Prolyl oligopeptidase inhibition ameliorates experimental pulmonary fibrosis both in vivo and in vitro

Background: Pulmonary fibrosis is a progressive disease characterized by lung remodeling due to excessive deposition of extracellular matrix. Although the etiology remains unknown, aberrant angiogenesis and inflammation play an important role in the development of this pathology. In this context, recent scientific research has identified new molecules involved in angiogenesis and inflammation, such as the prolyl oligopeptidase (PREP), a proteolytic enzyme belonging to the serine protease family, linked to the pathology of many lung diseases such as pulmonary fibrosis. Therefore, the aim of this study was to investigate the effect of a selective inhibitor of PREP, known as KYP-2047, in an in vitro and in an in vivo model of pulmonary fibrosis. Methods: The in vitro model was performed using human alveolar A549 cells. Cells were exposed to lipopolysaccharide (LPS) 10 μg/ml and then, cells were treated with KYP-2047 at the concentrations of 1 μM, 10 μM and 50 μM. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide colorimetric assay, while inflammatory protein expression was assessed by western blots analysis. The in vivo model was induced in mice by intra-tracheal administration of bleomycin (1 mg/kg) and then treated intraperitoneally with KYP-2047 at doses of 1, 2.5 and 5 mg/kg once daily for 12 days and then mice were sacrificed, and lung tissues were collected for analyses. Results: The in vitro results demonstrated that KYP-2047 preserved cell viability, reduced inflammatory process by decreasing IL-18 and TNF-α, and modulated lipid peroxidation as well as nitrosative stress. The in vivo pulmonary fibrosis has demonstrated that KYP-2047 was able to restore histological alterations reducing lung injury. Our data demonstrated that KYP-2047 significantly reduced angiogenesis process and the fibrotic damage modulating the expression of fibrotic markers. Furthermore, KYP-2047 treatment modulated the IκBα/NF-κB pathway and reduced the expression of related pro-inflammatory enzymes and cytokines. Moreover, KYP-2047 was able to modulate the JAK2/STAT3 pathway, highly involved in pulmonary fibrosis. Conclusion: In conclusion, this study demonstrated the involvement of PREP in the pathogenesis of pulmonary fibrosis and that its inhibition by KYP-2047 has a protective role in lung injury induced by BLM, suggesting PREP as a potential target therapy for pulmonary fibrosis. These results speculate the potential protective mechanism of KYP-2047 through the modulation of JAK2/STAT3 and NF-κB pathways.