ABSTRACT
Objective: To investigate the effects of thrombopoietin (TPO) on proliferation and collagen synthesis in pulmonary fibro-blasts induced by TGFβ1. Methods: Cultured human embryonic lung fibroblasts (HFLs) were treated with recombinant human TGF-β1 to induce myofibroblast differentiation. Different concentrations of recombinant human TPO were applied individually or in combina-tion. Cell proliferation rate was determined using the CCK8 assay. Q-PCR and immunofluorescence assay were employed to examine the mRNA and protein expression of α-smooth muscle actin (αSMA) and type I collagen (COL1)A2. Results: TGFβ1 treatment induced HFL transdifferentiation to myofibroblasts was determined by the expression of αSMA, a myofibroblast-specific marker. Cell prolifera-tion increased during the induction. COL1 gene and protein expression were upregulated by TGFβ1 induction (P<0.05). The TGFβ1-in-duced mRNA and protein expression of αSMA and COL1A2 was decreased by TPO treatment (P<0.05), as determined by reverse tran-scription quantitative polymerase chain reaction and immunofluorescence analysis, respectively. The inhibitory rate showed a dose de-pendent effect within a certain TPO concentration range. The CCK8 assay demonstrated that TPO downregulated the TGFβ1-induced proliferation (P<0.05). Furthermore, the expression of heme oxygenase-1 (HO-1) was downregulated in TGFβ1-induced lung fibro-blasts, and these effects were attenuated by TPO administration (P<0.05). Conclusions: TPO can inhibit the TGFβ1-induced prolifera-tion and differentiation of human lung fibroblasts. These effects may be mediated in part by HO-1-related signaling pathways.
ABSTRACT
Objective To study the inhibitory effect and mechanism of fluorofenidone on the proliferation of the rat lung fibroblasts induced by TGF-β1 . Methods Pulmonary fibroblasts cell were cultured and purified by using differentially adherent method, the third generation was used in the experiment. Cells were pretreated with 1,3,10 mmol?L-1 fluorofenidone, respectively. Pulmonary fibroblasts were identified by immunocytochemistry, and interfered by transient transfection. Cell proliferation was measured by BrdU marking. The mRNA and protein expression of eIF3α and p27 were analyzed by real-time PCR and Western blot. Results Immunocytochemistry staining identification of vimentin was positive in the third generation of pulmonary fibroblasts .TGF-β1 could obviously promote the eIF3α expression and proliferation of pulmonary fibroblasts. EIF3a siRNA was transfected with lipofectamine into cells, which obviously inhibit eIF3α expression induced by TGF-β1 , reversed cell proliferation induced by TGF-β1 , and could obviously promote p27mRNA expression induced by TGF-β1 . Fluorofenidone (3, 10 mmol?L-1 )could obviously inhibit cell proliferation induced by TGF-β1 and reduce eIF3α expression of pulmonary fibroblasts promoted by TGF-β1 . The inhibition rate was 66.7% and 78.8% respectively. Fluorofenidone (3 mmol?L-1 ,10 mmol?L-1 ) could also obviously reverse p27 expression inhibited by TGF-β1 , the reverse rate was 75. 5% and 71. 3% respectively. Conclusion eIF3α/ p27 signal pathway involves in pulmonary fibroblasts proliferation induced by TGF-β1 .Fluorofenidone can inhibit pulmonary fibroblasts proliferation induced by TGF-β1 by inhibiting eIF3α expression, enhancing p27 expression and/ or increasing p27 directly.