| Pulmonary fibrosis is a progressive life-threatening disease for which no effective therapy exists. Mechanisms leading to fibroblast accumulation during pulmonary fibrogenesis remain unclear. The initial hypothetical model suggested chronic inflammation as the cause of pulmonary fibrosis, whereas a subsequent hypothesis posited epithelial injury and impaired wound repair as the etiology of fibrosis without preceding inflammation. The early inflammatory phase is usually associated with the release of several cytokines and chemokines by activated resident cells and infiltrating cells which, in turn, help further recruit inflammatory mononuclear cells, contribute to extracellular matrix (ECM) and then eventually to the formation of pulmonary fibrosis. Now most researchers believe that TGF-β1 may play a key role in pulmonary fibrogenesis by promoting alveolar epithelial cell transition to form mesenchymal cells with a myofibroblast-like phenotype. This process, termed epithelial-mesenchymal transition (EMT), shows the differentiation of lung fibroblastst trans into the myofibroblast withα-smooth muscle actin (α-SMA) marker expression. Its essense is excessive deposition of extracellular matrix components (ECM) in lung tissue, which occurs due to an imbalance between the production and degradation of matrix. Collagen is the most important part of ECM. Therefore, one pivotal antifibrotic pathway is to effectively inhibit the synthesis of collagen or increase its degradation. Our previous work has shown that arsenic trioxide can reduce hydroxyproline content and collagen deposition in bleomycin-induced pulmonary fibrosis rat lung tissue. But by which way of arsenic trioxide to influence pulmonary fibrosis? This is what we want to explore. ObjectiveThe aim of this study is to investigate the effects of arsenic trioxide on the proliferation and expression of typeⅠa nd typeⅠcollagen of pulmonary myofibroblast induced by TGF-β1, and clarify its mechanism.Materials and methodsHealthy rats lung fibroblasts were isolated and cultured till the cells naturally purified to the fourth generation. Added TGF-β1 to induce the fibroblasts, used arsenic trioxide as intervention, and then observed fibroblasts after the intervention at 12h, 24h and 48h and compared with control group. The morphology and structure of each group cells were observed by inverted microscope, the cells number was counted by MTT method, and fluorescence quantitative PCR was done to check the expression of the mRNA ofα-SMA, typeⅠcollagen and typeⅢcollagen in different groups.Results(1)The primary cells showed round nucleus and fusiformis shape under microscope. The images of immunohistochemistry demonstrated that cultured lung cells had positive staining of vimentin, but negative of keratin. It proved that the cultured cells were lung fibrolasts. (2) The proliferation of pulmonary myofibroblast induced by TGF-β1 could be inhibited by Arsenic Trioxide. The cell growth of 1uM/L arsenic trioxide group and the 2uM/L arsenic trioxide group was significantly inhibited, with the maximum inhibition ratio up to 50% (p<0.05). And the expression ofα-SMA mRNA was also significantly decreased by 51.8% (p<0.05) at most. (3) Arsenic trioxide can inhibit the typeⅠand typeⅢcollagen which were secreted by rat lung fibroblast and induced by TGF-β1. The mRNA expression of both typeⅠand typeⅢcollagen of 1uM/L arsenic trioxide group and the 2uM/L arsenic trioxide group were significantly reduced, up to 50% (p<0.05). ConclusionsArsenic trioxide can inhibit the differentiation of myofibroblast induced by TGF-β1, mainly in cell morphology, cell proliferation and the change ofα-SMA which is the landmark product of myofibroblast. Also we find that arsenic trioxide can reduce the mRNA expression of typeⅠand typeⅢcollagen which are induced by TGF-β1. All the effects of cell proliferation and reduce expression by arsenic trioxide are showed direct-correlation with dose/time-effect relationship. These results suggested that arsenic trioxide can inhibit proliferation of myofibroblast induced by TGF-β1, and then reduced the products of typeⅠand typeⅢcollagen secreted by myofibroblast, finally play an effect of antifibrosis. |
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