CDDO-Me对缺氧诱导的肺动脉外膜成纤维细胞活化的影响

Effects of CDDO-Me on hypoxia-induced activation of human pulmonary adventitia fibroblasts

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)-αand interleukin(IL)-1βwere detected by ELISA assay.The expression ofα-smooth muscle actin(α-SMA)and nuclear translocation of NF-κB(p65)were detected by immunofluorescence assay.The protein levels ofα-SMA,collagenⅠ,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 hypoxiainduced migration and myofibroblast transformation of PAAF,which reflected in improvement of hypertrophy,decreases in expressions ofα-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,hypoxiainduced nuclear translocation of p65,phosphorylation of p65 protein,and the secretion of TNFαand IL-1βwere inhibited by CDDOMe 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.