| Background Pulmonary fibrosis is a progressive and often fatal condition characterized pathologically by mesenchymal cell proliferation and differentiation in the lung, expansion of the extracellular matrix, and remodeling of the lung parenchyma. Pulmonary fibrosis occurs in a number of clinical diseases, including idiopathic interstitial pneumonias, occupational diseases, as part of several systemic connective tissue diseases, and in response to many types of lung injury, including radiation and some chemo-therapeutic drugs. Transforming growth factor β1(TGF-β1) plays important roles in the process of pulmonary fibrosis. The Smad proteins are key intracellular signaling effectors of TGF-β1. Regulation of TGF-β1 signaling occurs at multiple levels.Ubiquitin proteasome pathway(UPP) is an important system to monitor the function and degradation of protein in eukaryotic cells. UPP is associated with a wide range of human biological processes such as cell cycle, DNA repair, transcriptional regulation, signal transduction, phagocytosis and so on. The dysfunction of UPP is involved in a variety of human diseases.Some recent studies have found that ubiquitin-proteasome inhibitor could reduce or inhibit disease such as liver fibrosis, renal fibrosis, skin fibrosis, pulmonary fibrosis, etc. However, the molecular mechanism of this process is still unclear. MG132 is one of the ubiquitin-proteasome inhibitors which could be used to restrain the activity of protease and prevent the target protein from degradation.Objective To investigate the effect of Ubiquitin-proteasome inhibitor MG132 on TGF-β1-induced activation in lung fibroblasts and its possible molecular mechanism.Methods(1)Cultivating the human embryonic lung fibroblasts(MRC-5) in vitro;(2) MTS assay was utilized to investigate the effects of TGF-β1 on the proliferative activity of the human embryonic lung fibroblasts(MRC-5);(3) The human embryonic lung fibroblasts(MRC-5) were randomly divided into three groups as follows: control group(cells cultured with DMEM without TGF-β1 or MG132); TGF-β1 group(cells cultured with 10 ng/ml TGF-β1) and MG132 group [TGF-β1(10 ng/mL) + MG132(0.5μmol/L)];(4) The protein levels of α-SMA and collagen type I alpha 1(COL1A1) were detected by Western blot;(5) The expressions of TβRI, Smad2, Smad3,Smad7 and Sno N(Ski-related novel gene N) mRNA were evaluated by RT-PCR,and the protein levels of TβRI, Smad2, Smad3,Smad7 and Sno N were detected by Western blot.Results(1) The proliferation of lung fibroblasts MRC- 5 could be promoted by the treatment of TGF-β1 within limits and the best concentration of TGF-β1 was 10 ng/ml.(2) TGF-β1 treatment of lung fibroblasts increased α-SMA and COL1A1 expression(P<0.05 as compared with control). MG132 inhibited TGF-β1-induced α-SMA and COL1A1 expression in lung fibroblasts(P<0.05 as compared with TGF-β1 group).(3) The mRNA levels of Smad7 and Sno N in TGF-β1 group and MG132 group were increased(P<0.05 as compared with control), and there were no significant difference between TGF-β1 group and MG132 group(P>0.05). Contrary to the mRNA levels, the protein levels of Smad7 and Sno N in TGF-β1 group were decreased(P<0.05 as compared with control). Compared with TGF-β1 group, the protein levels of Smad7 and Sno N in MG132 group were increased(P<0.05).(4) The protein and mRNA levels of TβRI in TGF-β1 group and MG132 group were increased(P<0.05 as compared with control), and there were no significant difference between TGF-β1 group and MG132 group(P>0.05).(5) There were no significant difference of the protein and mRNA levels of Smad2 and Smad3 among the three groups(P>0.05).Conclusion(1) The proliferation of lung fibroblasts MRC-5 could be promoted by the treatment of TGF-β1;(2) Ubiquitin-proteasome inhibitor MG132 could inhibit TGF-β1-induced activation in lung fibroblasts in vitro;(3) The effect of Ubiquitin-proteasome inhibitor MG132 in lung fibroblasts which may be associated with the inhibitory effect of MG132 on TGF-β/Smads signaling by preventing degradation of Smad7 and Sno N. |
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