The Role Of NLRP3 In Oxidized Low-density Lipoprotein-induced Damage To Endothelial Cell

Objective Atherosclerosis(AS)mainly manifests as vascular wall dysfunction caused by plaque accumulation,which is the main pathological process in the occurrence and development of various cardiovascular diseases.Dysfunction of vascular endothelial cells induced by oxidized low density lipoprotein(ox-LDL)is closely related to the development of AS.The increased morbidity and mortality of atherosclerosis has brought great medical and economic burden to the society.Nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome activation-related inflammatory responses has found to be involved in the development of AS.The purpose of this study was to explore the effect of NLRP3 on ox-LDL-induced damage to vascular endothelial cells.Methods Human umbilical venous endothelial cells(HUVEC)were incubated with different concentrations of ox-LDL for 12,24 or 48 hours,to establish ox-LDL-induced endothelial cell damage model,followed by incubation with ox-LDL and an NLRP3 inhibitor MCC950 to explore the role of NLRP3 on ox-LDL-induced endothelial cell damage.Cell counting kit-8(CCK-8)and optical microscope observation method were used to detect endothelial cell proliferation under different conditions.Flow cytometry method was used to detect cell apoptosis under different conditions.Lactic acid dehydrogenase(LDH)cytotoxicity detection kit and total superoxide dismutase(SOD)detection kit were applied to determine the expression levels of oxidative damage markers under different conditions.Western blot was used to determine the expression levels of cell apoptosis markers,NLRP3 and the supernatant apoptosis associated speck like protein containing a CARD(ASC),and the downstream inflammatory factors of NLRP3,cleaved cysteinyl aspartate specific proteinase 1(caspase-1),mature interleukin-1β(IL-1β)and IL-18 under different conditions.Results 25ug/ml,50 ug/ml and 100 ug/ml of ox-LDL inhibited HUVEC proliferation and promoted cell apoptosis with concentration dependence,which hold statistically significant difference compared to the blank group(P<0.05).In addition,ox-LDL induced oxidative damage to HUVEC,which was demonstrated as the increased LDH level and decreased SOD activity,and the difference is statistically significant compared to the blank group(P<0.05).In ox-LDL-induced HUVEC damage,the expression levels of NLRP3,supernatant ASC and its downstream inflammatory signaling molecules,cleaved caspase-1,mature IL-1βand IL-18 were significantly increased.MCC950,a NLRP3 targeted inhibitor reversed ox-LDL-induced proliferation inhibition,apoptosis promotion and oxidation damage to HUVEC.In addition,under the incubation of MCC950,the levels of supernatant ASC and the downstream inflammatory factors of NLRP3,cleaved caspase-1,mature IL-1β and IL-18 were found to be reduced in ox-LDL-incubated endothelial cells.Conclusion Ox-LDL induced HUVEC damage by activating the NLRP3inflammasome-related signaling pathway.While inhibition of NLRP3 and the downstream signal proteins could alleviate HUVEC damage.NLRP3 inflammasome hold the potential to serve as an effective target for atherosclerosis treatment.