MYO9B Deficiency Protects Mice From Bleomycin-induced Pulmonary Fibrosis By Regulating Fibroblast Differentiation

Rationale:Interstitial lung disease is a group of lung disease mainly characterized by pathological changes in the lungs.The end-stage disease can lead to respiratory failure,limited treatment options,and poor therapeutic effects and prognosis.Idiopathic pulmonary fibrosis（IPF）is the most common type of interstitial lung disease with a median survival of less than 5 years.The occurrence of fibrosis is closely related to repeated alveolar injury,and the abnormal activation and proliferation of fibroblasts is a key part in the progress of fibrosis.Cytoskeleton myosin IXB（MYO9B）is a heavy chain protein of actin-related molecules.The Rho GAP domain contained in it can regulate cell morphology,migration,and cell proliferation.And MYO9B can regulate the activity of Rho-related coiled coil kinase（ROCK）.Studies have confirmed that ROCK is involved in the differentiation of myofibroblasts.Therefore,changes in MYO9B activity may lead to the progression of fibrosis.Objective:To investigate the expression of MYO9B in lung tissue of IPF patients and bleomycin-induced pulmonary fibrosis in mouse models,and to preliminary explore the possible role and mechanism of MYO9B in pulmonary fibrosis.Methods:The expression of MYO9B was detected by Immunofluorescence and Western Blot in IPF patients and normal lung tissues.In animal experiments,a mouse lung fibrosis model was established by tracheal instillation of bleomycin（BLM）,and mice in the control group were instilled with saline.The Immunofluorescence staining was used to detect the expression of MYO9B in mouse lung tissue.WT mice and MYO9B^-/-mice were randomly divided into four groups:WT mouse+Saline group,WT mouse+BLM group,MYO9B^-/-mouse+Saline group,MYO9B^-/-mouse+BLM group.The lung tissue of each group of mice was stained with HE,Sirius Red and Masson,and the degree of lung injury and fibrosis was evaluated.q PCR and Western Blot were used to detect the expression of MYO9B,collagen I,fibronectin andα-SMA in lung tissues of WT mice and MYO9B^-/-mice,respectively.In cell experiments,primary fibroblasts from WT and MYO9B^-/-mice were taken and stimulated with TGF-βfor 48 hours,respectively,and the expression levels of MYO9B,fibronectin andα-SMA before and after stimulation were measured.Western Blot was used to detect the expression of TGF-β/Smad signaling pathway in lung tissue of BLM-induced wild type mice and MYO9B^-/-mice,respectively.Results:MYO9B was highly expressed in the lung tissue of patients with IPF,and Immunofluorescent staining showed that MYO9B was highly expressed in fibroblasts.Compared with the control group,the expression of MYO9B was increased in a mouse model of pulmonary fibrosis.Pathological analysis showed that after lung fibrosis of MYO9B^-/-and WT mice induced by BLM,the degree of fibrosis was lighter than that of WT mice;the expression levels of collagen I,fibronectin andα-SMA in lung tissue of MYO9B^-/-mice were lower than those of WT mice.The primary fibroblasts of WT mice and MYO9B^-/-mice were extracted.After stimulation with TGF-β,the expression levels of fibronectin andα-SMA in fibroblasts of MYO9B^-/-mice were lower than those of WT mice.The expression of TGF-β/Smad signaling pathway in BLM-induced MYO9B^-/-mice was lower than that in WT mice after BLM induction.The control group showed no statistical difference.Conclusion:MYO9B deletion may reduce BLM-induced lung fibrosis in mice by inhibiting fibroblast differentiation,and may be involved in the pathogenesis of pulmonary fibrosis.