【摘要】 目的 探讨沉默调节蛋白1(SIRT1)/核因子-κB(NF-κB)/核苷酸结合寡聚结构域样受体蛋白3(NLRP3)在染料木素抑制氧化低密度脂蛋白（ox-LDL）诱导人脐静脉内皮细胞（HUVEC）焦亡中的调控作用。方法 体外培养HUVEC，设置8组：空白对照组、ox-LDL(50 mg/L)组、ox-LDL(50 mg/L)+低剂量染料木素（200 nmol/L）组、ox-LDL(50 mg/L)+MCC950(10 nmol/L)组、ox-LDL(50 mg/L)+低剂量染料木素（200 nmol/L）+MCC950(10 nmol/L)组、ox-LDL(50 mg/L)+染料木素（1 000 nmol/L）组、SIRT1沉默（转染SIRT1小干扰RNA）+ox-LDL(50 mg/L)+染料木素（1 000 nmol/L）组、NLRP3过表达（转染NLRP3表达载体）+ox-LDL(50 mg/L)+染料木素（1 000 nmol/L）组。采用流式细胞术检测焦亡细胞比例；酶活性试剂盒测定半胱氨酸天冬氨酸蛋白酶（caspase）-1及乳酸脱氢酶（LDH）活性；酶联免疫吸附法检测人白细胞介素（IL）-1β、IL-18水平；免疫印迹法检测NF-κB、乙酰化NF-κB(Ac-NF-κB)、NLRP3、凋亡相关的斑点样蛋白（ASC）、caspase-1、切割型caspase-1(cleaved caspase-1)、消皮素D(GSDMD)及N端形成孔隙的消皮素D片段（NT-GSDMD）的蛋白水平。结果 与空白对照组相比，ox-LDL组IL-1β、IL-18水平，LDH、caspase-1活性及焦亡细胞比例升高（t=17.234、16.523、18.182、20.181、11.740，均P<0.05）。与ox-LDL组相比，ox-LDL+低剂量染料木素组、ox-LDL+MCC950组上述指标降低（t=6.958、7.993、6.171、7.251、4.971、5.938、7.199、6.773、6.743、5.402，均P<0.05）。与ox-LDL+低剂量染料木素组及ox-LDL+MCC950组相比，ox-LDL+低剂量染料木素+MCC950组上述指标降低（均P<0.05）。与ox-LDL+染料木素组相比，SIRT1沉默+ox-LDL+染料木素组、NLRP3过表达+ox-LDL+染料木素组上述指标升高（均P<0.05）。与空白对照组相比，ox-LDL组Ac-NF-κB、NLRP3、ASC、cleaved caspase-1、NT-GSDMD蛋白水平增高（t=6.424、5.258、6.178、5.149、6.536，均P<0.05）；与ox-LDL组相比，ox-LDL+染料木素组上述蛋白水平降低（t=5.777、4.177、5.782、4.018、5.847，均P<0.05）；与ox-LDL+染料木素组相比，SIRT1沉默+ox-LDL+染料木素组上述蛋白水平升高（均P<0.05）。结论 SIRT1/NF-κB/NLRP3通路在染料木素抑制ox-LDL诱导的HUVEC焦亡中发挥关键调控作用。更多还原

【Abstract】 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 oxLDL(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(NTGSDMD). 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 genisteinmediated inhibition of ox-LDL-induced pyroptosis in HUVEC.更多还原