BTSA1 Attenuates Pulmonary Fibrosis By Eliminating Senescent Myofibroblasts

Pulmonary fibrosis is a chronic progressive and aging-related interstitial lung disease in which senescent myofibroblasts and epithelial cells exist in fibrotic lung tissue.Apoptosis resistance of senescent myofibroblasts is closely related to the development of pulmonary fibrosis.Elimination of senescent myofibroblasts is an approach to reduce or even reverse pulmonary fibrosis.BAX is a member of the BCL-2 family of proteins,its function is regulated by both pro-apoptotic proteins and anti-apoptotic proteins,it’s a crucial"executors"involving in mitochondria-mediated apoptosis pathways.This topic mainly investigated the effect of BAX agonist-BTSA1 on bleomycin(BLM)-induced pulmonary fibrosis in mice,and the eliminating effect of BTSA1 on senescent myofibroblasts.The purpose of all this is to provide new enlightenment for the prevention and treatment of pulmonary fibrosis.Objective:1.To observe the expression level of BAX in senescent myofibroblasts in bleomycin-induced pulmonary fibrosis;2.To investigate the effect of BAX agonist-BTSA1 on bleomycin-induced pulmonary fibrosis in mice;3.To investigate the eliminating effect of BTSA1 on senescent myofibroblasts in vitro.Methods:1.HE staining,Masson staining and real-time fluorescence quantitative PCR experiments detected respectively the morphology of mouse lung tissue,the distribution of collagen in mouse lung tissue and the m RNA levels of type I collagen,type III collagen and fibronectin in mouse lung tissue,which confirmed the pulmonary fibrosis in the lung tissue of mice 21 days after bleomycin injury.We have also observed the senescent cells by immunofluorescence staining 2.The expression level of BAX and BCL-XL in senescent myofibroblasts in lung tissue of BLM-induced pulmonary fibrosis mice was observed by multiple tissue immunofluorescence staining.3.Using HE staining,Masson staining,immunohistochemistry,Western Blot and real-time quantitative PCR experiments demonstrated the effect of BTSA1 on pulmonary fibrosis mice.4.Extract mouse primary fibroblasts,and use X-ray irradiation to induce senescence;usingβ-galactosidase staining,cell immunofluorescence staining and Western Blot experiments to prove that senescent myofibroblasts can be obtained by the X-ray irradiation method.5.The expression of BAX and BCL-XL in mouse senescent myofibroblasts was observed by immunofluorescence staining.6.Using CCK-8 cell proliferation toxicity detection,cell immunofluorescence staining,Western Blot Western blotting,JC-1 mitochondrial membrane potential detection,Annexin V/PI double staining apoptosis detection,flow cytometry and other experimental methods,investigated the pro-apoptotic effect of BTSA1 on senescent myofibroblasts.Results:1.Compared with normal control mice,there are senescent myofibroblasts(p21~+/α-SMA~+)in the lung tissue of BLM-induced pulmonary fibrosis mice;and BAX(BCL-XL)level is upregulated in senescent myofibroblasts compared to non-senescent myofibroblasts.2.BTSA1 not only improves bleomycin-drived lung morphology and collagen deposition,but reduces the counts of senescent myofibroblasts.3.After X-ray irradiating fibroblasts,cell size,cell proliferation,β-galactosidase,and expression of p21,have obviously mutated,indicating that X-ray irradiation can successfully establish the model of senescent myofibroblasts.4.Compared with control fibroblasts,BAX expression was up-regulated in senescent myofibroblasts.5.After BTSA1 intervening control fibroblasts,the mitochondrial membrane potential of control fibroblasts did not decrease significantly(JC-1 staining),Annexin V/PI staining did not increase significantly;while BTSA1 interfered with senescent myofibroblasts,the mitochondrial membrane potential of senescent myofibroblasts was significantly reduced(JC-1 staining).Annexin V/PI double staining and flow cytometry results also showed that BTSA1 could significantly promote the apoptosis of senescent myofibroblasts.Conclusion:1.BAX agonist BTSA1 can reduce the degree of bleomycin-induced pulmonary fibrosis in mice;2 BTSA1 can promote the apoptosis of senescent myofibroblasts.