The Mechanism Of Vitamin A In The Inflammation Of Kawasaki Disease

Background: Kawasaki disease(KD)is one systemic self-limited vasculitis,and children aged between 3 and 5 years are the main risk groups.Coronary artery lesions(CALs)are the most predominant complications,which can lead to coronary artery aneurysm,thrombosis,stenosis,and myocardial infarction etc.The etiology and pathogenesis of KD are still unclear.Studies have reported that there are the uncontrolled inflammation and imbalance of immune response acting as a key role of the pathogenesis in KD and CALs.Vitamin A(VA)is an essential nutrient for human body,and acts as a senior role in inflammation,cell apoptosis and cardiovascular disease.But there are no reports about the relationship between VA and KD.Hence,the aim of this study is to probe the role of VA in the inflammation and the process of coronary artery inflammation in acute KD children.Methods:(1)Serum samples were collected from children with acute KD(KD group)and healthy control group(HC).KD group was divided into KD without CALs group(KD-NCALs)and KD with CALs group(KD-CALs).The serum VA level of each group was determined by high-performance liquid chromatography(HPLC),and the levels of TNFαand IL-6 were determined by ELISA.(2)At the same time,the inflammatory injury model of human umbilical vein endothelial cells(HUVEC)induced by TNFα(50ng/ml)was established and co-cultured with different concentrations of retinoic acid(RA).CCK8 and flow cytometry were used to measure the proliferation and apoptosis of HUVECs.The PCR and Western-blot were used to detect the gene and protein levels of RARα,AKT,NF-κB and Caspase-3.HUVECs were treated with RARα agonist(AM580),RARα inhibitor(Ro 41-5253),AKT inhibitor(LY294002),and NF-κB inhibitor(SN50)in vitro,and then co-cultured with TNFα and RA.The apoptosis of HUVECs was detected by flow cytometry.The protein expression levels of RARα,AKT,NF-κB and Caspase-3 were detected by Western-blot.(3)The intraperitoneal single injection of Lactobacillus casei wall extract was used to build the model of coronary arteritis.After daily administration of VA,HPLC was used to detect the serum VA level of mice,and the inflammatory infiltration of coronary artery was observed by HE staining.The m RNA and protein expression levels of RARα,AKT,NF-κB,and Caspase-3 were detected by PCR and Western-blot;VA combined with RARα agonist(AM580),RARαinhibitor(Ro 41-5253),AKT inhibitor(LY294002)and NF-κB inhibitor(SN50)were used to intervene the coronary arteritis model mice.HE and immunohistochemistry were used to observe the inflammatory infiltration of coronary artery.The m RNA and protein expression levels of RARα,AKT,NF-κB,and Caspase-3 were detected by PCR and Western-blot.Results:(1)The serum VA level of children with Kawasaki disease in acute stage was significantly lower than that of healthy children.Serum VA was positively correlated with L%,and negatively correlated with the WBC,N%,ESR,CRP,and Pct in KD group(P<0.05).Serum VA was negatively correlated with WBC,N%,and IL-6,and positively correlated with L% in KD-CALs group(P<0.05).(2)CCK8 and flow cytometry exhibited that there were increased proliferation rate and decreased apoptosis rate of HUVECs after treating with RA(1-10μmol/L)(P<0.05);There was a significantly decreased gene level of NF-κB(P<0.05);And there were reduced levels of Cleaved-caspase-3 protein and NF-κB phosphorylation protein(P<0.05);After adding RARα agonist and NF-κB inhibitor,there was a decreased apoptotic rate of HUVECs,while the apoptotic rate of HUVECs was increased after adding RARα inhibitor and AKT inhibitor;After adding RARα inhibitor,there was a significantly decreased expression of AKT phosphorylated protein(P<0.05);After adding RARαagonist,and the protein expression of phosphorylated NF-κB was significantly decreased(P<0.05);And after adding RARα inhibitor,the expression of Cleaved-caspase-3 was significantly increased(P<0.05).(3)After VA intervention in coronary arteritis mice,the serum VA concentration was significantly increased(P<0.05),and the coronary artery inflammatory infiltration was improved.After VA intervention,the RARαm RNA level and AKT m RNA and phosphorylation protein level were increased,while the m RNA level of NF-κB and protein level of Cleaved-caspase-3 were decreased(P<0.05);HE and immunohistochemical staining showed that the inflammatory infiltration of coronary artery was significantly increased by RARα inhibitor and AKT inhibitor,while the inflammatory infiltration of coronary artery was significantly decreased by RARα agonist and NF-κB inhibitor;The expression of AKT m RNA and phosphorylated protein were increased in coronary arteritis model mice treated with RARα agonist,and there was a significantly decreased expression of AKT m RNA after RARα inhibitor intervention(P<0.05);At the same time,there were significantly increased expression of NF-κB gene level and Caspase-3 gene level after AKT inhibitor intervention(P<0.05),and there were significantly decreased expression of Caspase-3 m RNA level and Cleaved-caspase-3 protein level after NF-κB inhibitor and RARα agonist intervention(P<0.05).Conclusion:(1)VA participates in the inflammatory reaction process of KD and can also improve the inflammatory infiltration of coronary artery:(2)VA can affect AKT/NF-κB signal transduction through RA signal,thus regulating the expression of apoptosis protein Caspase-3 and participates in KD and coronary arteritis.