Study On The Mechanism Of Idiopathic Pulmonary Fibrosis And Interleukin-31

Objective:In the process of idiopathic pulmonary fibrosis (IPF), TGF-β plays a major role at sites of inflammation activity, makes fibroblast proliferate, leading to severe pulmonary fibrosis, and TGF-β makes function through of Smads proteins’s signal transduction and regulation. Smad-3is a key molecule mediated by TGF-β to induce pulmonary fibrosis by regulating target genes and affecting the IL-6family, especially the expression of IL-31, and further to affect the JAKs/STATs pathway, causing pulmonary fibrosis. This is a possible mechanism of pulmonary fibrosis.Therefore, this project aims at using cultured cells and BLM-induced pulmonary fibrosis in mice models to get this TGF-β-Smad-3-IL-31-JAKs/STATs pathway. Studies of this pathway are a possible mechanism of pulmonary fibrosis, which can provide some clues to the targeted therapy of idiopathic pulmonary fibrosis.Methods:Seventy-five mice were divided into three groups randomly. One group were bleomycin-induced pulmonary fibrosis model (intratracheal injection of the BLM5mg/kg0.1mL), and one was dexamethasone treatment group (DXM group), n=25. One day after modeling, the dexamethasone group mice were intraperitoneally injected of DXM (5mg/kg O.1mL), once daily for28days. Another group was normal control group (NC group,25mice). At3,7,14,21,28days of administration,5mice were sacrificed in each group. By routine histopathology we observed the lung tissue; we used immunohistochemical detection to detect transforming growth factor-1(TGF-β1), Smad-3content; Then RT-PCR assay was used to detect the expression levels of IL-31, JAKs/STATs mRNA; Western blot was measured to check IL-31protein content; Chromatin Immunoprecipitation (CHIP) assay was used to test the binding of Smad-3to IL-31promoters. At the same time,the primary lung fibroblasts was cultured for3generations. Using SB431542to inhibit the expression of TGF-β, the expression of Smad-3and IL-31decreased. And using siRNA to knockdown the expression of IL-31, the expression level of STAT-1also decreased.We viewed the form of lung fibroblasts, lung fibroblasts proliferation characteristics and lung fibroblast collagen synthesis; lung tissue hydroxyproline (HYP) content was tested.Results:(1) Generally observing in the lungs of mice and the lung tissue, we could find the lung tissue of the experimental mice showed the dynamic change from alveolitis to fibrosis. The treatment group also showed similar pathological changes, but lighter than the experimental group, significantly worse than the control group. The control group showed slight pathological changes, no significant alveolar inflammation and fibrosis change.(2) TGF-β1was weakly expressed in normal control group, and in model group7d-14d it arrived peak, with statistical significance (p<0.01). At28d, the TGF-β1expression significantly reduced. After7d-14d, TGF-β1of treatment group was significantly lower than the model group, dexamethasone significantly reduced the expression of TGF-β1. In model mice, the Smad-3expression of alveolar macrophages and alveolar epithelial cells increased and was significantly higher at day7than control group (dark brown-yellow staining), then declined, on the28day was still significantly higher. The Smad-3expression in treatment group mice at every time points was significantly lower than the model group.(3) Compared with the control group, in the BLM model group, the IL-31and STAT-1mRNA began to increase from the third day, reached a peak on the14day, then gradually declined, but still higher than the28days. There are statistically significant (p<0.05). In control group, there was a small amount of the STAT-1protein expression by Western blot detection. In model mice, at the lesions early (the7days), STAT-1protein levels increased, and tended to increase with time, peaked at the14day, then decreased at the28day. The difference was statistically significant (P <0.05).(4) Using cross-linked with anti-Smad-3antibody to detect immunoprecipitated protein-DNA complexes, and using RT-PCR to detect IL-31promoter, compared with the control group, in model group, IL-31promoter was significantly increased. At the14day, it reached a peak, was still significantly higher at the28day, and there was statistically significant (P<0.05).(5) Using SB431542to inhibit the expression of TGF-β, the expression of Smad-3and IL-31decreased.(6) And using siRNA to knockdown the expression of IL-31, the expression level of STAT-1also decreased.(7) Compared with NC control group, in the BLM group lung tissue hydroxyproline content began to rise at the14day, reached the peak at the21to28day (p<0.01). The content of HYP in DXM treatment group obviously decreased during1428d.(p<0.05).Conclusion:The experiment proved the existence of a new pulmonary fibrosis signaling pathway from TGF-β-mad-3-IL-31--JAKs/STATs, which made a foundation for the study of the mechanism of pulmonary fibrosis, and provided a starting point for further study on the molecular mechanisms of interstitial fibrosis, and also provided a new therapeutic targets for pulmonary fibrosis. The results also speculated that in the bleomycin model dexamethasone treatment effect may be related to TGF-β, Smad-3and STAT-JAK pathway.