CDDO-Me对缺氧诱导的肺动脉外膜成纤维细胞活化的影响
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1.南京医科大学第一附属医院呼吸与危重症学科;2.中国药科大学新药研究中心天然药物活性组分与药效国家重点实验室

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国家科技重大专项(2018ZX10722301);国家自然科学基金(81273571,81870054);江苏省卫生厅重点项目(H201601)


Effects of CDDO-Me on hypoxia-induced activation of human pulmonary adventitia fibroblasts
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Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital of Nanjing Medical University

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    摘要:

    目的:探索CDDO-Me对缺氧诱导的人肺动脉外膜成纤维细胞(pulmonary artery adventitia fibroblasts, PAAFs)活化的影响及其相关机制。方法:体外培养人PAAFs,随机分为常氧组(21% O2)、缺氧组(1% O2)、缺氧+ CDDO-Me组和常氧+CDDO-Me组。采用CCK8法检测细胞活力;Transwell实验检测细胞迁移;DCFH-DA荧光探针检测细胞活性氧( reactive oxygen species, ROS)水平,ELISA试剂盒检测细胞内谷胱甘肽(glutathione, GSH)、丙二醛( malondialdehyde, MDA) 及超氧化物歧化酶( superoxide dismutase, SOD) 含量以及细胞上清液中TGF-β1、TNFα、IL-1β表达水平;免疫荧光法观察细胞内α-SMA表达以及NF-κB(p65)核转位情况;Western blot检测细胞内α-SMA、I型胶原蛋白、波形蛋白表达以及Smad3、p65磷酸化水平。结果:CDDO-Me(62.5、125、250、500 nmol/L)可以浓度依赖性地降低缺氧诱导的PAAFs细胞活力的升高,并在250 nmol/L和500 nmol/L浓度时具有显著抑制作用。CDDO-Me(500 nmol/L)可以抑制缺氧诱导的PAAFs迁移以及肌成纤维细胞转化,恢复细胞形态,抑制缺氧诱导的PAAFs中α-SMA、I型胶原蛋白和波形蛋白表达水平的升高。在缺氧条件下,CDDO-Me(500 nmol/L)显著降低PAAFs中ROS和MDA水平,升高GSH和SOD含量,提高细胞抗氧化应激能力。CDDO-Me(500 nmol/L)可以抑制缺氧诱导的PAAFs中细胞因子TGF-β1的分泌,抑制TGF-β1/Smad3信号通路的激活。此外,CDDO-Me(500 nmol/L)还可以抑制缺氧诱导的PAAFs中NF-κB(p65)的核转位及其磷酸化,并抑制其下游炎症因子TNFα和IL-1β的表达。结论:CDDO-Me可以抑制缺氧诱导的PAAFs增殖、迁移以及向肌成纤维细胞转化,并抑制相关炎症因子的释放,其机制可能与CDDO-Me提高PAAFs抗氧化应激能力,抑制TGF-β1/Smad3信号通路和NF-κB信号通路相关。

    Abstract:

    Objective: To investigate the effect of CDDO-Me on the hypoxia-induced activation of human pulmonary artery adventitia fibroblasts (PAAFs) and potential mechanism. Methods: Human PAAFs were cultured in vitro and randomly divided into four groups: the normoxia group (21% oxygen), the hypoxia group (1% oxygen), the hypoxia plus CDDO-Me group and the normoxia plus CDDO-Me group. Cell viability was determined by CCK8 assay. Transwell assay was carried out to assess cell migration. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were detected to evaluate the level of oxidative stress. The levels of TGF-β1, TNFα and IL-1β were detected by ELISA assay. The expression of α-SMA and nuclear translocation of NF-κB (p65) were detected by immunofluorescence assay. The protein levels of α-SMA, collagen I, vimentin, phospho-Smad3 and Smad3, phospho-p65 and p65 were determined by Western blot. Results: CDDO-Me treatment (62.5, 125, 250, 500 nmol/L) decreased hypoxia-induced elevations of cell activity in a concentration dependent manner, which showed significant inhibition at concentration of 250 nmol/L and 500 nmol/L. CDDO-Me (500 nmol/L) remarkably inhibited hypoxia-induced migration and myofibroblast transformation of PAAFs, which reflected in improvement of hypertrophy, decrease in expressions of α-SMA, collagen I and vimentin. In addition, CDDO-Me (500 nmol/L) significantly inhibited hypoxia-induced increased levels of ROS and MDA, decreased levels of GSH and SOD, and improved the ability of antioxidation. Hypoxia increased the secretion of TGF-β1 and activate TGF-β1/Smad3 signaling pathway in PAAFs, which was attenuated by CDDO-Me (500 nmol/L). Besides, hypoxia-induced nuclear translocation of p65, phosphorylation of p65 protein, and the secretion of TNFα and IL-1β were inhibited by CDDO-Me treatment. Conclusion: CDDO-Me can inhibit hypoxia-induced PAAFs proliferation, migration, myofibroblast transformation and the secretion of inflammatory cytokines, which may be related to the improvement of antioxidation ability, and the inhibition of TGF-β1/Smad3 and NF-κB signaling pathway in PAAFs.

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  • 收稿日期:2020-04-14
  • 最后修改日期:2020-12-08
  • 录用日期:2021-09-28
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