Role And Mechanism Of NLRP3 Inflammasome In Smooth Muscle-derived Foam Cell Formation

Background and Objective: The instability of atherosclerotic(AS)plaques is the pathological basis affecting the occurrence of cardiovascular events,and the formation of foam cells,which are derived from macrophages and vascular smooth muscle cells(VSMC),is the core of the instability of AS plaques.Most previous studies focused on the involvement of macrophage-derived foam cells in AS,but in recent years,foam cells in advanced AS plaques have been found.50% came from VSMC,which changed the traditional understanding.Previous studies have shown that NLRP3 can promote the formation of macrophage-derived foam cells,but whether it has an effect on the formation of VSMC-derived foam cells has not been reported.In this study,a VSMC overexpressed cell model was constructed by lentivirus transfection and a NLRP3 gene knockout cell model was constructed by si RNA.The effect of NLRP3 expression on the formation of VSMC-derived foam cells was observed at the cellular level,and the NLRP3 specific inhibitor MCC950 was applied for intervention.To observe whether inhibition of NLRP3 activation can reverse the effect on smooth muscle derived foam cell formation to some extent.This study aims to further explore the role and mechanism of NLRP3 inflammasome on smooth muscle-derived foam cell formation and find potential intervention targets,providing evidence for promoting the development of NLRP3 AS an inflammatory targeting drug.Methods:In this study,NLRP3 overexpressed stable cells were constructed from human aorta smooth muscle cells using lentiviral vectors,NLRP3 knockdown was performed with small interfering RNA(si RNA),NLRP3 specific inhibitor MCC950 and ox-LDL was stimulated with a final concentration of 100ug/ml.The cells were divided into the following 7 groups: Blank group,ox-LDL group,NLRP3(+)group +ox-LDL,vehicle control group +ox-LDL,si-NLRP3(-)group +ox-LDL,si-NC group +ox-LDL,Mcc950 inhibitor group +ox-LDL.The migration capacity of smooth muscle cells stimulated by ox-LDL was observed by cell scar assay,the uptake capacity of smooth muscle cells to Dil-ox LDL was observed under fluorescence microscope,the lipid load of cells and the degree of cell foam were observed by oil red O staining,and cholesterol efflux rate was detected by cholesterol efflux kit to determine whether lipid efflux was free.NLRP3 and downstream inflammatory cytokines caspase-1,IL-1β and IL-18 were detected by Western blot and q PCR.The above methods were used to investigate the effects of NLRP3 inflammasome on the migration,lipid uptake,accumulation and outflow of vascular smooth muscle cells,and to reveal the role of NLRP3 inflammasome activation in the formation of smooth muscle derived foam cells.Semi-quantitative analysis of relevant data was performed by using software such as Image J and Graph Pad Prism 9.0.Result:(1)After 48 h stimulation with ox-LDL(100ug/ml),smooth muscle cells increased their migration ability,enhanced lipid uptake ability,and reduced cholesterol efflux rate,resulting in intracellular lipid accumulation and gradually foaming into smooth muscle derived foam cells.(2)Compared with the control group,the migration capacity and lipid uptake capacity of smooth muscle cells in NLRP3(+)group were increased,while the cholesterol outflow rate was decreased.The expression of downstream inflammatory cytokines was higher than that of control group.In si-NLRP3 group,ox-LDL was also treated,and the migration capacity of vascular smooth muscle cells and lipid uptake capacity were inhibited,while the outflow of cholesterol was increased,and the expression of downstream inflammatory factors was inhibited.These results suggest that NLRP3 inflammasome may be involved in the formation of smooth muscle derived foam cells.(3)The application of NLRP3 specific inhibitor Mcc950 can inhibit ox-LDL-induced smooth muscle cell migration,lipid uptake and inflammatory activation to a certain extent,and increase lipid outflow,suggesting that Mcc950 has an inhibitory effect on smooth muscle derived foam cells and a certain protective effect on smooth muscle cells.Conclusion:(1)ox-LDL induced the formation of smooth muscle derived foam cells(2)NLRP3 inflammasome play an important role in ox-LDL-induced smooth muscle derived foam cell formation(3)NLRP3 specific inhibitor MCC950 may play an important protective role in smooth muscle derived foam cell formation(4)NLRP3 is expected to be an important intervention target for AS control...