ovalbumin and pyrazolanthrone

Airway fibrosis is one of the pathological characteristics of severe asthma. Transforming growth factor (TGF)-β has been known to promote epithelial-mesenchymal transition formation and to play a role in the progression of tissue fibrosis. Cellular communication network factor 2 (CCN2) and fibronectin (FN) are well-known markers of EMT and fibrosis. However, whether AREG is involved in TGF-β-induced CCN2 and FN expression in human lung epithelial cells is unknown.. AREG and FN were analyzed by immunofluorescence staining on ovalbumin-challenged mice. CCN2 and FN expression were evaluated in human lung epithelial (A459) cells following TGF or AREG treatment for the indicated times. Secreted AREG from A549 cells was detected by ELISA. Cell migration was observed by a wound healing assay. Chromatin immunoprecipitation was used to detect the c-Jun binding to the CCN2 promoter.. AREG and FN expression colocalized in lung tissues from mice with ovalbumin-induced asthma by immunofluorescence staining. Moreover, TGF-β caused the release of AREG from A549 cells into the medium. Smad3 siRNA down-regulated AREG expression. AREG also stimulated CCN2 and FN expression, JNK and c-Jun phosphorylation, and cell migration in A549 cells. AREG small interfering (si) RNA inhibited TGF-β-induced expression of CCN2, FN, and cell migration. Furthermore, AREG-induced CCN2 and FN expression were inhibited by EGFR siRNA, a JNK inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). EGFR siRNA attenuated AREG-induced JNK and c-Jun phosphorylation. Moreover, SP600125 downregulated AREG-induced c-Jun phosphorylation.. These results suggested that AREG mediates the TGF-β-induced EMT in human lung epithelial cells through EGFR/JNK/AP-1 activation. Understanding the role of AREG in the EMT could foster the development of therapeutic strategies for airway remodeling in severe asthma.

Topics: Amphiregulin; Animals; Asthma; Epithelial Cells; Epithelial-Mesenchymal Transition; ErbB Receptors; Fibronectins; Fibrosis; Humans; Lung; Mice; Ovalbumin; RNA, Small Interfering; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

c-Jun N-terminal kinase (JNK) relays extracellular stimuli through phosphorylation cascades that lead to various cell responses. In the present study, we aimed to investigate the effect of the JNK inhibitor SP600125 on the resolution of airway inflammation, and the underlying mechanism using a murine acute asthma model.. Female C57BL/6 mice were sensitized with saline or ovalbumin (OVA) on day 0, and challenged with OVA on day 14-20. Meanwhile, some of the mice were treated with SP600125 (30 mg/kg) intraperitoneally 2 h before each challenge. The airway inflammation was evaluated by counting the numbers of various types of inflammatory cells in bronchoalveolar lavage fluid (BALF), histopathology, cytokines production and mucus secretion in individual mouse. In addition, we analyzed the protein levels of phosphorylated JNK and TLR9 in the lung tissues.. SP600125 markedly reduced the invasion of inflammatory cells into the peribronchial regions, and decreased the numbers of eosinophils, monocytes, neutrophils and lymphocytes in BALF. SP600125 also reduced the level of plasma OVA-specific IgE, lowered the production of pro-inflammatory cytokines in BALF and alleviated mucus secretion. Meanwhile, SP600125 inhibited OVA-induced, increased expression of p-JNK and TLR9 in the lung tissues.. Collectively, our data demonstrated that SP600125 promoted resolution of allergic airway inflammation via TLR9 in an OVA-induced murine acute asthma model. The JNK-TLR9 pathway may be a new therapeutic target in the treatment for the allergic asthma.

Topics: Acute Disease; Animals; Anthracenes; Asthma; Bronchoalveolar Lavage Fluid; Cell Count; Cytokines; Disease Models, Animal; Female; Hypersensitivity; Immunoglobulin E; Inflammation; JNK Mitogen-Activated Protein Kinases; Lung; Mice, Inbred C57BL; Mucus; Ovalbumin; Phosphorylation; Toll-Like Receptor 9

Jun N-terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9-cd] pyrazol-6 (2H)-one), a novel inhibitor of JNK in a model of asthma. Brown-Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen-induced, increased activity of phosphorylated c-jun but not of phosphorylated-MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0.04), lymphocyte (P < 0.05), eosinophil (P < 0.04) and neutrophil (P < 0.005) numbers in bronchoalveolar lavage. Eosinophil and T-cell accumulation in the airways, mRNA expression for interleukin-1beta, tumour necrosis factor-beta, interleukin-3, interleukin-4 and interleukin-5, serum levels of allergen-specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen-activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.

Topics: Allergens; Animals; Anthracenes; Asthma; Bronchoalveolar Lavage Fluid; Cell Count; Cytokines; Enzyme Activation; Eosinophils; JNK Mitogen-Activated Protein Kinases; Leukocyte Count; Leukocytes, Mononuclear; Lung; Macrophages; Male; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Animal; Neutrophils; Ovalbumin; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Inbred BN

Chronic cellular inflammation and airway wall remodelling with subepithelial fibrosis and airway smooth muscle (ASM) cell hyperplasia are features of chronic asthma. Jun N-terminal kinase (JNK) may be implicated in these processes by regulating the transcriptional activity of activator protein (AP)-1. We examined the effects of an inhibitor of JNK, SP600125 (anthra [1,9-cd] pyrazole-6 (2 H)-one), in a model of chronic allergic inflammation in the rat. Rats sensitised to ovalbumin (OA) were exposed to OA-aerosol every third day on six occasions and were treated with SP600125 (30 mg kg-1 b.i.d; 360 mg in total) for 12 days, starting after the second through to the sixth OA exposure. We measured eosinophilic and T-cell inflammation in the airways, proliferation of ASM cells and epithelial cells by incorporation of bromodeoxyuridine (BrdU), and bronchial responsiveness to acetylcholine. SP600125 significantly reduced the number of eosinophils (P<0.05) and lymphocytes (P<0.05) in bronchoalveolar lavage fluid, suppressed eosinophilic (P<0.05) and CD2+ T-cell (P<0.05) infiltration within the bronchial submucosa, and the increased DNA incorporation in ASM (P<0.05) and epithelial cell incorporation (P<0.05). SP600125 did not alter bronchial hyper-responsiveness observed after chronic allergen exposure. Pathways regulated by JNK positively regulate ASM cell proliferation and allergic cellular inflammation following chronic allergen exposure.

Topics: Actins; Animals; Anthracenes; Bronchi; Bronchoalveolar Lavage Fluid; Cell Count; Cell Division; Chronic Disease; Eosinophils; Hypersensitivity; Immunohistochemistry; Inflammation; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; Myocytes, Smooth Muscle; Ovalbumin; Rats; Rats, Inbred BN; Respiratory Mucosa