坏死细胞释放IL-1对血管平滑肌细胞氧化应激的影响

Effect of IL-1 released by necrotic cells on oxidative stress of vascular smooth muscle cells

目的研究坏死细胞对正常血管平滑肌细胞氧化应激的影响及其主要作用因子。方法体外培养的血管平滑肌细胞以无糖低氧条件诱导细胞坏死,收集坏死细胞培养上清液。体外培养且生长正常的血管平滑肌细胞根据不同的干预方式分为对照组(不予任何干预)、坏死上清组(NCS组,以坏死细胞培养上清液进行干预)和NCS+白介素1受体拮抗剂(IL-1RA)组(NCS+IL-1RA组)。对各组氧化应激指标细胞活性氧(ROS)生成以及培养细胞上清液中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性进行检测;RT-PCR和Western blotting检测各组细胞内皮型一氧化氮合成酶(eNOS)、诱导型一氧化氮合酶(iNOS)和氧化酶亚基p47phox mRNA和蛋白的表达。结果与对照组比较,NCS组ROS生成和MDA含量明显增多(P<0.05),SOD活性显著降低(P<0.05);与NCS组比较,NCS+IL-1RA组ROS生成和MDA含量明显减少(P<0.05),SOD活性显著升高(P<0.05)。RT-PCR和Western blotting检测结果显示:与对照组比较,NCS组和NCS+IL-1RA组细胞中iNOS、eNOS和p47phoxmRNA和蛋白的表达均显著上调(P<0.05),且NCS+IL-1RA组较NCS组显著下调(P<0.05)。结论坏死细胞可诱导正常血管平滑肌细胞的氧化应激反应,抑制IL-1信号通路能减轻由坏死细胞引发的氧化应激反应。

Objective To investigate the effect of necrotic cells on oxidative stress of normal vascular smooth muscle cells, and explore the main active factors. Methods Necrosis of vascular smooth muscle cells was induced by glucose-free hypoxic conditions, and the supernatants were collected. Normal vascular smooth muscle cells were divided into control group （without any treatment）, necrotic cell snpernatants group （ NCS group, treated with cuhure supernatants of necrotic cells） and NCS ＋ intedeukin 1 receptor antagonist （IL-1RA） group （treated with NCS ＋ IL-1RA）. The production of reactive oxygen species （ROS） of cells and the content of malondialdehyde （MDA） and activity of superoxide dismutase （SOD） in cuhure supernatants were determined, and the expression of endothelial nitric oxide synthase （eNOS）, inducible nitric oxide synthase（iNOS） and oxidase subunit p47phox mRNA and protein was detected by RT-PCR and Western blotting. Results The production of ROS and content of MDA in NCS group were significantly higher than those in control group （P 〈 0.05）, and the activity of SOD in NCS group was significantly lower than that in control group （P 〈 0.05）. The production of ROS and content of MDA in NCS ＋ IL-1RA group was significantly lower than those in NCS group （P 〈0.05）, and the activity of SOD in NCS ＋ IL-1RA group was significantly higher than that in NCS group （P 〈 0.05）. RT-PCR and Western blotting revealed that the expression of iNOS, eNOS and p47phox mRNA and protein in cells in NCS group was significantly higher than that in control group （ P 〈 0. 05）, and the expression of iNOS, eNOS and p47phox mRNA and protein in cells in NCS ＋ IL-1RA group was significantly lower than that in NCS group （ P 〈 0.05）. Conclusion Necrotic cells can induce oxidative stress of normal vascular smooth muscle cells, and the inhibition of IL-1 signaling pathway can alleviate the oxidative stress.