沉默调节蛋白1介导染料木素抑制氧化低密度脂蛋白诱导人脐静脉内皮细胞焦亡的作用OA

Objective To investigate the regulatory role of sirtuin-1(SIRT1)/nuclear factor kappa-B(NF-κB)/NOD-like receptor protein 3(NLRP3)in genistein-mediated inhibition of oxidized low-density lipoprotein(ox-LDL)-induced pyroptosis in human umbilical vein endothelial cell(HUVEC).Methods HUVECs were cultured in vitro and divided into eight groups:the blank control group;the ox-LDL(50 mg/L)group;the ox-LDL(50 mg/L)+low-dose genistein(200 nmol/L)group;the ox-LDL(50 mg/L)+MCC950(10 nmol/L)group;the ox-LDL(50 mg/L)+low-dose genistein(200 nmol/L)+MCC950(10 nmol/L)group;the ox-LDL(50 mg/L)+genistein(1 000 nmol/L)group;the SIRT1 knockdown(transfected with SIRT1 small interfering RNA)+ox-LDL(50 mg/L)+genistein(1 000 nmol/L)group;and the NLRP3 over-expression(transfected with NLRP3 expression vector)+ox-LDL(50 mg/L)+genistein(1 000 nmol/L)group.The proportion of pyroptotic cells was analyzed by flow cytometry.Caspase-1 and lactate dehydrogenase(LDH)activities were detected using enzyme activity kits.The levels of human interleukin(IL)-1β and IL-18 were quantified by enzyme-linked immunosorbent assay(ELISA).Western blotting was used to determine the protein expression levels of NF-κB,acetylated NF-κB(Ac-NF-κB),NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),caspase-1,cleaved caspase-1,gasdermin D(GSDMD)and its N-terminal pore-forming fragment(NT-GSDMD).Results Compared with the blank control group,the ox-LDL group exhibited significantly elevated levels of IL-1β and IL-18,increased activities of LDH and caspase-1,and a higher pyroptotic cell ratio(t=17.234,16.523,18.182,20.181,11.740;all P<0.05).These indicators were significantly reduced in both the ox-LDL+low-dose genistein group and the ox-LDL+MCC950 group compared to the ox-LDL group(t=6.958,7.993,6.171,7.251,4.971,5.938,7.199,6.773,6.743,5.402;all P<0.05).The ox-LDL+low-dose genistein+MCC950 group showed a further significant decrease in these indicators compared to either the ox-LDL+low-dose genistein group or the ox-LDL+MCC950 group(all P<0.05).Compared with the ox-LDL+genistein group,the SIRT1 knockdown+ox-LDL+genistein group and the NLRP3 overexpression+ox-LDL+genistein group demonstrated significant increases in the aforementioned indicators(all P<0.05).Regarding protein expression,the ox-LDL group showed significantly higher levels of Ac-NF-κB,NLRP3,ASC,cleaved caspase-1,and NT-GSDMD than the blank control group(t=6.424,5.258,6.178,5.149,6.536;all P<0.05).These protein levels were significantly decreased in the ox-LDL+genistein group compared to the ox-LDL group(t=5.777,4.177,5.782,4.018,5.847;all P<0.05).Furthermore,these protein levels were significantly elevated in the SIRT1 knockdown+ox-LDL+genistein group compared to the ox-LDL+genistein group(all P<0.05).Conclusion The SIRT1/NF-κB/NLRP3 pathway plays a crucial role in genistein-mediated inhibition of ox-LDL-induced pyroptosis in HUVEC.