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）
目的：探索甲基巴多索隆（bardoxolone methyl，CDDO-Me）对缺氧诱导的人肺动脉外膜成纤维细胞（pulmonary artery adventitia fibroblast，PAAF）活化的影响及其相关机制。方法：体外培养人PAAF，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CCK-8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转化生长因子（transforming growth factor，TGF）-β1、肿瘤坏死因子（tumor necrosis factor，TNF）-α、白细胞介素（interleukin，IL）-1β表达水平；免疫荧光法观察细胞内α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）表达以及核因子κB（nuclear factor kappa-B，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、125.0、250.0、500.0 nmol/L）可以浓度依赖性地降低缺氧诱导的PAAF细胞活力的升高，并在250.0 nmol/L和500.0 nmol/L时具有显著抑制作用。CDDO-Me（500.0 nmol/L）可以抑制缺氧诱导的PAAF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PAAF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（500.0 nmol/L）显著降低PAAF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（500.0 nmol/L）可以抑制缺氧诱导的PAAF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（500.0 nmol/L）还可以抑制缺氧诱导的PAAF中NF-κB（p65）的核转位及其磷酸化，并抑制其下游炎症因子TNF-α和IL-1β的表达。结论：CDDO-Me可以抑制缺氧诱导的PAAF增殖、迁移以及向肌成纤维细胞转化，提高PAAF抗氧化应激能力，并抑制TGF-β1/Smad3信号通路和NF-κB信号通路。
Objective：This study aims to investigate the effect of bardoxolone methyl （CDDO-Me） on the hypoxia-induced activation of human pulmonary artery adventitia fibroblasts（PAAF）and potential mechanism. Methods：Human PAAF 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 CCK-8 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 transforming growth factor（TGF）-β1，tumor necrosis factor（TNF）-α and interleukin（IL）-1β were detected by ELISA assay. The expression of α-smooth muscle actin （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（62.5，125.0，250.0，500.0 nmol/L）decreased hypoxia-induced elevations of cell viability in a concentration dependent manner，which showed significant inhibition at concentration of 250.0 nmol/L and 500.0 nmol/L. CDDO-Me（500.0 nmol/L）remarkably inhibited hypoxia-induced migration and myofibroblast transformation of PAAF，which reflected in improvement of hypertrophy，decreases in expressions of α-SMA，collagenI and vimentin. In addition，CDDO-Me（500.0 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 PAAF，which was attenuated by CDDO-Me（500.0 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 proliferation，migration，myofibroblast transformation of PAAF，improve the antioxidation ability，and inhibit TGF-β1/Smad3 and NF-κB signaling pathway in PAAF.
彭丽瑶,魏桂红,黄张建,孔 辉,解卫平. CDDO⁃Me对缺氧诱导的肺动脉外膜成纤维细胞活化的影响[J].南京医科大学学报（自然科学版）,2021,(9):1273-1280
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