ABSTRACT
BACKGROUND: Airway remodeling is one of the reasons for severe steroidresistant asthma related to HMGB1/RAGE signaling or Th17 immunity. OBJECTIVE: Our study aims to investigate the relationship between the HMGB1/RAGE signaling and the Th17/IL-17 signaling in epithelial-mesenchymal transformation (EMT) of airway remodeling. METHODS: CD4+ T lymphocytes were collected from C57 mice. CD4+ T cell and Th17 cell ratio was analyzed by flow cytometry. IL-17 level was detected by ELISA. The Ecadherin and α-SMA were analyzed by RT-qPCR and immunohistochemistry. The Ecadherin, α-SMA, and p-Smad3 expression were analyzed by western blot. RESULTS: The HMGB1/RAGE signaling promoted the differentiation and maturation of Th17 cells in a dose-dependent manner in vitro. The HMGB1/RAGE signaling also promoted the occurrence of bronchial EMT. The EMT of bronchial epithelial cells was promoted by Th17/IL-17 and the HMGB1 treatment in a synergic manner. Silencing of RAGE reduced the signaling transduction of HMGB1 and progression of bronchial EMT. CONCLUSION: HMGB1/RAGE signaling synergistically enhanced TGF-ß1-induced bronchial EMT by promoting the differentiation of Th17 cells and the secretion of IL-17.
ABSTRACT
BACKGROUND: Natural cytotoxicity receptor negative innate lymphoid cell (NCR- ILC3) involves into mucosal homeostasis, inflammation regulation and tissue remodeling. The proportion of NCR- ILC3 is increased in the lung of smokers with chronic obstructive pulmonary disease (COPD). However, there's still few understandings on the role of NCR- ILC3 in COPD pathogenesis. METHODS: COPD mice were induced by cigarette smoking. The pathology in lung was detected in histology. The frequency of NCR- ILC3 (CD3-CD45+RORγt+NkP46-) from murine lung was detected using flow cytometry. IL-17+RORγt+ double positive cells in lung were assessed by double immunofluorescence staining. The protein expressions of epithelial-to-mesenchymal transition (EMT) markers, namely E-cadherin and Vimentin, were assessed using immunohistochemistry staining and western blotting. RESULTS: The frequency of NCR- ILC3 in lung was higher in COPD group than controls. The IL-17+RORγt+ cells in lung from COPD mice were more than controls. E-cadherin expression was decreased but Vimentin expression was increased in lung of COPD mice, when compared with controls. The frequency of NCR- ILC3 in lung tissues were positively correlated with mean linear intercept in lung, destructive index in lung and EMT, respectively. CONCLUSIONS: NCR- ILC3 could contribute to emphysema and EMT in lung of cigarette smoking-induced COPD, which will provide further understanding on COPD pathogenesis of immune response.
Subject(s)
Cigarette Smoking , Pulmonary Disease, Chronic Obstructive , Mice , Animals , Interleukin-17/metabolism , Lymphocytes , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Vimentin/metabolism , Immunity, Innate , Cigarette Smoking/adverse effects , Pulmonary Disease, Chronic Obstructive/etiology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Lung/pathology , Cadherins/metabolismABSTRACT
Background: Atrial natriuretic peptide (ANP) inhibits TGF-ß1-induced epithelial-mesenchymal transition (EMT) in human airway cells. We aim to explore the role and mechanism of ANP on EMT of bronchial epithelial cells from murine model of allergic asthma in vitro. Methods: Murine model of allergic asthma was established with BALB/c mice using ovalbumin (OVA). Bronchial epithelial cells were isolated from OVA-exposed mice, and then were cocultured with TGF-ß1, ANP, natriuretic peptide receptor A antagonist, cGMP analog, cGMP inhibitor or/and protein kinase G (PKG) inhibitor, respectively. We assessed expressions of E-Cadherin, α-SMA, cGMP, Smad3 and p-Smad3 in the murine cells before and after Smad3 silence. Results: Compared with bronchial epithelial cells from controls and OVA-exposed mice without additional stimulation, the mRNA and protein expressions of E-Cadherin were decreased but α-SMA expressions were increased in cells with TGF-ß1 stimulation from OVA-exposed mice in vitro. That could be reversed by ANP. The effect of ANP could be mimicked by the cGMP analog, which could be reversed by cGMP or PKG inhibitor. Moreover, the phosphorylated Smad3 expression was consistent with that of α-SMA. When Smad3 was silenced, Smad3 was mostly expressed in cytoplasm. In contrast, it is mostly expressed in nucleus of non-silenced cells during EMT. Conclusions: In a murine model of allergic asthma, ANP could inhibit TGF-ß1-induced EMT of bronchial epithelial cells through cGMP/PKG signaling, targeting TGF-ß1/Smad3 via attenuating phosphorylation of Smad3 in vitro, which may provide potential of ANP in treating allergic asthma with airway remodeling.
Subject(s)
Asthma/drug therapy , Atrial Natriuretic Factor/pharmacology , Bronchi/drug effects , Epithelial Cells/drug effects , Epithelial-Mesenchymal Transition/drug effects , Signal Transduction/drug effects , Smad3 Protein/metabolism , Airway Remodeling/drug effects , Animals , Asthma/metabolism , Bronchi/metabolism , Cadherins/metabolism , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Epithelial Cells/metabolism , Female , Hypersensitivity/drug therapy , Hypersensitivity/metabolism , Mice , Mice, Inbred BALB C , Ovalbumin/pharmacology , Phosphorylation/drug effects , Transforming Growth Factor beta1/metabolismABSTRACT
Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-ß1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA expressions were increased after induction by TGF-ß1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and α-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased α-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with α-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-ß1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-ß1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.
