The Role And Mechanism Of Huc-MSCs In Repairing Coronary Endothelial Injury By Inhibiting The Pyroptosis Pathway In Kawasaki Disease

Objective At the in vivo and external levels,studying the function and molecular mechanisms of human umbilical cord mesenchymal stem cells(HucMSCs)involved in repairing KD coronary artery endothelial damage through regulating Kawasaki disease(KD)endothelial cells pyroptosis-related pathway of KD coronary artery endothelial injury,which provides an important theoretical and experimental basis for the development of related KD treatment methods in the future.Methods(1)The peripheral blood samples and clinical data of KD and healthy children were collected from department of pediatrics and health physical examination center of affiliated hospital of Nantong University.The differences in lactate dehydrogenase(LDH)were analyzed through the clinical data,and the concentrations of IL-1β and IL-18 in peripheral blood plasma were determined by ELISA.The m RNA levels of pyroptosis related proteins in peripheral blood mononuclear cells(PBMCs)were detected by RT-q PCR.(2)HE staining and immunohistochemical staining were used to observe the changes of inflammatory cells and macrophages around coronary artery and the localization of NLRP3 and IL-1β of pyroptosis pathways before and after intervention after the coronary artery injury model of KD mice treated with Huc-MSCs.Western blot was used to detect the protein expression levels of key molecules in the pyroptosis pathway of Huc-MSCs before and after intervention.(3)The co-culture system of human myeloid leukemia mononuclear cells(THP-1 cells)and human umbilical vein endothelial cells(HUVECs)were stimulated by inactivated serum in the acute stage of KD to constructe the cell model of KD in vitro,and then co-cultured with Huc-MSCs after 24 h;The cell biology function experiments such as cell migration,cell invasion,and tubule formation experiments were investigated before and after Huc-MSCs intervention changes in the biological function of HUVECs,and analysis of changes in cellular inflammatory status before and after Huc-MSCs intervention by Western blot,cellular immunofluorescence,TUNEL,LDH release experiments.KD cell models and Huc-MSCs treatment models were constructed after pretreatment interventions with NLRP3 inhibitors and agonists respectively to investigate the biological function of HUVECs and the changes in cellular inflammatory status.Results(1)Compared with HC group,the concentrations of IL-1β,IL-18 and LDH in the plasma of children with acute KD were also significantly higher than that in HC group(P < 0.001);The m RNA expression of pyroptosis pathway molecules in PBMCs was significantly higher than that in HC group(P < 0.001).(2)In vivo model,there was significant inflammatory infiltration around the coronary artery wall of KD mice,the area of positive expression of F4/80 around the coronary artery was obvious,and the positive expression of NLRP3 and IL-1β could be observed around the blood vessel wall,while the inflammatory infiltration was significantly reduced after Huc-MSCs intervention and the deposition and positive expression areas of the above molecules were significantly reduced;the expression of key molecular proteins in the KD group of the pyroptosis pathway was significantly increased compared with that of group N(P < 0.01 or P < 0.001),and the expression of Huc-MSCs was significantly reduced compared with that before intervention(P< 0.05 or P < 0.001);(3)In the cell model,the area of migration,the number of invading and migrating,the number of lumen and branches,and the total length of the tubes in the KD group of HUVECs were significantly reduced compared with the N group(P < 0.01 or P < 0.001);the biological function of HUVECs was significantly improved compared with the KD group after co-culture with Huc-MSCs(P < 0.05),and the proportion of TUNEL-positive cells and the concentration of LDH in the KD group were significantly increased compared with the N group(P < 0.05);After co-culture treatment with Huc-MSCs and HUVECs,the proportion of TUNEL-positive cells decreased significantly(P <0.05)and the concentration of LDH decreased significantly(P < 0.01);the expression trend of key molecular proteins in the KD group pyroptosis pathway was consistent with that of animals.Compared with the KD group,the area of migration,the number of invading and migrating cells,the number of lumen and branches,and the total length of the tube after treating HUVECs with NLRP3 agonists were significantly reduced(P < 0.05 or P < 0.01 or P < 0.001),and the expression of key molecule proteins in pyrotosis pathway,and the proportion of TUNEL-positive cells,the concentration of LDH were significantly increased(P < 0.05 or P < 0.01 or P < 0.001;the above results could be partially reversed after co-culture with Huc-MSCs;After the treatment of HUVECs by NLRP3 inhibitors,the experimental results showed a contrary trend with the agonist group,and the trend of co-culture with HucMSCs was more significant.Conclusions(1)Pyroptosis is involved in the inflammatory processes in the acute phase of KD.(2)Intervention with Huc-MSCs can reduce inflammatory cell infiltration of coronary arteries in KD animal models.(3)Huc-MSCs intervention can reduce the inflammatory state of HUVECs and improve endothelial dysfunction of HUVECs by inhibiting NLRP3-dependent pyroptosis pathway in KD cell model.