SIRT1对低氧诱导肺动脉内皮细胞损伤的保护作用及机制

SIRT1 ameliorates hypoxia-induced pulmonary artery endothelial cell injury

目的探讨沉默信息调节因子1相关酶(SIRT1)对低氧诱导肺动脉内皮细胞损伤的保护作用及其机制。方法体外培养人肺动脉内皮细胞,将细胞分为8组:对照组、低氧组、SRT1720(SIRT1激动剂)组、EX-527(SIRT1抑制剂)组、低氧+SRT1720组、低氧+EX-527组、低氧+TEMPOL(mitoROS抑制剂)组和低氧+NAC(ROS抑制剂)组。正常组细胞置于正常细胞培养箱中培养,低氧模型细胞置于3%O2,5%CO_(2)低氧舱中处理24 h,其余组分别予SRT1720、EX-527、TEMPOL和NAC处理24 h后低氧处理。DHE和MitoSOX染色法检测细胞内及线粒体活性氧水平,试剂盒检测细胞MDA水平,免疫荧光法检测细胞沉默信息调节因子相关酶1(SIRT1)、核苷酸结合寡聚化片段结构域样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白水解酶1(Caspase-1)、发动蛋白相关GTP酶(Drp1)、线粒体融合蛋白2(Mfn2)水平,JC-1染色法检测细胞线粒体膜电位水平,ELISA法检测细胞炎症因子IL-1β和IL-18水平,免疫印迹法检测细胞Gasdermin D(GSDMD)蛋白水平。结果与对照组相比,低氧组肺动脉内皮细胞的SIRT1蛋白表达降低(P<0.01),胞内ROS、mitoROS、MDA、NLRP3、Caspase-1、GSDMD、Drp1蛋白表达和炎症因子IL-18和IL-1β水平均升高(P均<0.01),线粒体膜电位(MMP)水平、Mfn2蛋白表达均降低(P均<0.01)。与低氧组相比,低氧+SRT1720组、低氧+TEMPOL组和低氧+NAC组人肺动脉内皮细胞的SIRT1蛋白表达增加(P均<0.05),胞内ROS、mitoROS、MDA、NLRP3、Caspase-1、GSDMD、Drp1蛋白表达和炎症因子IL-18和IL-1β水平均降低(P均<0.05),MMP水平、Mfn2蛋白表达均增加(P均<0.01);而低氧+EX-527组人肺动脉内皮细胞的胞内ROS、mitoROS、MDA、NLRP3、Caspase-1、GSDMD、Drp1蛋白表达和炎症因子IL-18和IL-1β水平均升高(P均<0.05),MMP水平、Mfn2蛋白表达均降低(P均<0.05)。结论SIRT1可通过抑制线粒体ROS的形成和NLRP3介导焦亡发生,对低氧诱导肺动脉内皮细胞损伤有保护作用。

Objective To investigate the effect of SIRT1 on hypoxia-induced pulmonary artery endothelial cell injury and its mechanism.Methods Human pulmonary artery endothelial cells were cultured in vitro and divided into eight groups:the control group,hypoxia group,SRT1720(SIRT1 agonist)group,EX-527(SIRT1 inhibitor)group,hypoxia+SRT1720 group,hypoxia+EX-527 group,hypoxia+TEMPOL(mitoROS inhibitor)group,and hypoxia+NAC(ROS inhibitor)group.The cells in the control group were cultured in the normal cell incubator,and the hypoxic model cells were treated with 3%O2 and 5%CO2 in the hypoxic chamber for 24 h.Cells in the others group were treated with SRT1720,EX-527,TEMPOL,and NAC for 24 h before hypoxic treatment.The levels of intracellular and mitochondrial reactive oxygen species were detected by DHE and MitoSOX staining,the levels of MDA were detected by the kit,the levels of SIRT1,NLRP3,Caspase-1,Drp1 and Mfn2 were detected by immunofluorescence,and the levels of mitochondrial membrane poten‑tial were detected by JC-1 staining.The levels of inflammatory cytokines IL-1βand IL-18 were detected by ELISA,and the level of GSDMD protein was detected by Western blotting.Results Compared with the control group,the protein ex-pression of SIRT1 in pulmonary artery endothelial cells decreased,but the protein expression levels of ROS,mitoROS,MDA,NLRP3,Caspase-1,GSDMD,Drp1,and the levels of inflammatory cytokines IL-18 and IL-1βincreased,and the levels of mitochondrial membrane potential(MMP)and Mfn2 protein expression decreased in the hypoxia group(all P<0.05).Compared with the hypoxia group,the expression of SIRT1 protein in pulmonary artery endothelial cells in-creased,but the protein expression levels of ROS,mitoROS,MDA,NLRP3,Caspase-1,GSDMD,Drp1 and inflammato-ry cytokines IL-18 and IL-1βdecreased,and the MMP level and Mfn2 protein expression increased in the hypoxia+SRT1720 group,hypoxia+TEMPOL group,and hypoxia+NAC group(all P<0.05);the protein expression levels of ROS,mitoROS,MDA,NLRP3,Caspase-1,GSDMD,Drp1 and inflammatory cytokines IL-18 and IL-1βincreased in the pulmonary artery endothelial cells,and the levels of MMP and Mfn2 protein expression decreased in hypoxia+EX-527 group(all P<0.05).Conclusion SIRT1 can ameliorate pulmonary endothelial cell injury induced by hypoxia by inhibit-ing the formation of mitochondrial ROS and NLRP3-mediated pyroptosis.