The Role of TSP-1-CD47 in ROS-mediated Pulmonary Fibrosis Induced by Paraquat

Abstract

Objective: Taking human A549 cells as the research object, to construct the paraquat-induced pulmonary fibrosis model in vitro, and to explore the role of TSP-1 (Thrombospondin-1, TSP-1) and its receptor CD47 in PQ-induced pulmonary fibrosis. Methods: Human A549 cells were cultured in vitro, divided into normal control group, PQ group, Anti-TSP1 group (PQ with neutralizing anti-TSP1 antibody at a final concentration of 10 μg/ml) . A549 cells were stimulated with different concentrations (50, 100, 200, 400, 600, 800, 1 000 μmol/L) for different time (12, 24, 48 h) , and then CCK8 method was used to detect the cell viability to screen out the concentration and time of half cell viability. The subsequent test will be performed at this concentration point.The morphology of the cells was observed under inverted microscope. The expression levels of Fibronectin (FN) and type I collagen were determined by Enzyme Linked Immunosorbent Assay (ELISA) . Immunocytochemistry (ICC) and Immunofluorescence (IF) were used to observe the expression of TSP-1 and CD47 protein and the co-expression.The mRNA expression of TSP-1 and CD47 was detected by Real Time PCR (RT-PCR) . The protien expression of TSP-1 and CD47 was detected by Western Blot (WB) . The levels of Reactive Oxygen Species (ROS) were measured by flow cytometry. Results: Before neutralizing anti-TSP1 antibody intervention: (1) When the time of PQ was constant, the cell viability decreased with the increase of PQ concentration. (2) The cells in the control group were closely connected, cobble-like, arranged neatly; with the increase of PQ concentration, the cell gap of PQ group gradually increased, spindle shape or long spindle shape. (3) With the increase of PQ concentration, the relative expression of FN and I collagen in PQ group was gradually increased compared with the control group in a concentration-dependent manner, and 200 μmol/L is the most obvious. (4) Compared with the control group, the mRNA level and the protein expression of TSP-1 and CD47 in PQ group was significantly increased, and 200 μmol/L is the most obvious, and Immunofluorescence showed they were co-expression in cytoplasm. (5) Compared with the normal group, the level of ROS in A549 cells was significantly increased at 24 h after PQ stimulation. (6) Compared with PQ group, the cell viability of Anti-TSP1 group was significantly increased, and the morphology changed to normal cell morphology, and the mRNA level and the protein expression of TSP-1 and CD47 decreased, and the overexpression of ROS was inhibited, and the relative expression of FN and I collagen decreased. Conclusion: PQ stimulation induced morphological changes of A549 cells, increased expression of TSP-1, CD47, FN and type I collagen, and increased production of ROS.Neutralizing anti-TSP1 antibodies against TSP-1 can partially improve the above lesions. TSP-1-CD47 may be associated with oxidative stress-mediated PQ-induced pulmonary fibrosis.