锌对尿毒症血清诱导的内皮细胞氧化应激的保护作用及其机制

Protective effect of Zinc on oxidative stress in vascular endothelial cells induced by uremic serum and its possible mechanism

目的:探讨锌对尿毒症血清诱导的血管内皮细胞氧化应激的保护作用及可能机制。方法:收集尿毒症患者及健康正常人血清,以15%的血清浓度作用于血管内皮细胞。分为4组进行实验:正常血清组(N)、尿毒症血清组(U)、尿毒症血清+10μmol/L硫酸锌组(U+10)、尿毒症血清+30μmol/L硫酸锌组(U+30)。荧光素DCFH-DA探针法测定细胞内活性氧含量,采用总抗氧化能力检测试剂盒检测细胞总抗氧化能力,实时荧光定量PCR检测细胞还原型烟酰胺腺嘌呤二核苷酸磷酸:醌氧化还原酶1(nicotinamide adenine dinucleotidephosphate:quinone oxidoreductase 1,NQO-1)及诱导Ⅰ型血红素氧合酶(heme oxygenase,HO-1)m RNA的表达,蛋白印迹检测细胞NQO-1、HO-1及核因子E2相关因子2(nucleus factor-E2 related factor 2,Nrf2)的表达。结果:与N组比较,U组内皮细胞的总抗氧化能力明显降低(N vs.U:452.8±19.5 vs.321.4±27.8,P<0.001),活性氧水平升高(22.9±2.7 vs.75.1±11.2,P<0.001);而锌干预处理(U+10和U+30)则增加了细胞总抗氧化能力(U vs.U+10 vs.U+30:321.4±27.8 vs.455.7±16.0 vs.462.9±18.6,均有P<0.001),减少了活性氧产生(75.1±11.2 vs.45.2±7.6 vs.49.3±8.3,P<0.001);同时,尿毒症血清组及锌干预组NQO-1、HO-1 m RNA及蛋白表达增高(NQO-1蛋白:N vs.U vs.U+10:0.40±0.05 vs.0.65±0.07 vs.1.09±0.08;HO-1蛋白:0.21±0.07 vs.0.47±0.08 vs.1.09±0.11,P<0.05),且锌干预组较尿毒症血清组增加更明显(P<0.05);锌干预组细胞核Nrf-2表达较正常血清组及尿毒症血清组增加(N vs.U vs.U+10 vs.U+30:0.23±0.07 vs.0.25±0.07 vs.0.65±0.07 vs.0.69±0.06,均有P<0.001);但上述指标在不同浓度的锌处理组间均无统计学差异。结论:锌可能通过促进Nrf-2核转位,增加抗氧化酶NQO-1及HO-1表达,进而增加血管内皮细胞的总抗氧化能力和削弱尿毒症血清诱导的氧化应激反应。

Objective：To investigate the protective effect of Zinc on oxidative stress in vascular endothelial cells induced by uremic serum and its possible mechanism. Methods：Serum of uremic patients and healthy controls were collected and the endothelial cells were treated with serum concentration of 15%. The pas- saged endothelial cells were divided into four groups：normal serum group（N）,uremia serum group（U）,uremia serum with 10μmol/L zinc sulfate（U＋10）,uremia serum with 30 μmol/L Zinc sulfate（U＋30）. DCFH-DA as fluorescence probe was used to detect reactive oxygen species level by fluorescence microplate reader. Total antioxidant capacity was examined by assay kit with a rapid ABTS method. The expression of NQO-1 and HO-I mRNA was measured by real-time quantitative RT-PCR. The total protein expression of HO-1, NQO-1 and the Nrf-2 was measured by Western blot. Results：Compared with those in N group, the total antioxidant capacity of endothelial cells in U group was decreased（P〈0.001 ） and the ROS was significantly increased（P〈0.001 ）;while the Zinc pretreat- ment（the U＋10 and U＋30 two group） increased the total antioxidant capacity of cells and decreased the production of reactive oxygen species（all P〈0.001 ）. Both mRNA and protein expressions of HO-1 and NQO-1 were significantly increased in U and Zinc pretreat- ment groups（P〈0.05）,more prominent in Zinc pretreatment group（P〈0.05）. There was no significant difference between U and N groups in terms of the protein expression of Nrf-2,while the Nrf-2 nucleus expression was significantly increased in Zinc pretreat- ment group compared with the N and U（P〈0.001 ）. Conclusion ：Zinc can improve the total antioxidant capacity and attenuate the ox- idative stress induced by uremia serum in endothelial cells through promoting the Nrf-2 nuclear translocations and increasing the expression of HO-1, NQO-1.