A Study Of Oxidative Phosphorylation Regulating The Expression Of E-selectin In Coronary Artery Lesion Of Kawasaki Disease

PART I EFFECTS OF SERA FROM CHILDREN WITH KAWASAKI DISEASE ON EXPRESSION OF E-SELECTIN IN HUMAN CORONARY ARTERY ENDOTHELIA CELLSObjective:To investigate the expression of E-selectin in human coronary artery endothelial cells stimulated by serafrom patients with different coronary outcomes of Kawasaki disease in vitro,and to seek the relationshipbetween E-selectinand coronary artery lesionof Kawasaki diseaseMethods:(1)Human coronary artery endothelial cells were cultured in vitro and divided into normal group(HC group),coronary artery lesion group(CAL+group)and no coronary artery lesion group(CAL-group).Sera of normal children,children with Kawasaki disease accompanied by coronary artery lesion and children without coronary artery lesion were added for intervention for 4h,respectively;(2)The expression level of E-selectin mRNA was detected by RT-qPCR;(3)The expression level of E-selectin protein was detected by WB;(4)The expression position and expression level of E-selectin were detected by immunofluorescence.Results:(1)After intervention for 4h,the expression level of E-selectin mRNA in the CAL+group was significantly higher than that in the CAL-group and the healthy control group(P<0.01).The expression level of E-selectin mRNA in the CAL-group was significantly higher than that in the HC group(P<0.01);(2)After 4h intervention,the expression level of E-selectin in the CAL+group was significantly higher than that in the CAL-group and the normal group(P<0.01).The expression level of E-selectin in the CAL-group was significantly higher than that in the HC group(P<0.01);(3)Immunofluorescence results showed that after intervention for 4h,E-selectin was mainly expressed in the cell membrane and cytoplasm of the CAL+group and the CAL-group.The expression level of E-selectin in the CAL+group was significantly higher than that in the CAL-group and the HC group(P<0.01),and the expression level of E-selectin in the CAL-group was significantly higher than that in the HC group(P<0.01).Conclusion:E-selectin was increased in all human coronary artery endothelial cells after sera intervention in children with Kawasaki disease,but the expression level was higher after sera intervention with coronary artery lesion,suggesting that the high expression of E-selectin may be related to the coronary artery lasion in Kawasaki disease.PART Ⅱ OVER-EXPRESSION OF E-SELECTIN MEDIATED ENDOTHELIA-MONOCYTE ADHESION IN KAWASAKI DISEASEObjective:To explore the importance of E-selectin in the adhesion between THP-1 cells and the sera-treated coronary endothelia cells.Methods:(1)Human coronary endothelia cells were cultured in vitro and divided into negative control group and small interfering RNA(E-selectin specific siRNA)group.Negative control siRNA and E-selectin specific siRNA were added,respectively,for 72h;(2)After intervention,human coronary endothelia cells in each group were further divided into CAL+groupand CAL-group.Sera of children with Kawasaki disease with coronary artery lesion and children without coronary artery lesion were added for 4 hours after intervention,respectively;(3)THP-1 cells labeled with BCECF-AM were co-incubated with human coronary endothelia cells after sera intervention for 1h;(4)The adhesion of THP-1 cells to human coronary endothelia cells was recorded by fluorescence microscopy;(5)The fluorescence value of THP-1 cells that adhered to human coronary endothelial cells was detected by enzyme-plate analyzer.Results:(1)In the Negtive control group,thefluorescence intensity of THP-1 cells adhered to endothelial cells in the CAL+group was higher than that in the CAL-group(P<0.01).(2)In the E-selectin-siRNA group,there was no significant difference in the fluorescence intensity of THP-1 cells attached to endothelial cells between the CAL+group and the CAL-group(P>0.05).(3)The fluorescence intensity of THP-1 cells adhering to endothelial cells in E-selectin-siRNA group was significantly decreased when the sera of CAL+group or CAL-group was added compared with the same seraintervention in Negtive Control group(P<0.01).Conclusion:Over-expression of E-selectin plays an important role in endothelia-monocyte adhesion of coronary artery lesion in Kawasaki disease.PART Ⅲ OXIDATIVE PHOSPHORYLATION UP-REGULATIONMEDIATEDTHE EXPRESSION OF E-SELECTIN IN ENDOTHELIA CELLS OFKAWASAKIDISEASEObjective:To explore the mechanism of E-selectin over-expression and endothelia-monocyte adhesion in endothelial cells treatedwith serafrom children with coronary artery lesionof Kawasaki disease.Methods:(1)Human coronary artery endothelial cells(HCAEC)were cultured in vitro and divided into HC group,CAL+group and CAL-group.Sera of healthy children,children with Kawasaki disease accompanied by coronary artery lesion and without coronary artery lesion were added for intervention for 4h,respectively;(2)High-throughput RNA sequencing was performed on the cells of CAL+groupand CAL-group,and difference analysis and enrichment analysis were performed.Results:(1)There were 1532 differential genes in the CAL+group compared with the CAL-group,among which 767 genes were up-regulated and 765 genes were down-regulated;(2)GO analysis showed that the oxidative phosphorylation related genes were significantly up-regulated in the CAL+group compared with the CAL-group(P<0.05);(3)KEGG analysis showed that the oxidative phosphorylation related pathway genes were significantly up-regulated in the CAL+group compared with the CAL-group(P<0.05);(4)The ATP content,Complexi activity and mitochondrial DNA copy number of human coronary endothelial cells in the CAL+group were significantly higher than those in the CAL-group(P<0.01).The ATP content,Complexi activity and mitochondrial DNA copy number of human coronary endothelial cells in the CAL-group were significantly higher than those in the normal group(P<0.01);(5)Oligomycin significantly decreased the expression of E-selectin at mRNA and protein levels in the CAL+group and the CAL-group(P<0.01).Conclusion:The up-regulation of oxidative phosphorylation wasrelatedto the high expression of E-selectin in human coronary endothelial cells treated by Kawasaki disease coronary artery lesion seraand promoted the adhesion of endothelia cells to monocytes,which may be involved in the process of Kawasaki disease coronary artery lesion.PART Ⅳ EFFECTS OF OLIGOMYCIN ON VASCULITIS AND EXPRESSION OF E-SELECTIN IN A MOUSE MODEL OF KAWASAKI DISEASEObjective:To investigate the effect of oligomycin on vasculitis and the expression of E-selectin in a mouse model of Kawasaki disease established by LCWEMethods:(1)A mouse model of Kawasaki disease was established in C57 male mice aged 4-6 weeks.The mice were divided into LCWE group,LCWE plus oligomycin group and PBS group,and were intraperitoneally injected with LCWE,LCWE+oligomycin and PBS,respectively.(2)Observe the general condition of mice;(3)After 14 days,the hearts of mice were stained with HE to understand the vasculitis of mice;(4)The expression of E-selectin was identified by cardiac histochemical staining.Results:(1)Compared with the other two groups,LCWE group showed less activity and irritability,and there was no significant difference in body weight between the three groups.(2)Compared with PBS group,the inflammation index of LCWE group was significantly increased by HE staining(P<0.01),and the inflammation index of LCWE plus oligomycin group was significantly decreased compared with LCWE group(P<0.05).(3)The expression of E-selectin in LCWE group was significantly increased compared with PBS group(P<0.01),and the expression of E-selectin in LCWE+oligomycin group was significantly decreased compared with LCWE group(P<0.05).Conclusion:Oligomycin,an oxidative phosphorylation inhibitor,could reduce the severity of vasculitis and the expression level of E-selectin in Kawasaki disease mice model,suggesting that oxidative phosphorylation may regulate the expression of E-selectin and participate in coronary artery lesion in Kawasaki disease mice model.