Tian X R, Tian X L, Wang H F, Chang Q, Huo R J, Ying D L, Zheng G P
Department of Pulmonary Medicine, the Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001, China.
Zhonghua Yi Xue Za Zhi. 2016 Jun 28;96(24):1929-33. doi: 10.3760/cma.j.issn.0376-2491.2016.24.013.
To investigate the regulation effect of β-catenin pathway on transforming growth factor beta1 (TGF-β1) induced pulmonary pro-fibrosis.
The rat alveolar typeⅡ cells (RLE-6TN) were divided into four groups: A1.control group; B1.TGF-β1 group was treated with 5 μg/L TGF-β1; C1.pcDNA+ TGF-β1 group was transiently transfected with eukaryotic expression vector pcDNA3.0 (pcDNA) and followed by TGF-β1 treatment (5 μg/L); D1.F-(β-TrCP)-Ecad+ TGF-β1 group was transiently transfected with β-catenin protein knockout vector [F-(β-TrCP)-Ecad] and followed by TGF-β1 treatment (5 μg/L). After 24 hours, cells were observed under the inverted phase contrast microscope, then the expressions of E-cadherin, α-smooth muscle actin (α-SMA) and fibronectin (Fn) in each group were measured by Western blot and the mRNA levels of Snail which was the downstream profibrotic transcription production in cell culture supernatants of each group were detected by real-time fluorescence quantification-polymerase chain reaction (RT-PCR) .The rat alveolar macrophages (CRL-2192) were divided into five groups: A2.control group; B2.Interferon gamma (IFN-γ) group was treated by 20 μg/L IFN-γ; C2.TGF-β1+ IFN-γ group was treated by 20 μg/L IFN-γ with 10 μg/L TGF-β1; D2.F-(β-TrCP)-Ecad+ TGF-β1+ IFN-γ group was transfected with F-(β-TrCP)-Ecad and other dispose was the same as group C2; E2.WTβ-catenin+ TGF-β1+ IFN-γ group was transfected with WTβ-catenin and other dispose was the same as group C2.After 24 hours, protein levels of β-catenin in group A2, B2, C2 were determined by Western blot.Inducible nitric oxide synthase (iNOS) mRNA levels of each group were detected by RT-PCR.
The RLE-6TN cells of group B1, C1 showed a change in morphology to spindle-shaped cells, the cells of group D1 maintained a cobblestone morphology. Protein expressions of the fibroblast markers α-SMA and Fn, and mRNA expressions of the downstream profibrotic transcription production Snail of group B1, C1 were significantly higher than group A1, while protein expressions of the epithelial marker E-cadherin were significantly lower.The protein expressions of α-SMA, Fn and mRNA expressions Snail of group D1 were significantly lower than group C1 (0.352±0.076 vs 0.937±0.303, 0.319±0.072 vs 0.903±0.211, 3.675±0.642 vs 9.708±2.031), while the protein expressions of E-cadherin were significantly higher (1.482±0.227 vs 0.604±0.121) (all P<0.05). The steady state protein levels of β-catenin in CRL-2192 cells was low and β-catenin protein expressions of CRL-2192 cells in group A2, B2 and C2 had no significantly statistical differences.The mRNA expressions of iNOS of group B2 cells were significantly higher than group A2, C2, D2, E2 (64.95±4.47 vs 9.87±0.73, 21.32±2.41, 18.35±3.61, 22.87±3.14) (all P<0.01), the expressions of iNOS of group C2, D2, E2 were all higher than group A2 (all P<0.05), but there were no significant differences among group C2, D2 and E2.
Inhibition of β-catenin pathway inhibits TGF-β1-induced epithelial-mesenchymal transition (EMT) and has no effect on its anti-inflammation effect.Therefore, β-catenin pathway regulates the pulmonary pro-fibrosis effect of TGF-β1.
探讨β-连环蛋白通路对转化生长因子β1（TGF-β1）诱导的肺纤维化的调控作用。
将大鼠Ⅱ型肺泡上皮细胞（RLE-6TN）分为四组：A1.对照组；B1.TGF-β1组，用5μg/L TGF-β1处理；C1.pcDNA+TGF-β1组，用真核表达载体pcDNA3.0（pcDNA）进行瞬时转染，然后用TGF-β1（5μg/L）处理；D1.F-(β-TrCP)-Ecad+TGF-β1组，用β-连环蛋白蛋白敲除载体[F-(β-TrCP)-Ecad]进行瞬时转染，然后用TGF-β1（5μg/L）处理。24小时后，在倒置相差显微镜下观察细胞，然后用蛋白质免疫印迹法检测各组中E-钙黏蛋白、α-平滑肌肌动蛋白（α-SMA）和纤连蛋白（Fn）的表达，并用实时荧光定量聚合酶链反应（RT-PCR）检测各组细胞培养上清液中作为促纤维化转录产物下游的Snail的mRNA水平。将大鼠肺泡巨噬细胞（CRL-2192）分为五组：A2.对照组；B2.干扰素γ（IFN-γ）组，用20μg/L IFN-γ处理；C2.TGF-β1+IFN-γ组，用20μg/L IFN-γ和10μg/L TGF-β1处理；D2.F-(β-TrCP)-Ecad+TGF-β1+IFN-γ组，用F-(β-TrCP)-Ecad转染，其他处理同C2组；E2.WTβ-连环蛋白+TGF-β1+IFN-γ组，用野生型β-连环蛋白转染，其他处理同C2组。24小时后，用蛋白质免疫印迹法检测A2、B2、C2组中β-连环蛋白的蛋白水平。用RT-PCR检测各组诱导型一氧化氮合酶（iNOS）的mRNA水平。
B1、C1组的RLE-6TN细胞形态变为梭形细胞，D1组细胞保持鹅卵石样形态。B1、C1组成纤维细胞标志物α-SMA和Fn的蛋白表达以及促纤维化转录产物下游的Snail的mRNA表达均显著高于A1组，而上皮标志物E-钙黏蛋白的蛋白表达显著降低。D1组α-SMA、Fn蛋白表达及Snail mRNA表达均显著低于C1组（0.352±0.076对0.937±0.30；0,319±0.072对0.903±0.211；3.675±0.642对9.708±2.031），而E-钙黏蛋白的蛋白表达显著升高（1.482±0.227对0.604±0.121）（均P<0.05）。CRL-2192细胞中β-连环蛋白的稳态蛋白水平较低，A2、B,2、C2组CRL-2192细胞中β-连环蛋白的蛋白表达无显著统计学差异。B2组细胞中iNOS的mRNA表达显著高于A2、C2、D2、E2组（64.95±4.47对9.87±0.73、21.32±2.41、18.35±3.61、22.87±3.14）（均P<0.01），C2、D2、E2组iNOS的表达均高于A2组（均P<0.05），但C2、D2、E2组之间无显著差异。
抑制β-连环蛋白通路可抑制TGF-β1诱导的上皮-间质转化（EMT），且对其抗炎作用无影响。因此，β-连环蛋白通路调控TGF-β1的肺纤维化作用。
