Role Of TSP-1 And Its Receptor CD47 In Paraquat-Induced Murine Pulmonary Fibrosis Model And Its Mechanism: A Preliminary Study

Object:To explore the relationship between TSP-1-CD47 and oxidative stress, and analyze TSP-1-CD47 roles in the pathogenesis of pulmonary fibrosis via observing the expression of TSP-1 and its ligand CD47 in lung tissue, the changes of ROS of erythrocyte and oxidative stress indicators of lung tissues after establishing a successful paraquat-induced murine pulmonary fibrosis model.Method:54 adult male Sprague-Dawley rats were randomly divided into normal group (n=6) and PQ group (n=48). PQ group was divided into six time points:2hours,12hours, 1day,3days,7days and 14days, each time point 8 rats. PQ was administrated by gavage at a dose of 50mg/kg to the rats in PQ group to establish a paraquat-induced murine pulmonary fibrosis model, and the control group was given the same volume of saline. During the experiment, observe the general status and the mortality of rats. Rats were sacrificed at each time point. Blood was reserved to detect the ROS of erythrocyte by flow cytometry. Lungs were excised from rat body and weighed lung wet to calculate lung coefficient. Then lung speciments were reserved for later evaluation. Observe the concentration of OH-, malondialdehyde(MDA) and hydroxyproline(HYP) though enzyme-linked immunosorbent assay. Observe the pathological changes though HE and Masson trichrome staining. Observe the express of TSP-1 and CD47 in the lung tissues though immunohistochemical analyses. Data were statistically analyzed using SPSS17.0.Results:Compared with normal group, the differences were statistically significant that the weight significant decreased and lung coefficient significantly increased in PQ poisoning rats (p<0.05). Along with the extension of the time after PQ poisoning, lung tissue can appear vary degrees of inflammatory cell infiltration, leakage of red blood cells, alveolar fluid leakage and other changes. The difference was statistically significant that the HE staining semi-quantitative alveolitis pathology score in PQ groups was higher than which of normal group. After exposured 2h, the ratios of the positive rate of ROS fluorescence intensity in erythrocyte and which in lung tissue were gradually increased as well as the concentration of OH" and MDA, then gradually reduced to the lowest after exposured 7d (p<0.05). A small amount of filamentous formation of collagen fibers in the lung tissue was showed though Masson staining after exposured 2h. Along with the extension of the time after PQ poisoning, it was significantly increased that the semi-quantitative pulmonary fibrosis pathology score and the concentration of HYP, which were reached the peak at day14 (p<0.05). After exposured 2h, the expression of TSP-1 was significantly increased as well as CD47. After exposured 12h, the expression of TSP-1 and CD47 was weakly positive. After exposured 3d, the expression of TSP-1 and CD47 was significantly increased as the time goes. Correlation analysis found that, the expression of TSP-1 and CD47 were positively correlated with the ratio of the positive rate of ROS fluorescence intensity, the same to the concentration of OH" and MDA within 2h, and the expression of TSP-1 and CD47 were positively correlated with the concentration of hydroxyproline after 1d.Conclusions:1. It can be successfully established murine pulmonary fibrosis model by gavage 20%PQ at a dose of 50mg/kg to the rats.2. PQ can induce systemic oxidative stress as well as lung tissues.3. PQ can induce the expressions of TSP-1 and CD47 in lung tissues.4. The expressions of TSP-1 and CD47 were closely related to the oxidative stress and pulmonary fibrosis in paraquat-induced murine pulmonary fibrosis.5. TSP-1-CD47 may be involved in the early alveolitis and the progress of pulmonary fibrosis in paraquat-induced murine pulmonary fibrosis.