Abstract
Belumosudil (SLX-2119) inhibits rho-associated, coiled-coil containing protein kinase 2 (ROCK2), which mainly functions by regulating inflammatory and adipogenic pathways; however, the effects of belumosudil and its mechanism of action toward pulmonary fibrosis (PF) remain unclear. We conducted in vivo investigations and examined belumosudil effects on PF using an intratracheal bleomycin (BLM) infusion rat model. We then examined mechanisms whereby belumosudil inhibited in vitro-induced PF by determining its effects on transforming growth factor (TGF)-β1-induced mouse lung fibroblasts (proliferation) and lipopolysaccharide-(LPS) or interleukin (IL)-4-induced mouse mononuclear macrophage (RAW264.7) polarization. Continuous belumosudil (20 mg/kg) gavage for 2 weeks elicited protective effects in BLM-induced PF rats, as shown by changes in Hounsfield units, which indicated overall pulmonary function, and lung coefficient and lung microscopic pathology scores, which indicated gross pulmonary pathology. ROCK2, p-Smad3, connective tissue growth factor (CTGF), arginase-1 (Arg-1), and inducible nitric oxide synthase (iNOS) protein levels were also altered. In vitro, belumosudil (100 nM) reduced LPS or IL-4-induced macrophage polarization, lowered Arg-1 and iNOS levels, reduced TGF-β1-induced fibroblast proliferation, and generated lower ROCK2, p-Smad3, and CTGF levels. Belumosudil appeared to alleviate experimental PF by reducing fibroblast proliferation and macrophage polarization by inhibiting ROCK2/Smad3/CTGF signaling.