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Background Emerging evidence provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), and rare anti‐PF therapeutic method has promising effect in its treatment. Rho‐associated coiled‐coil kinases (ROCK) inhibition significantly ameliorates bleomycin‐induced PF and decreases macrophage infiltration, but the mechanism remains unclear. We established bleomycin and radiation‐induced PF to identify the activity of WXWH0265, a newly designed unselective ROCK inhibitor in regulating macrophages. Methods Bleomycin‐induced PF was induced by intratracheal instillation and radiation‐induced PF was induced by bilateral thoracic irradiation. Histopathological techniques (haematoxylin and eosin, Masson's trichrome and immunohistochemistry) and hydroxyproline were used to evaluate PF severity. Western blot, quantitative real‐time reverse transcription‐polymerase chain reaction and flow cytometry were performed to explore the underlying mechanisms. Bone marrow‐derived macrophages (BMDMs) were used to verify their therapeutic effect. Clodronate liposomes were applied to deplete macrophages and to identify the therapeutic effect of WXWH0265. Results Therapeutic administration of ROCK inhibitor ameliorates bleomycin‐induced PF by inhibiting M2 macrophages polarisation. ROCK inhibitor showed no significant anti‐fibrotic effect in macrophages‐depleted mice. Treatment with WXWH0265 demonstrated superior protection effect in bleomycin‐induced PF compared with positive drugs. In radiation‐induced PF, ROCK inhibitor effectively ameliorated PF. Fibroblasts co‐cultured with supernatant from various M2 macrophages phenotypes revealed that M2 macrophages stimulated by interleukin‐4 promoted extracellular matrix production. Polarisation of M2 macrophages was inhibited by ROCK inhibitor treatment in vitro. The p‐signal transducer and activator of transcription 3 (STAT3) in lung tissue and BMDMs was significantly decreased in PF in vivo and vitro after treated with ROCK inhibitors. Conclusion Inhibiting ROCK could significantly attenuate bleomycin‐ and radiation‐induced PF by regulating the macrophages polarisation via phosphorylation of STAT3. WXWH0265 is a kind of efficient unselective ROCK inhibitor in ameliorating PF. Furthermore, the results provide empirical evidence that ROCK inhibitor, WXWH0265 is a potential drug to prevent the development of PF.

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