| Silicosis is one of the most serious occupational lung diseases in the world, which is well-known to be associated with long term exposure to silica particles. The basic process of this disease is due to silica induced lung injury and alveolar inflammation, and then leading to tissue repair, recruitment and proliferation of stromal cells, and excessive deposition of extracellular matrix(ECM). Silicon nodules and interstitial fibrosis are the most significant features of silica induced lung fibrosis, which are irreversible respiratory dysfunction. However, the exact molecular mechanism of this pathological process is still largely unknown, which limits the effective treatments for silicosis.Cytokines are a class of small molecular polypeptide secreted by immune cells, which are responsible for the maintenance of tissue homeostasis and coordinate with the response to injury. Numerous studies have shown that the pathogenesis of lung fibrosis was related to a dysregulation in various cytokines, study in the function of specific cytokine can help provide clues for clarifying molecular mechanism in silicosis. In addition, proteases is also an important mediator during fibrogenetic process, which not only can degrade components of ECM, but also can active other proteins. The large number of mediators involved and the complexity of their interaction makes it difficult to clarify the pathology of fibrogenesis. Thus, sorting out the role for specific mediator in this complicated event is very necessary to help reveal the pathological processes and provide clues for effective therapy. Here we focus on the expression of matrix metalloproteinases (MMPs) and their role in the pathogenesis of silica-induced lung fibrosis.MMP-3 is a member of the MMP family which mainly degrades components of the extracellular matrix. The degradative function of MMP-3 are important in tissue repair, on the other hand, MMP-3 is also able to activate cytokines, growth factors and cell surface receptors via regulating proteolytic processing. According to its multiple biological activities, MMP-3 is thought to be implicated in a diverse range of pathological processes, including rheumatoid arthritis, Parkinson’s disease, osteoarthritis, etc. To the best of our knowledge, there is no study reporting the role of MMP-3 in silica induced lung fibrosis.Objective:To investigate a broad spectrum of mediators implicated in the fibrotic process, then according to reported literatures, choosing MMP-3 to validate its expression at different time-point in silica induced lung fibrosis and clarify its role in this pathological process, which may ultimately lead to the development of novel therapeutic approaches for lung fibrosis.Methods:(1) Set up the silica induced lung fibrosis mice model by the intrastracheal administration of a single dose of SiO2 suspension. The dynamic changes of lung tissue structure were observed in pathological section stained with hematoxylin-esin. (2) Lung tissue samples were collected at days 0,3,7,14,28,56 after silica instillation. Detecting the changes in the expression of cytokines by RayBio Mouse Cytokine Antibody Array G Series 2000. (3) Analysing the expression trends of dis-regulated cytokines which are higher or lower than 2 fold changes in at least two treatment groups compared with the control group. (4) Validating the expression of the MMP-3 in vivo by Western blot and Immunohistochemistry. Identifying the expression of MMP-3 and other fibrotic related proteins in vitro experiments via Western blot test. (5) The HBE cells and the 3T3 cells were transfected with the siRNA of MMP-3, then the expression of MMP-3, TGF-β1, a-SMA and FN were detected by Western blot.Results:(1) Mice gradually developed to lung fibrosis, (silicosis) as determined by HE staining on day 28 after silica admistration. Silica induced marked inflammation within two weeks before developing into fibrosis. (2) we detected 144 cytokines and found 66 of them were dis-regulated more than 2 folds in at least 2 treatment groups compared with the control group. According to the literature, the fibrotic process and the observed dis-regulated cytokines which had significant change with the high original signal, we speculate that the cytokines closely involved in fibrosis were the MMP family, Interleukin family and Chemokine family. (3) MMP-3 was selected for the further study and found:compared to the control group, the expressions of MMP-3, TGF-β1, FN and α-SMA were increased in the lung tissues of mice which were silica-treated, whereas, the expression of E-cadherin declined with treated time. And we observed the changes in vitro experiment were consistent with the results mentioned above. In the RAW cell model, the expression of MMP-3, TGF-β1 were increased after the silica-treated. The same changes were observed in the HBE cell model, accompany with the increase of a-SMA and the decrease of E-cadherin. In the 3T3 cell model, after treating with exogenous TGF-β1, the expression of MMP-3 and FN both are increased. (4) In the HBE cell model, down-regulate MMP-3 can reduce the expression of TGF-β1, α-SMA as well as FN in the silica-treated group. On the other hand, in the 3T3 cell model, down regulate MMP3 could reduce TGF-β1 expression, but the expression of FN still higher, which indiceted that MMP3 could degradate FN and at the meantime it may promote EMT by activating TGF-β1.Conclusion:A broad spectrum of mediators are implicated in the lung fibrotic process, including growth factors, chemokines and proteolytic enzymes. MMP-3 is one of the important mediators which contributes to the development of fibrosis. It may trigger activation of the TGF-β1 leading to EMT of lung epithelial cells, on the other hand, EMT induced more synthesis of ECM and resulted in more secretion of MMP-3. Taken together, MMP-3 affects the process of fibrosis via this positive feedback loop. Thus, the selective targeting of MMP-3 may represent a potential target for future therapeutic strategies. |
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