Anti-inflammatory Effect And Mechanism Of 1α,25-dihydroxyvitaminD₃ In Kawasaki Disease

Background: Kawasaki disease（KD）is the leading cause of acquired heart disease in developed countries.At present,there is still a lack of good adjuvant treatment for children with KD combined with coronary artery damage.Vitamin D is an important immunomodulator in the human body and plays a strong anti-inflammatory role in a variety of diseases.However,there are few studies on the anti-inflammatory effect and mechanism of vitamin D in Kawasaki disease.Part Ⅰ Investigating the effect of 1,25-（OH）2D3 on the inflammatory response of Kawasaki disease serum-induced THP-1 cellsObjective: To elucidate the role of Toll-like receptor 4（TLR4）signaling pathway in coronary artery injury in acute Kawasaki disease.To elucidate the effect of 1,25-（OH）2D3 on TLR4 signaling pathway.Elucidating the anti-inflammatory effect of 1,25-（OH）2D3 in coronary artery injury in Kawasaki disease.Methods: Serum was collected from 73 children with Kawasaki disease treated in our hospital,30 children with general upper respiratory tract infection and 30 children examined in the department of child care.The change of 25-（OH）D3 level in each group was detected by the kit.ROC curve analysis was used to determine the diagnostic cut-off values.In vitro cell experiments,the collected serum was used to induce macrophages to construct an innate immune model of Kawasaki disease.The experimental groups were 15% normal serum group,15% KD serum group,15% KD serum +1,25-（OH）2D3group（1n M 、 10 n M 、 100 n M）.1,25-（OH）2D3 was added for pre-protection prior to serum stimulation.After 24 hours of stimulation with serum,the expression levels of TLR4,IL-1β and TNF-α m RNA were detected by RT-PCR and the content of TNF-α in cell culture supernatant was detected by ELISA;the protein expression levels of TLR4 and p-P65 were detected by Western blot,and the protein expression levels of TLR4 in different groups was detected by immunofluorescence.Results:1.Leukocyte count,neutrophil count,platelet count,ALT and C-reactive protein were significantly higher in Kawasaki disease children than in normal group（P<0.05）.2.Compared with the control group and the infected group,the level of 25-（OH）D3 in children with Kawasaki disease was significantly lower（P<0.001）,the diagnostic cut-off point was 27.55ng/m L,the area under ROC curve was 0.922,the predictive sensitivity was 93.3%,the specificity was 75%.3.In vitro cell test showed that compared with normal serum group,Kawasaki disease serum significantly promoted the m RNA expression of inflammatory factors TLR4,IL-1β and TNF-α in macrophages（P<0.05）,and increased the content of TNF-α secreted by macrophages（P<0.05）.1,25-（OH）2D3preprotection inhibits serum-induced inflammatory cytokine production.4.Western blot and immunofluorescence detection showed that Kawasaki disease serum significantly promoted the expression of TLR4 and p-P65 protein in macrophages（P<0.05）,while 1,25-（OH）2D3 significantly inhibited the expression of TLR4 and p-P65 protein（P<0.05）.Part II Investigating the effect of 1,25-（OH）2D3 on TNF-α-induced inflammatory damage in coronary endothelial cellsObjective: To investigate the role of NF-κB inflammatory pathway in coronary artery injury in Kawasaki disease,and to elucidate the molecular mechanism of 1,25-（OH）2D3 in improving the inflammatory injury of coronary endothelial cells in Kawasaki disease.Methods: The vascular model of Kawasaki disease was constructed using HCAECs induced by TNF-α.Pre-protection with different concentrations（1n M,10 n M,and 100 nm）of 1,25-（OH）2D3 was used as the drug intervention group.CCK-8 assay to detect TNF-α drug concentration and 1,25-（OH）2D3 toxicity concentration.The m RNA expression levels of inflammatory factors IL-6,IL-8,ICAM-1 and E-selectin were detected by RT-PCR.The expression of endothelial cell connexin was detected by immunofluorescence.The expression of NF-κB inflammatory protein was detected by immunofluorescence.Results:1.CCK-8 viability assay shows significant inhibition of coronary endothelial cell viability by TNF-α at 50ng/m L.However,1,25-（OH）2D3 at the maximum concentration of 10μM still had no significant effect on the viability of coronary endothelial cells.2.Compared with the blank control group,TNF-α significantly promoted the m RNA expressions of such proinflammatory cytokines as IL-6,IL-8,ICAM-1,and E-selectin in coronary endothelial cells（P<0.05）.Compared with the TNF-α group,1,25-（OH）2D3 pre-protection could significantly inhibit the expression of IL-8,ICAM-1 and E-selectin（P<0.05）.However,1,25-（OH）2D3 did not appear to inhibit IL-6 significantly.3.Compared with the blank control group,TNF-α significantly reduced the immunofluorescence intensity of VE-cadherin and ZO-1（P<0.05）,damaging the integrity of endothelial junction.In contrast,1,25-（OH）2D3 application significantly enhanced the fluorescence intensity of endothelial ligand proteins（P<0.05）and improved endothelial junction.4.Immunofluorescence results showed that TNF-α significantly up-regulated the expression of NF-κB inflammatory protein（P<0.05）,while 1,25-（OH）2D3 pre-protection significantly down-regulated the expression of endothelial NF-κB protein（P<0.05）.Conclusion: The level of 25-（OH）D3 in children with Kawasaki disease is significantly reduced.Supplementation with 1,25-（OH）2D3 inhibits the development of inflammatory responses in Kawasaki disease macrophages and endothelial cells by modulating key inflammatory pathways,thereby reducing coronary inflammatory injury.Vitamin D is a potential adjuvant treatment for Kawasaki disease.