Regorafenib prevents the development of emphysema in a murine elastase model

Abstract

Emphysema is a chronic obstructive lung disease characterized by inflammation and enlargement of the air spaces. Regorafenib, a potential senomorphic drug, exhibited a therapeutic effect in porcine pancreatic elastase (PPE)-induced emphysema in mice. In the current study we examined the preventive role of regorafenib in development of emphysema. Lung function tests and morphometry showed that oral administration of regorafenib (5 mg/kg/day) for seven days after instillation of PPE resulted in attenuation of emphysema. Mechanistically, regorafenib reduced the recruitment of inflammatory cells, particularly macrophages and neutrophils, in bronchoalveolar lavage fluid. In agreement with these findings, measurements using a cytokine array and ELISA showed that expression of inflammatory mediators including interleukin (IL)-1β, IL-6, and CXCL1/KC, and tissue inhibitor of matrix metalloprotease-1 (TIMP-1), was downregulated. The results of immunohistochemical analysis confirmed that expression of IL-6, CXCL1/KC, and TIMP-1 was reduced in the lung parenchyma. Collectively, the results support the preventive role of regorafenib in development of emphysema in mice and provide mechanistic insights into prevention strategies. [BMB Reports 2023; 56(8): 439-444].

Fig. 1

Preventive effect of regorafenib on development of PPE-induced emphysema in mice. (A) Experimental scheme. In the PPE + regorafenib (Reg) group, regorafenib (5 mg/kg) was administered orally for one day for sensitization before and consecutive seven days after treatment with PPE via the intranasal route. At three weeks after treatment with PPE, mice were analyzed to measurement of Lm and lung function. (B) Representative H&E stained images. Scale bar indicates 100 μm. (C) Quantification of Lm. N = 6/group. (D) Lung function test. Cst (static compliance), A (total lung capacity) and K (form of deflating the PV-loop) parameters are shown. N = 6/group. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, t-test.

Fig. 2

Effect of regorafenib on PPE-induced inflammatory cells in BAL fluid. (A) Experimental scheme. Regorafenib (5 mg/kg) was sensitized and administered orally in PPE + Reg group for two days after treatment with PPE via the intranasal route. (B) Representative images of stained BAL cells. Scale bars indicate 100 μm. (C-G) Measurement of cell numbers in BAL cells (N = 3 per group). Total number of cells (C), macrophages (D), neutrophils (E), eosinophils (F), and lymphocytes (G) were counted on five areas of stained slides. Error bars indicate the mean ± SEM. **P < 0.01, ****P < 0.0001, One-way ANOVA.

Fig. 3

Effect of regorafenib on PPE-induced in inflammatory mediators in BAL fluid. (A) Representative membrane images of the antibody array. (B) Quantification of relative optical density for IL-1β, IL-6, CXCL1/KC, and TIMP-1 in the array membranes. (C-F) Measurement of IL-1β (C), IL-6 (D), CXCL1/KC (E), and TIMP-1 (F) levels in BAL fluid by ELISA. Error bars indicate the mean ± SEM. N = 8 per group, *P < 0.05, **P < 0.01, ****P < 0.0001, t-test.

Fig. 4

Effect of regorafenib on PPE-induced expression of inflammatory mediators in lung parenchyme. (A) Experimental scheme. Mice were sensitized by treatment with regorafenib (5 mg/kg) for one day in the only PPE + Reg group. After PPE treatment, regorafenib was administered orally to mice for six days. On the seventh day, lung tissues were removed from mice, prepared in paraffin sections and stained with antibodies for inflammatory mediators. (B) Representative images of stained paraffin sections. Red arrows indicate the macrophages in inserted boxes. Scale bars indicate 50 μm. (C-E) Quantification of Fig. 4B. IL-6 (C), CXCL1/KC (D), and TIMP-1 (E). Error bars indicate the mean ± SEM. N = 5 per group, *P < 0.05, **P < 0.01, t-test.