Effect of miR-21-5p on pyroptosis of endothelial cells in Kawasaki disease mouse model and its mechanism
Objective To explore the effect and mechanism of miR-21-5p on focal death of endothelial cells in Kawasaki disease(KD)mouse model.Methods According to the random number table method,16 C57BL/6 mice were divided into KD group and PBS group,with 8 mice in each group.KD model was induced by intraperitoneal injection of lactobacillus casei cell wall extract(LCWE)in mice.Mouse coronary artery endothelial cells(MCAECs)were treated with LCWE to establish a KD cell model.On the basis of LCWE treatment,MCAECs were transfected with miR-21-5p inhibitor or the negative control of the inhibitor(inhibitor-NC),miR-21-5p mimics or the negative control of the mimics(miR-NC),and pretreated with or without NLRP3 inhibitor MCC950.HE staining was used to observe the histopathological changes of coronary artery;qRT-PCR was used to detect the expression of miR-21-5p;Western blot was used to detect the protein expression of recombinant NLR family,Pyrin domain containing protein 3(NLRP3),apoptosis-related speckle-like protein(ASC),cysteinyl aspartate specific proteinase(caspase-1),pro-caspase-1,gasdermin D(GSDMD)p30,IL-1β,and IL-18.Cell vitality was determined with cell counting kit-8 test and lactate dehydrogenase(LDH)activity was determined with LDH test kits.Results Compared with the PBS group,the KD mice had obvious coronary artery tissue lesions,and the level of pyroptosis mediated by NLRP3 inflammasomes and the expression of miR-21-5p were increased in the coronary artery tissue of KD mice(all P<0.01).Compared with the control group,the activity of MCAECs cells in LCWE group was significantly decreased and the expression of miR-21-5p was significantly increased(both P<0.01).Compared with LCWE+inhibitors-NC group,the expression of miR-21-5p,the protein expression of NLRP3,ASC,caspase-1,GSDMD p30,IL-1 β,IL-18 and LDH release quantity in LCWE+miR-21-5p inhibitor group were significantly lower(all P<0.01),and the cell vitality increased significantly(P<0.05).Compared with LCWE group,LDH release and the protein expression of NLRP3,ASC,caspase-1,GSDMD p30,IL-1 β and IL-18 in LCWE+MCC950 group were significantly decreased(all P<0.01),and the cell activity increased significantly(P<0.01).Further transfection of miR-21-5p mimics significantly reversed the above changes(all P<0.01).Conclusion In KD,miR-21-5p regulates vascular endothelial cell pyroptosis by activating NLRP3 inflammasome to aggravate vascular endothelial cell injury.These results suggest that inhibition of miR-21-5p can partially ameliorate vascular endothelial cell injury in KD,and miR-21-5p might be used as a potential intervention target for the treatment of KD.
Kawasaki diseaseInflammasomePyroptosismiR-21-5pRecombinant NLR family,Pyrin domain containing protein 3