Role Of TGF-β/Smad Signaling Pathways In The Pathogenesis And The Expression Of Fibronectin And Osteopontin In Mice With Pulmonary Fibrosis

Objective:Using the bleomycin-induced C57BL/6 mice model of pulmonary fibrosis, we observe the role of TGF-β/Smad signaling pathways in the pathogenesis and expression of fibronectin and osteopontin in mice with pulmonary fibrosis, and further explore the underlying mechanisms of pulmonary fibrosis.Methods:Three randomized groups of healthy male C57BL/6 mice(20g±2g)(N=72): control group(group NS), model group(group M), SB431542 bocking group(group SB). Each group contained 24 mice. On the initial day the experimental pulmonary fibrosis models induced by bleomycin(BLM, 3.5mg/kg) injection through tracheal were carried in the mice, while Control group was drop into saline through tracheal. SB431542 treatment group received intraperitoneal injection of SB431542 dissolved in 10% ethanol solution at3, 4 and 5 days after bleomycin treatment, while the rest of two groups received intraperitoneal injection of 10% ethanol solution as the same volume. Eight mice in each group were randomly killed by abeominal aorta bloodletting method on days 7, 14 and 28,and collect the lung tissue specimens; Alveolitis and pulmonary fibrosis were evaluated with pathological slides stained by HE and Masson.Pulmonary fibrosis was measured by content of lung tissue hydroxyproline(HYP);the expression of FN, OPN and p-Smad3 in lung tissue were determined by immunohistochemistry, comparing with each group.Results:1.In group NS, the mice had normol lung tissue structure. Compared with the control group, a great deal of inflammatory cell infiltration into the alveolar was observed in model group on day 7, and the fibrosis began to appear on day 14, while on day 28 the alveolitis was alleviated and the fibrosis became serious; In group SB, the alveolitis and the fibrosis in mice were significantly allevilated than group M. 2. the content of HYP in lung tissues at three time point had no obvious change in group NS; However, the content of HYP in other two group increased gradually over time, and peaked on day 28; On day14 and 28, the content of HYP in group M and group SB were higher than that in group NS(P<0.05), while the content of HYP in group SB decreased obviously than that in group M(P<0.05). 3. p-Smad3 was mainly expressed in the cytoplasm of macrophages, fibroblasts,alveolar epithelial cells and bronchial epithelial cells in lung tissue. FN mainly located in pulmonary vasculature, bronchi, alveolar interval and hyperplastic interstitial cells in lung tissue. A spot of p-Smad3 and FN were located in lung tissue, while the expression of p-Smad3 and FN in group M significantly increased compared with those in the same period of control group(P<0.05). And p-Smad3 reached their peaks on day 28, and FN reached its peak on day 14. OPN maily were distributed in smooth muscle of trachea paries and vessel wall, pulmonary interstitial macrophages and alveolar epithelial cells. The expression of OPN in group M significantly increased than that in group NS(P<0.05), and it peaked on day 28. Compared with group M, the expression of p-Smad3, FN and OPN were significantly reduced in group SB(P<0.05).Conclusion:FN may have an effect on the occurrence and development of the early alveolar inflammatory injury in mice with pulmonary fibrosis, while OPN may be throughout in the whole process of pulmonary fibrosis. Both play an important role in the process of pulmonary fibrosis in mice. Blocking TGF-β/Smad signaling pathway may suppress the expression of FN, OPN, and then it may suppress the development of pulmonary fibrosis,so it suggests that TGF-β/Smad is a crucial signaling pathway for pulmonary fibrosis progression.