内源性一氧化氮对血管内皮细胞超氧化物歧化酶1活性及细胞凋亡的影响

Effect of endogenous nitric oxide on superoxide dismutase-1 activity and apoptosis of vascular endothelial cells

目的探讨内源性一氧化氮(NO)对人脐静脉内皮细胞(HUVECs)超氧化物歧化酶1(SOD1)活性及内皮细胞凋亡的调节作用。方法以HUVECs为研究对象,采用内皮型一氧化氮合酶(eNOS)短发夹RNA(shRNA)慢病毒转染对内皮细胞eNOS进行敲低,HUVECs分为4组:空载体组(scramble)、转染eNOS shRNA组(eNOS shRNA)、转染eNOS shRNA+硝普钠(SNP)组(eNOS shRNA+SNP)及转染eNOS shRNA+SNP+三(2-羧乙基)膦盐酸盐(TCEP)组(eNOS shRNA+SNP+TCEP)。采用Western blot法检测eNOS蛋白表达和SOD1二聚体/单体蛋白表达,采用酶联免疫吸附法检测SOD酶活性,采用NO荧光探针法检测内皮细胞NO水平,采用二氢乙锭(DHE)检测内皮细胞超氧化物阴离子水平,采用原位末端转移酶标记技术检测内皮细胞凋亡情况。结果与空载体组相比,eNOS shRNA组中内源性NO水平(2.690±0.420比15.029±2.193,P<0.01)、eNOS蛋白表达(1.000±0.778比3.141±0.199,P<0.01)、SOD1二聚体/单体比例(4.6±1.0比7.6±2.0,P<0.05)和SOD活性[(0.432±0.254)Carmen′s unit/10^(4) cell比(1.000±0.116)Carmen′s unit/10^(4) cell,P<0.01]均显著降低,细胞内超氧阴离子水平(11.180±1.560比6.146±1.007,P<0.01)和HUVECs凋亡水平[75.0(55.0,100.0)%比0(0,0)%,P<0.01]均显著增加。与eNOS shRNA组相比,eNOS shRNA+SNP组中内源性NO含量(16.705±0.116比2.690±0.420,P<0.01)、SOD1二聚体/单体比例(7.3±2.0比4.6±1.0,P<0.05)和SOD活性[(0.737±0.060)Carmen′s unit/10^(4) cell比(0.432±0.254)Carmen′s unit/10^(4) cell,P<0.05]均显著增加,细胞内超氧阴离子水平(6.897±1.648比11.180±1.560,P<0.01)和HUVECs凋亡水平[0(0,0)%比75.0(55.0,100.0)%,P<0.01]均显著下降。与eNOS shRNA+SNP组相比,eNOS shRNA+SNP+TCEP组中SOD1二聚体/单体比例(4.4±0.9比7.3±2.0,P<0.05)和SOD活性[(0.214±0.084)Carmen′s unit/10^(4) cell比(0.737±0.060)Carmen′s unit/10^(4) cell,P<0.01]均显著降低,超氧阴离子水平(10.917±1.552比6.897±1.640,P<0.01)和HUVECs凋亡水平[63.6(55.0,90.0)%比0(0,0)%,P<0.01]均显著增加;但NO水平(16.112±0.926比16.705±0.116,P>0.05)无明显变化。结论内源性NO通过上调SOD1二聚体/单体比例,增强SOD活性,抑制活性氧积累,从而有效对抗人脐静脉内皮细胞凋亡。

Objective To investigate the regulatory effects of endogenous nitric oxide(NO)on the activity of superoxide dismutase-1(SOD1)and apoptosis of human umbilical vein endothelial cells(HUVECs).Methods HUVECs were taken as the research object.The endothelial NO synthase(eNOS)short hairpin RNA(shRNA)lentivirus was employed to transfect HUVECs to knock down eNOS.HUVECs were divided in 4 groups:the scramble group,the eNOS shRNA group,the eNOS shRNA+sodium nitroprusside(SNP)group and the eNOS shRNA+SNP+tris(2-carboxyethyl)phosphine hydrochloride(TCEP)group.The protein expressions of eNOS and SOD1 dimer/monomer in cells were detected by western blot.The activity of SOD was detected by the enzyme-linked immunosorbent assay.The NO content in cells was detected with NO fluorescence probe.The level of superoxide anion in HUVECs was detected with dihydropyridine(DHE).The terminal deoxynucleotidyl transferase(TdT)dUTP nick-end labeling(TUNEL)assay was adopted to detect the apoptosis of HUVECs in situ.Results Compared with the scramble group,the endogenous NO content(2.690±0.420 vs.15.029±2.193,P<0.01),eNOS protein expression(1.000±0.778 vs.3.141±0.199,P<0.01),SOD1 dimer/monomer ratio(4.6±1.0 vs.7.6±2.0,P<0.05)and SOD activity[(0.432±0.254)Carmen′s unit/10^(4) cell vs.(1.000±0.116)Carmen′s unit/10^(4) cell,P<0.01]were significantly decreased,while the level of intracellular superoxide anion(11.180±1.560 vs.6.146±1.007,P<0.01)and HUVECs apoptosis[75.0(55.0,100.0)%vs.0(0,0)%,P<0.01]were significantly increased in the eNOS shRNA group.Compared with the eNOS shRNA group,the content of endogenous NO(16.705±0.116 vs.2.690±0.420,P<0.01),the ratio of SOD1 dimer/monomer(7.3±2.0 vs.4.6±1.0,P<0.05)and the activity of SOD[(0.737±0.060)Carmen′s unit/10^(4) cell vs.(0.432±0.254)Carmen′s unit/10^(4) cell,P<0.05]were significantly increased,while the level of superoxide anion(6.897±1.648 vs.11.180±1.560,P<0.01)and the HUVECs apoptosis[0(0,0)%vs.75.0(55.0,100.0)%,P<0.01]were significantly decreased in the eNOS shRNA+SNP group.Compared with the eNOS shRNA+SNP group,the ratio of SOD1 dimer/monomer(4.4±0.9 vs.7.3±2.0,P<0.05)and the activity of SOD[(0.214±0.084)Carmen′s unit/10^(4) cell vs.(0.737±0.060)Carmen′s unit/10^(4) cell,P<0.01]were significantly decreased,while the level of superoxide anion(10.917±1.552 vs.6.897±1.640,P<0.01)and the apoptosis level of HUVECs[63.6(55.0,90.0)%vs.0(0,0)%,P<0.01]were significantly increased in the eNOS shRNA+SNP+TCEP group.However,there was no significant difference in the NO content(16.112±0.926 vs.16.705±0.116,P>0.05).Conclusions Endogenous NO could effectively antagonize the apoptosis of endothelial cells by increasing the cysteine-dependent SOD1 dimer/monomer ratio,enhancing SOD activity and inhibiting the accumulation of reactive oxygen species.