4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Idiopathic-Pulmonary-Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a highly fatal disease that lacks appropriate treatments and highly effective drugs. Many reported indicated that the TGF-β1/Smad3 signaling pathway played a pivotal role in development of IPF. In this case, it was hypothesized that discovery novel compounds to block the TGF-β1/Smad3 signaling pathway might be useful for treatment of IPF. Therefore, a high-throughput screening system based on stably transfected CAGA-NIH3T3 cells was established for discovering lead compounds which could validly suppress the TGF-β1/Smad3 signal path. In this study, a series of novel Pleuromutilin derivatives were prepared and quickly evaluated by high-throughput assay. The lead compound 32 was discovered to be able to remarkably suppress the TGF-β1/Smad3 pathway in vitro. Further biological evaluation revealed that compound 32 could remarkably decrease the myofibroblast stimulation and extracellular matrix (ECM) deposition. More importantly, compound 32 could remarkably mitigate bleomycin (BLM)-triggered lung fibrosis in mice models. Additionally, the lead compound possess excellent pharmacokinetics properties, good oral availability and low toxicity. In general, our study has demonstrated the potency of a novel Pleuromutilin derivative (compound 32), which might be a prospective candidate for developing anti-IPF medicines by suppress the TGF-β1/Smad3 signal pathway.

Topics: Animals; Bleomycin; Diterpenes; Fibroblasts; High-Throughput Screening Assays; Idiopathic Pulmonary Fibrosis; Lung; Mice; Mice, Inbred C57BL; NIH 3T3 Cells; Pleuromutilins; Polycyclic Compounds; Transforming Growth Factor beta1

Idiopathic pulmonary fibrosis (IPF) is a deadly, progressive lung disease with very few treatment options till now. Bleomycin-induced pulmonary fibrosis (BIPF) is a commonly used mice model in IPF research. TGF-β1 has been shown to play a key role in pulmonary fibrosis (PF). Dendritic cell (DC) acts as a bridge between innate and adaptive immune systems. The coexistence of chronic inflammation sustained by mature DCs with fibrosis suggests that inflammatory phenomenon has key importance in the pathogenesis of pulmonary fibrosis. Here, we investigated the modulation of DCs phenotypic maturation, accumulation in lung tissue, and expression of other lung DC subsets in respect to TGF-β in PF. First, we established BIPF model in mice and blocked TGF-β expression by the use of inhibitor SB431542. Accumulation of lung CD11c+ DCs is significantly higher in both inflammatory and fibrotic phases of the disease but that percentages got reduced in the absence of TGF-β. TGF-β initiates up-regulation of costimulatory molecules CD86 and CD80 in the inflammatory phases of the disease but not so at fibrotic stage. Expression of lung DC subset CD11c+CD103+ is significantly increased in inflammatory phase and also in fibrotic phase of BIPF. Blocking of TGF-β causes decreased expression of CD11c+CD103+ DCs. Another important lung DC subset CD11c+CD11b+ expression is suppressed by the absence of TGF-β after bleomycin administration. CD11c+CD103+ DCs might have anti-inflammatory as well as anti-fibrotic nature in PF. All these data demonstrate differential modulation of CD11c+ lung DCs by TGF-β in experimental PF.

Topics: Animals; Antigens, CD; Benzamides; Bleomycin; CD11c Antigen; Dendritic Cells; Dioxoles; Disease Models, Animal; Fibroblasts; Idiopathic Pulmonary Fibrosis; Integrin alpha Chains; Male; Mice; Mice, Inbred C57BL; Transforming Growth Factor beta; Up-Regulation

To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis.. Lung cell suspension was prepared from β-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-β inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed.. The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-β receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%.. During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

Topics: Actins; Animals; Benzamides; Cell Movement; Cell Proliferation; Cells, Cultured; Dioxoles; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; Lung; Mice; MicroRNAs; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Fibroblast Growth Factor; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-β1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-β type I receptor (TβRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-β1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis.

Topics: Benzamides; Cell Movement; Cells, Cultured; Connective Tissue Growth Factor; Dioxoles; Fibroblasts; Humans; Idiopathic Pulmonary Fibrosis; Lung; Phosphatidylinositol 3-Kinases; RNA, Messenger; Signal Transduction; Sirolimus; Smad2 Protein; Smad3 Protein

Our objective was to investigate the pathogenesis pathways of idiopathic pulmonary fibrosis (IPF). Bleomycin (BLM) induced animal models of experimental lung fibrosis were used. CHIP assay was executed to find the link between Smad3 and IL-31, and the expressions of TGF-β1, Smad3, IL-31 and STAT1 were detected to find whether they were similar with each other. We found that in the early injury or inflammation of the animal model, BLM promoted the development of inflammation, leading to severe pulmonary fibrosis. Then the expression of TGF-β1 and Smad3 increased. Activated Smad3 bound to the IL-31 promoter region, followed by the activation of JAK-STAT pathways. The inhibitor of TGF-β1 receptor decreased the IL-31 expression and knocking-down of IL-31 also decreased the STAT1 expression. We conclude that there is a pathway of pathogenesis in BLM-induced mouse model that involves the TGF-β, IL-31 and JAKs/STATs pathway.

Topics: Animals; Antibiotics, Antineoplastic; Benzamides; Bleomycin; Cells, Cultured; Dioxoles; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Idiopathic Pulmonary Fibrosis; Interleukins; Lung; Lung Injury; Mice; Signal Transduction; STAT1 Transcription Factor; Time Factors; Transforming Growth Factor beta1

Epithelial-mesenchymal transition (EMT) has been considered to be involved in idiopathic pulmonary fibrosis (IPF). However, the EMT process in vivo is much more complex and controversial. Studies regarding the opposite process, mesenchymal-epithelial transition (MET) in fibroblasts, are limited. Therefore, the aim of this study was to verify the involvement of the transforming growth factor (TGF)-β1-dependent EMT network in the process of pulmonary fibrosis and to explore the possibility of MET. Fibrotic lung tissues were obtained from patients with IPF with histological evidence of usual interstitial pneumonia at the time of surgical lung biopsy. For the controls, histologically normal lung tissues were obtained from patients with primary spontaneous pneumothorax at the time of thoracoscopy with stapling of any air leaks. Real-time RT-PCR and western blot analysis revealed that the mRNA and protein levels of TGF-β1, TGF-β1 receptor type I/II/III (TβRI/II/III), Smad2/3/4 and Snail1/2 were significantly upregulated in the fibrotic lung tissue. Inhibitors of various kinases implicated in EMT, including TGF-β1/Smad, Rho kinase (ROCK), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH-terminal kinase (JNK) were used to determine the MET potential in fibroblasts from fibrotic lung tissue. Western blot analysis or indirect immunofluorescence staining revealed that Smad inhibitor, as well as other kinase inhibitors failed to induce the MET process, determined by cellular morphology and protein markers. Our data suggest that the MET process may not be the exact reversal of EMT. In addition to using kinase inhibitors, other intervention measures should be used to explore the possibility of the MET process in fibroblasts from fibrotic lung tissue.

Topics: Adult; Benzamides; Case-Control Studies; Cell Culture Techniques; Dioxoles; Epithelial-Mesenchymal Transition; Fibroblasts; Gene Expression Regulation; Humans; Idiopathic Pulmonary Fibrosis; JNK Mitogen-Activated Protein Kinases; Male; Middle Aged; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pulmonary Fibrosis; Receptors, Transforming Growth Factor beta; rho-Associated Kinases; RNA, Messenger; Transforming Growth Factor beta1; Young Adult