Cromolyn sodium and masitinib combination inhibits fibroblast-myofibroblast transition and exerts additive cell-protective and antioxidant effects in a bleomycin-induced in vitro fibrosis model

Abstract

Background: Several in vitro and in vivo studies suggest that tyrosine kinase inhibitors may be effective in the treatment of idiopathic pulmonary fibrosis. In our previous study, we reported that combined treatment with masitinib, a tyrosine kinase inhibitor used to treat mastocytoma, and cromolyn sodium, a mast cell stabilizer drug used in allergic diseases, exerts synergistic cell-protective and antioxidant effects on an in vitro neurodegenerative model. In this study, we aimed to investigate the anti-fibrotic, cell-protective, and antioxidant effects of this drug combination on a bleomycin-induced in vitro model of pulmonary fibrosis. Methods: HFF-1 fibroblast cells were cultured and were divided into groups according to bleomycin and hydrogen peroxide (H2O2) applications, followed by treatment with cromolyn sodium/masitinib. The cell viability rates and metabolic activity were assessed with an MTT assay. Cell groups were compared morphologically under an inverted microscope. Additionally, Hoechst staining was performed for apoptotic nuclear assessment. Triple-immunofluorescence labeling (alpha-smooth muscle actin, CD44, DAPI) was performed to observe fibroblast-myofibroblast transition. Total oxidant and antioxidant capacities were measured in conditioned media of cell groups.Results: Cromolyn sodium and masitinib mono treatments significantly increased the cell viability rate of fibroblasts which was decreased by bleomycin and H2O2. Combined treatments of cromolyn sodium and masitinib increased cell viability rates more than monotherapy. Combined cromolyn sodium and masitinib exerted a protective effect on bleomycin-induced changes by preserving cell morphology with fewer apoptotic cells and a lower rate of fibroblast to myofibroblast transition, and improved arrangement of fibroblasts. Masitinib, cromolyn sodium, and their combination reduced the mean number of apoptotic cells. Combined cromolyn sodium and masitinib decreased the expression of α-SMA that was induced by bleomycin and increased the total antioxidant capacity while significantly decreasing the total oxidant capacity level that was increased by bleomycin and H2O2.Conclusions: We observed the additive anti-fibrotic, cell-protective and antioxidant effects of masitinib and cromolyn sodium combination in an in vitro fibrosis model. New combination therapies may offer an innovative treatment approach for pulmonary fibrosis, with the advantages of lower doses of both drugs, fewer side effects, and different mechanisms of action at the same time.