The Dynamic Expression Of UGBP In Bleomycin-induced Pulmonary Fibrosis Model In Mice And Primary Mechanism

Uteroglobin-binding protein(UGBP), which is also known as UG receptor, may be associated with Clara cell-binding protein (CCSP or UG) to play its multi-functional biological activity, but the physiological functions and the exact expression pattern of UGBP in lung fibrosis are still remain elusive. In the early experimental studies, we know TGF-β1 plays a central role in the development of pulmonary fibrosis. Based on it, we think that TGF-β1 may be connected with the change of UGBP in bleomycin-induced pulmonary fibrosis. The aim of study was to understand the dynamic expression pattern of UGBP by observing the spatial and temporal expressional diversification in bleomycin-induced pulmonary fibrosis model in mice, and the primary mechanism were investigated by observing the change of UGBP after inhibition of the TGF-(31 signaling pathway in vitro. It is anticipated to provide new insights to illustrate the molecular mechanism and transcriptional regulation of UGBP.Methods:1. The dynamic expression of UGBP in bleomycin-induced pulmonary fibrosis model in mice 50 healthy male KM mice were randomly divided into the control group(Con) and the bleomycin-induced pulmonary fibrosis group (D1,D6,D12,D18). Bleomycin were injected intraperitoneally (15mg/kg) for 10 consecutive days in the pulmonary fibrosis groups, then the mice were killed from every group on the 1st,6th,12th,18th day respectively after last treatment. The tissue microarray which made by the left lung tissue were for observing pathological changes by hematoxylin-eosin(HE) staining and Masson staining, and for UGBP protein expression by immunohistochemistry, the right lung tissue were applied Mas son staining to analyse collagen content, and real-time quantitative PCR (RT-PCR) were used to analyse the mRNA expressions of UGBP,TGF-β1 and collagen in lung tissue.2. Study on the mechanism of UGBP changes in vitro20 healthy male KM mice were randomly divided into the control group (Con), N+TGF-β1 receptor antagonist group (Con+R0158), the bleomycin-induced group (B), TGF-β1 receptor antagonist group (B+R0158). B and B+R0158 group were injected BLM intraperitoneally (15mg/kg) for 10 consecutive days. the mice were killed on the 6th day after last treatment. The lung tissue were then cut into small pieces and cultured in the Transwell board. In Con+R0158 and B+R0158 group, TGF-β1 receptor antagonist were applied, then cultured four days and gathered these pieces to analysis the expressions of UGBP,TGF-β1 and collagen in lung tissure by using real-time quantitative PCR(RT-PCR).Results:1. The model of the intraperitoneal bleomycin-induced pulmonary fibrosis was successful. The fibrosis was gradually significant.different pathological changes were observed in the lung tissue from different time points.2. Immunohistochemical staining of UGBP positive signals were significantly stronger than control group on airway epithelial cells at Day 1 and there were no significant changes throughout the time course, but still more than control group. The positive cells also revealed small and heterogeneous in the site of fibrosis at Day 12. While the expression became significant strong in the area of pulmonary fibrosis at Day 18.3. Real-time PCR analysis of TGF-β1 expression was significantly higher (P<0.05) at Day 6 than the control group and much higher (P<0.01) at Day18.The expression of collagen was significantly higher (P<0.01) at Day 12 and still remain high expression at Day 18 (P<0.01). The expression of UGBP was a little higher at Day 1,Day6 and Day 12 than the control group, and then the UGBP expression was significant higher (P<0.01)at Day 18.4. Adding TGF-(31 receptor antagonist to break the signal transduction.The results showed that the mRNA expressios of TGF-β1 in B and B+R0158 group is significant higher (P<0.01) than Con and Con+R0158 group. But B and B+R0158, Con and Con+R0158 had no statistics differences. The expression of collagen in B group is significanlyt higher (P<0.01) than the control group and the expression of collagen in B+R0158 group is significantly lower (P<0.01) than B group, but also higer (P<0.05) than the control group. The expression of UGBP in B group is significantly higher (P<0.01) than the control group and the expression of UGBP in B+RO158 group is significantly lower (P<0.01) than B group and there were no statistics differences between B+R0158 group and the control group.Conclusion:1. The pulmonary fibrosis was gradually significant after injecting bleomycin intraperitoneally. UGBP expression is high in the fibrosis stage, and different cells have different pattern.2. The change of the UGBP expression is associated with the TGF-β1 signaling pathway in vitro. The TGF-β1 signaling pathway may play an important role in the regulation of UGBP expression.