M₃ Muscarinic Acetylcholine Receptor Antagonist Darifenacin Protects against Pulmonary Fibrosis through ERK/NF-κB/miR-21 Pathway  

作　　者：Ying Liu Yanan Jiang Chao Wang Haiying Zhang Yan Liu Ying Liu;Yanan Jiang;Chao Wang;Haiying Zhang;Yan Liu(Key Laboratory of Tropical Translational Medicine of Ministry of Education, College of Pharmaceutical, Hainan Medical University, Haikou, China;Department of Pharmacology, Harbin Medical University, Harbin, China;Sanya Central Hospital, Sanya, China)

机构地区：[1]Key Laboratory of Tropical Translational Medicine of Ministry of Education, College of Pharmaceutical, Hainan Medical University, Haikou, China [2]Department of Pharmacology, Harbin Medical University, Harbin, China [3]Sanya Central Hospital, Sanya, China

出　　处：《American Journal of Molecular Biology》2022年第2期11-22,共12页美国分子生物学期刊（英文）

摘　　要：Idiopathic pulmonary fibrosis is an untreatable lethal lung disease, which is related to the aberrant proliferation of fibroblasts. M₃ muscarinic acetylcholine receptor (M₃-mAChR) activation exerts proliferative effect on various kinds of cells. However, whether M₃-mAChR inhibition has a protective effect on pulmonary fibrosis remains unexplored. A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Darifenacin was used to block M₃-mAChR. Histological changes were observed using Masson’s Trichrome and hematoxylin and eosin (HE) staining. Hydroxyproline was measured by Hydroxyproline detection kit. Transforming growth factor β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, pulmonary fibroblasts were isolated from lungs of neonatal rat. After treatment, the cell viability, Hydroxyproline level was measured by MTT and Hydroxyproline detection kit respectively. The expression level of extracellular signal-regulated kinase (ERK), nuclear factor kappa-B (N-NF-κB), and microRNA-21 (miR-21) was detected by western blot or quantitative real-time PCR (qRT-PCR). Darifenacin relieved the fibrotic effects provoked by bleomycin. The expression level of hydroxyproline, TGF-β1 and TNF-α level was all downregulated after darifenacin treatment. In lung fibroblasts, darifenacin decreased cell viability and hydroxyproline level induced by bleomycin. Besides, phosphorylation-ERK and nuclear N-NF-κB protein level was downregulated, as well as miR-21 level. M₃-mAChR antagonist darifenacin attenuates bleomycin-induced pulmonary fibrosis in rats, which may relate to the ERK/NF-κB/miRNA-21 signaling pathway.Idiopathic pulmonary fibrosis is an untreatable lethal lung disease, which is related to the aberrant proliferation of fibroblasts. M₃ muscarinic acetylcholine receptor (M₃-mAChR) activation exerts proliferative effect on various kinds of cells. However, whether M₃-mAChR inhibition has a protective effect on pulmonary fibrosis remains unexplored. A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Darifenacin was used to block M₃-mAChR. Histological changes were observed using Masson’s Trichrome and hematoxylin and eosin (HE) staining. Hydroxyproline was measured by Hydroxyproline detection kit. Transforming growth factor β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, pulmonary fibroblasts were isolated from lungs of neonatal rat. After treatment, the cell viability, Hydroxyproline level was measured by MTT and Hydroxyproline detection kit respectively. The expression level of extracellular signal-regulated kinase (ERK), nuclear factor kappa-B (N-NF-κB), and microRNA-21 (miR-21) was detected by western blot or quantitative real-time PCR (qRT-PCR). Darifenacin relieved the fibrotic effects provoked by bleomycin. The expression level of hydroxyproline, TGF-β1 and TNF-α level was all downregulated after darifenacin treatment. In lung fibroblasts, darifenacin decreased cell viability and hydroxyproline level induced by bleomycin. Besides, phosphorylation-ERK and nuclear N-NF-κB protein level was downregulated, as well as miR-21 level. M₃-mAChR antagonist darifenacin attenuates bleomycin-induced pulmonary fibrosis in rats, which may relate to the ERK/NF-κB/miRNA-21 signaling pathway.

关 键 词：Pulmonary Fibrosis M₃ Muscarinic Acetylcholine Receptor DARIFENACIN 

分 类 号：R98[医药卫生—药品]