same time,IL-18 stimulated vascular smooth muscle in vitro system was established at different concentations and at different times.The levels of MMP-9 gene and protein were detected by quantitative PCR and Western-blotting.The vascular smooth muscle cells were treated with Tumor necrosis factor a(TNF-a)combined with IL-18 to detect the MAPK pathway,whether the PI3K pathway and the NF-?B pathway are involved in the action of IL-18,and the utilization Inhibitors of the MAPK pathway,the PI3K pathway,and the NF-?B pathway verify whether the relevant pathway is involved in IL-18-mediated vascular smooth muscle migration.Results:IL-18 treatment significantly increased migration of human aortic vascular smooth muscle cells,a effect that was reversed by IL-18 antibody neutralization(The values of the four groups were100±5?300± 15?290±15?150±8.).Compared with control group P<0.05.IL-18 stimulation increases the level of MMP-9 gene and increases the activity of MMP-9 in a dose-and time-dependent manner(The values of the five groups were 1±0.05?2.5±0.15?5±0.25?10±0.5?12±0.6.),while the addition of IL-18 neutralizing antibody reverses the increase in active MMP-9 levels caused by IL-18(The values of the six groups were 1?1 ±0.05?1.2±0.06?5±0.25?8±0.4?10±0.5.).Compared with control group P<0.05.Compared with control group P<0.05.In the case,simultaneous MMP9 inhibition or silencing significantly reduced IL-18-induced increases in human aortic vascular smooth muscle cell migration.IL-18 stimulation can increase the levels of p-JNK,pERK,p-p38 and p-AKT in human aortic smooth muscle cells,and can be down-regulated by IL-18 after addition of inhibitors of JNK,ERK,p38 and AKT.Increased migration of vascular smooth muscle cells(The values of the seven groups were 100±5?300±25?285±20?200±10?190±10?195 ±10?180±10.).Compared with control group P<0.05.Inhibition of the NF-?B pathway was able to down-regulate the migration of human aortic vascular smooth muscle cells induced by IL-18,and the NF-?B inhibitor Bayll-7082 significantly inhibited the increase in cell migration induced by IL-18(The values of the four groups were 100±5?300±25?285 ±20?180± 10.).Compared with control group P<0.05.Conclusions:The promotion of IL-18 on vascular smooth muscle migration may be achieved by affecting MMP9,MAPK,PI3K and NF-?B related pathways.Part III IL-18 related circRNA-001175 regulates endothelial cell function in high glucose conditionObjective:Circular RNA(circRNA)is a novel type of RNAs.Studies have found that circRNA is rich in miRNA binding sites and can play competitive endogenous RNA roles.In recent years,circRNA has been used as a new regulatory molecule to regulate the function of miRNA as a miRNA sponge to inhibit its target genes.It may regulate gene expression in cells.It is also involved in various biological processes.The high glucose stress is one of the major risk factors for cardiovascular diseases.It can induce vascular endothelial cell apoptosis.Through literature retrieval,circma-001175 showed differential expression in diabetic AS.However,the role and biological function of circRNA-001175 is still unclear under high glucose.And there were few studies on its mechanism.The purpose of this study is to investigate the role of circRNAs in human umbilical vein endothelial cells(HUVECs)induced by high glucose.Methods:Correlation analysis was performed by Pearson test by injecting diabetes patients and detecting cir RNA-001175 levels in diabetic patients while detecting serum IL-18 levels.Human Umbilical Vein Endothelial Cells(HUVEC)were established in vitro,and the expression pattern of circRNA-001175 was observed after treatment with high glucose and normal sugar,respectively.The circRNA-001175 mimetic was transfected by gene manipulation,the cell proliferation was analyzed by MTS and EdU method,and the apoptosis of endothelial cells was detected by Annexin V-PI staining.Results:Spearman correlation method was used to analyze the results and found that the serum IL-18 level of type 2 diabetes patients was correlated with the circrna-001175 level,with the correlation coefficient r=0.51 and p=0.02.Real-time PCR results showed that glucose treatment gradually reduced the expression of circRNA-001175 in a concentration-dependent manner.Compared with control cells P<0.05.The CCK-8 assay showed that high glucose treatment significantly reduced cell viability,and this decrease in cell viability was reversed by upregulation of circRNA-001175.Compared with NC group P<0.05.Furthermore,circRNA-001175 transfection showed a protective effect on the proliferation induced by high glucose treatment.Hoechst staining and flow cytometry analysis showed that upregulation of circRNA-001175 inhibited apoptosis of HUVECs induced by high glucose treatment.Compared with NC group P<0.05.Furthermore,the results of in vitro angiogenesis experiments observed that up-regulation of circRNA-001175 increased the angiogenic capacity of HUVECs under high glucose.Conclusions:CircRNA-001175 may play a key role of protection on HUVECs from high glucose stress.Circ RNA-001175 has great potential to become diagnostic or predictive biomarkers for high glucose disease and provide new insights into the treatment of diseases. |
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