| Background:Kawasaki disease(KD)is a systemic self-limited vasculitis that mostly occurs in children under 5 years of age.The main complication of the disease is coronary artery abnor-mality(CAA),which is considered the leading cause of acquired heart disease Globally,China has the third highest incidence of KD,second only to Korea and Japan.Although the etiology is not well known,specific infection agents may cause genetic susceptibility in certain children.An aberrant immune response is also believed to be key in the preclinical and acute stages of this disease.Treatment with high-dose intravenous immunoglobulin(IVIG)along with moderate to high doses of aspirin is the current gold standard.While the mechanisms underlying the beneficial effects of IVIG remain unknown,its broad anti-inflammatory effect is believed to be an important factor.However,approximately 10-20%patients do not respond to such treatment and presented with prolonged changes in presence of prolonged fever.Moreover,alternative treatments after response failure to initial IVIG treatment are usually unsatisfactory,with a remarkable higher incidence of CAA.Therefore,it is crucial to improve the therapy of KDEETs exert a variety of biological functions in normal and pathophysiological conditions,including anti-inflammatory,anti-apoptptic,anti-fibrotic,and anti-oxidant,acting in both autocrine and paracrine manners.Furthermore,accumulating evidence demonstrates that EETs exert protective effects on various cardiovascular diseases,including attenuation of heart injuries,anti-hypertension and promoting vascular repair.However,in vivo,EETs are rapidly degraded to the less active dihydroxyeicosatrienoic acids(DHETs)by soluble epoxide hydrolase(sEH).Inhibition of sEH may lead to elevated levels of EETs,which in turn could elicit a variety of biological functions.12-(3-adamantan-1-yl-ureido)-dodecanoic acid(AUDA)is a soluble epoxide hydrolase inhibitor,which can elicit the effect of anti-inflammatory and vascular repair of EETs by inhibiting sEH.Meanwhile,in addition to its sEH inhibitory properties,AUDA has now been proved to possess other activities.However,little is known about the effect of AUDA on KD.Objective:1.To investigate the effects and mechanisms of AUDA on migration,adhesion,proliferation and angiogenesis of human coronary artery endothelial cells(HCAECs),and to explore whether AUDA can promote vascular repair.2.To study the effect of AUDA on cardiac histopathology and inflammatory factors in mice with KD,and to explore whether AUDA has anti-vasculitis effect.3.Collect clinical samples to study the expression and significance of EETs in peripheral blood of children with KD.Methods:1.Effects of AUDA on cellular proliferation,migration,adhesion and tube formation ability in HCAECs.1.1 Different concentrations of AUDA(0,1,10,50,100,100μmol/L+5μmol/L GW9662)-treated HCAECs were added to the transwell plate,and the effect of AUDA on the migration of HCAECs was examined.1.2 Different concentrations of AUDA(0,1,10,50,100,100μmol/L+5μmol/L GW9662)-treated HCAECs were added to human fibronectin-coated 96-well culture plates to examine the effect of sEHi on cell adhesion of HCAECs.1.3 The effect of different concentrations of AUDA(0,1,10,50,100,100μmol/L+5μmol/L GW9662)on the proliferation of HCAECs cells was detected by CCK-8 kit.1.4 Different concentrations of AUDA(0,1,10,50,100,100μmol/L+5μmol/L GW9662)-treated HCAECs were added to a Matrigel-coated 96-well culture plate to detect the effect of AUDA on angiogenesis in HCAECs.1.5 Quantitative RT-PCR and ELISA assay were performed to detect the expression of PPARy after 100 μmol/L AUDA treatment.The over-expression plasmid of PPARy/PPARy-specific siRNA was transfected into HCAECs cells.The proliferation of HCAECs cells was detected by CCK-8 kit to explore the targets and signaling pathways of AUDA in HCAECs.2.Building a mouse model of KD induced by LCWE,and examining the levels of TNF-α,IL-1β,and MMP-9 expression in the hearts of different groups of mice by ELISA and qRT-PCR2.1 The mice were divided into four groups:PBS group,LCWE group,LCWE+AUDA group,and LCWE+AUDA+GW9662 group.2.2 The heart tissue sections of mice were taken for HE staining to observe the inflammatory responses at 3,14,and 28 days.2.3 QRT-PCR and ELISA assay were performed to detect the levels of 14,15-EET in heart tissues of mice in each group.2.4 The mice at 14 days after the injection of each group of drugs were taken as subjects,and the cardiac tissue of individual mice were stained with hematoxylin and eosin(H&E)and the inflammatory responses was evaluated.2.5 The mice at 14 days after the injection of each group of drugs were taken as subjects,and the protein levels of TNF-α,IL-1β,and MMP-9 in mice cardiac tissue were assayed using commercial ELISA kits.2.6 The mice at 14 days after the injection of each group of drugs were taken as subjects,and the gene levels of TNF-α,IL-1β,and MMP-9 in mice cardiac tissue were assayed using QRT-PCR.3.Detection of EETs expression in peripheral blood of children with KD and its relationship with coronary artery injury3.1 Peripheral blood samples of children with KD and healthy control group were collected(all signed informed consent),and clinical data of each group were analyzed by statistical method.3.2 QRT-PCR and ELISA were used to detect the content of 14,15-EET in each group of samples,and the relationship between 14,15-EET level and coronary artery injury was analyzed.3.3 QRT-PCR and ELISA were used to detect the levels of TNF-α,IL-1β and MMP-9 in each group of samples,and to analyze the relationship between them and coronary artery injury.Results:1.Effects of AUDA on cellular proliferation,migration,adhesion and tube formation ability in HCAECs.1.1 AUDA augmented the migratory activity of HCAECs in a dose-dependent manner,compared with the control group(AUDA:0μmol/L),1,10,50,100μmol/L of AUDA increased the migration activity of HCAECs with statistical significance(P<0.05).1.2 AUDA increased the activity of adhesion in a dose-dependent manner,compared with the control group(AUDA:0μmol/L),1,10,50,100μmol/L of AUDA increased the adhesion activity of HCAECs with statistical significance(P<0.05).1.3 AUDA increased the activity of in vitro capillary tube formation in a dose dependent manner,compared with the control group(AUDA:0μmol/L),1,10,50,100μmol/L of AUDA increased the activity of in vitro capillary tube formation of HCAECs with statistical significance(P<0.05).1.4 AUDA increased the activity of proliferation in a dose dependent manner,compared with the control group(AUDA:Opmol/L),1,10,50,100μmol/L of AUDA increased the proliferation activity of HCAECs with statistical significance(P<0.05).1.5 100 μmol/L AUDA significantly enhanced the expression of PPARy(P<0.01).The proliferation activity of HCAECs of PPARy knockdown group was significantly decreased(P<0.05),while the overexpression of PPARy had the opposite effect(P<0.01).After knocking down PPARy in 100 μmol/L AUDA-treated cells,the cell proliferation returned to normal level(P<0.05).These results suggested that AUDA promoted the proliferation of human coronary endothelial cells by up-regulating PPARy.1.6 Treatment of 100μmol/L AUDA or overexpression of PPARy in human coronary endothelial cells decreased the level of STAT1 significantly(P<0.05),while the level of STAT1 mRNA increased significantly in PPARy knockdown group(P<0.01).After knocking down PPARy in 100 μmol/L AUDA-treated cells,the expression of STAT1 increased to the level of control group(P<0.05).These results suggested that AUDA played an anti-inflammatory role by up-regulating PPARy and inhibiting JAK/STAT1 signaling pathway.2.Building a mouse model of KD induced by LCWE to observe the pathological changes of mice myocardial tissue.2.1 There was no detectable inflammatory infiltration in the PBS-injected mice.In contrast,inflammatory infiltrates around the myocardial tissue were detected in the mice with LCWE injection.The spleen structure of the mice in the PBS group was clear and the spleen structure was intact.The spleen bodies of the LCWE group were proliferated and fused,which was most obvious in 14 days.2.2 QRT-PCR and ELISA results showed that the expression level of 14,15-EET increased significantly on the 3rd day,decreased on the 14th day and decreased to the normal level on the 28th day after LCWE injection(P<0.05).2.3 On the 14th day after injection,there was no detectable inflammatory infiltration in the PBS-injected mice,while inflammatory infiltrates around the myocardial tissue were detected in the LCWE group.In the LCWE+AUDA group,inflammatory infiltrates around the myocardial tissue were lower than the LCWE group(P<0.05),while in the LCWE+AUDA+GW9662 group,inflammatory reaction was severely higher in contrast to the LCWE+AUDA group(P<0.05).2.4 On the 14th day after injection,the protein levels of TNF-α,IL-1β and MMP-9 in the heart tissue of LCWE group were higher compared with PBS group.The protein levels of TNF-α,IL-1β and MMP-9 of LCWE+AUDA group were lower than those of the LCWE group.Compared with the LCWE+AUDA group,the protein levels of TNF-α,IL-1β and MMP-9 in the LCWE+AUDA+GW9662 group increased(P<0.05).2.5 On the 14th day after injection,the gene levels of TNF-α,IL-1β and MMP-9 in the heart tissue of the LCWE group were significantly higher than those in the PBS group.The gene levels of TNF-α,IL-1β and MMP-9 in the LCWE+AUDA group were lower than those in the LCWE group.Compared with the LCWE+AUDA group,the levels of TNF-α,IL-1β,and MMP-9 in the LCWE+AUDA+GW9662 group increased.3.The expression and clinical significance of EETs in peripheral blood of children with KD.3.1 Compared with healthy controls,14,15-EET were highly expressed in peripheral blood of children with KD(P<0.01).3.2 Compared with KD children without coronary artery injury,EETs were highly expressed in peripheral blood of children with KD without coronary artery injury(P<0.05).3.3 Compared with healthy controls,the expression levels of TNF-α,IL-1β and MMP-9 in peripheral blood of children with KD were significantly increased;compared with children without coronary artery injury,the expression levels of TNF-α,IL-1β and MMP-9 in children with KD without coronary artery injury were abnormally high.Conclusion:1.AUDA positively modulates the functions of HUVECs and promotes vascular repair;2.AUDA can reduce the inflammatory response of heart tissue in mouse KD model;3.AUDA may act in a PPARy dependent fashion.4.The expression of EETs in peripheral blood of children with Kawasaki disease increases;the higher expression level of EETs in children with coronary artery disease suggests that EETs may have protective effect on children with KD. |
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