| Part I Effect of newly identified compound RCM1 on FOXM1 in airway club cells.Objectives: To confirm the inhibitory effect of RCM1, which can inhibit the expression of FOXM1 in vitro, on FOXM1 in airway club cells in vivo.Methods: The transgenic CCSP-rtTA,tg/-/tetO-FOXM1 tg/-(GFP-FOXM1) mice was generated. Mice were given doxycycline (Dox) in food chow to induce GFP-FOXM1 transgene. HDM extract was diluted in 100?l of saline and given by intra-nasal administration on days 0 and 14. RCM-1 was given to mice intraperitoneally (i.p.) on days 13, 15 and 16. Twenty-four hours after the last HDM challenge, lungs were harvested for preparation of frozen sections. RNA and total protein. Fluorescent images were obtained using a Zeiss Axioplan2 microscope equipped with an Axiocam MRm digital camera and Axiovision 4.3 software (Carl Zeiss). Real-time PCR,immunohistology (IHC) staining and Western Blot were performed. Animal studies were approved by the Animal Care and Use Committee of Cincinnati Children's Research Foundation.Results: FOXM1 was overexpressed in airway epithelial cells of GFP-FOXM1 transgenic mice after HDM challenge and Dox. Application of RCM-1 resulted in the exclusion of GFP-FOXM1 from the nuclei of bronchiolar epithelial cells. RCM-1 decreased the amounts and nuclear localization of GFP-FOXM1 transgenic protein in mice exposed to the aeroallergen house dust mite extract (HDM) as shown by immunostaining for FOXM1, Western blot and Real-time PCR.Conclusions: RCM-1 effectively decreased the abundance of human FOXM1 proteins in airway club cells of transgenic mice.Part ? Study of FOXM1 inhibitor RCM1 on goblet cell metaplasia and pulmonary inflammation in asthmatic mice.Objectives: To explore the effect of RCM1 on goblet cell metaplasia (GCM),pulmonary inflammation and airway hyperreactivity (AHR) in HDM-induced asthmatic mice.Methods: HDM extract was given into the airway of Balb/c mice by intra-nasal.RCM-1 was given to mice intraperitoneally (i.p.). Mice were divided into 4 groups:saline+vehicle, saline+RCM1, HDM+vehicle, HDM+RCM1. Twenty-four hours after the last HDM challenge, lungs were harvested for preparation of paraffin sections,total RNA. bronchial alveolar lavage fluid (BALF). The performed tests were as follows. (1) The expression of GCM related genes, including Fxom1, Spdef, Foxa2,Foxa3, Scgbla1, Agr2, Muc5ac,and inflammation related genes, including Ccl1,Ccl11, Ccl24, Ccr2, Ccr3, Cx-3cl1, Cx3cr1, IL-4, IL-5, IL-12p35, IL-13, IL-33, Acta2.Ptgs2. Ltc4s, were measured. (2) H&E, Alcian Blue and IHC staining were performed to assess GCM, mucus secretion, airway inflammation and the expression of FOXM1,SPDEF, MUC5AC, FOXA2 and CCSP in airway epithelial cells. Histological assessment and scoring were done using H&E staining slides. (3) Cells in BALF were counted and analyzed by Flow cytometry. (4) IFNy, IL-4, IL-5 and IL-13 in BALF were measured by Luminex Multiplex xMAP bead-based antibody assay. (5)FlexiVent system was used to measure airway mechanics. Methacholine was delivered using an Aeroneb nebulizer (SCIREQ).Results: (1) RCM1 effectively inhibited HDM induced GCM, reduced the mucous secretion and decreased the expression of Fxoml, Spdef, Foxa3, Agr2 and Muc5ac.RCM1 intended to upregulate the transcription of Foxa2 and Scgb1a1 and expression of FOXA2 and CCSP in airway epithelial cells. (2) RCM1 relieved HDM-induced pulmonary inflammation and reduced the expression of chemokines, including Ccl2,Ccl11, Ccl24, Ccr2, Ccr3, and cytokines, including IL-5, IL-13, IL-33. RCM1 also decreased the level of IL-4, IL-5, IL-13 and increased IFN? in BALF. Moreover,RCM1 reduced the HDM-induced high histology scores. (3) RCM-1 did not influence the total number of inflammatory cells in BALF, but the numbers of neutrophils and T lymphocytes were decreased after RCM-1 treatment. (4) RCM-1 decreased airway resistance ana increased the compliance in response to HDM exposure.Conclusions: RCM-1, the FOXM1 inhibitor , effectively prevented goblet cell metaplasia, reduced lung inflammation and decreased airway resistance in response to HDM. The results of our studies suggest that RCM1 maybe a potential compound to treat asthmatic patients.Part ? The FOXM1 Inhibitor RCM-1 Protects lungs from Goblet Cell Metaplasia by inhibting IL-13/STAT6 SignalingObjectives: To find out the mechanism of the therapeutical effect of RCM1 on airway goblet cell metaplasia, pulmonary inflammation and AHR.Methods: 1. Experiments in vivo: Because the IL-13/STAT6 signaling pathway induces goblet cell metaplasia and IL-13 is a key cytokine in the pathogenesis of allergic asthma, we tested IL-13/STAT6 signaling pathway. Recombinant mouse IL-13 was given into the airway of Balb/c mice by intra-nasal. RCM-1 was given to mice intraperitoneally (i.p.). Mice were divided into 4 groups: saline+vehicle,saline+RCM1, IL-13+vehicle, IL-13+RCM1. Twenty-four hours after the last HDM challenge, lungs were harvested for preparation of paraffin sections, total RNA and protein, bronchial alveolar lavage fluid (BALF). The performed tests were as follows.(1) The expression of GCM related genes, including Fxoml, Spdef, Foxa2, Foxa3,Scgh1a1, Agr2, Muc5ac, and inflammation related genes, including Ccl2, Ccl11,Ccl20,Ccl24, Ccr3. Ccr6, Cx3cll, Cx3crl,Ifng,IL-4, IL-5, IL-12p35, IL-13,IL-25, IL-33,Acta2, Ptgs2, Ltc4s were measured. (2) H&E, Alcian Blue and IHC staining were performed to assess GCM, mucus secretion, airway inflammation and the expression of FOXM1, SPDEF, MUC5AC and FOXA2 in airway epithelial cells. (3) Cells in BALF were counted and analyzed. IFNy, IL-4, IL-5 and IL-13 in BALF were measured by Luminex Multiplex xMAP bead-based antibody assay. (4) FOXM1,STAT6, p-STAT6, ERK1/2, p-ERK1/2, AKT, p-AKT, ERK5, p-ERK5, 14-3-3 in total lung were measured by Western blot. (5) FlexiVent system was used to measure airway mechanics. Methacholine was delivered using an Aeroneb nebulizer(SCIREQ). (6) Systemic toxicity of RCM-1 was assessed by intestinal morphology and measurement of serum concentrations of total protein, albumin, liver enzymes AST and ALT, alkaline phosphatase (ALK), blood urea nitrogen (BUN) and creatine phosphokinase (CPK). 2, Experiments in vitro: Normal human bronchial epithelial cells (NHBE) were cultured in air-liquid interface. Cells were grown submerged in growth basal medium, After 2 days, cells were exposed to air, and B-ALITM differentiation medium was added to the basal chamber. All cultures were maintained at 37? in a humidified atmosphere of 5% C02. One hour after RCM-1 treatment (1?M), cells were treated with recombinant human IL-13 (lOng/ml) and harvested 2-24 hours later. FOXM1,STAT6, p-STAT6, ERK1/2, p-ERK1/2, AKT,p-AKT,ERK5,p-ERK5, 14-3-3 were measured by Western blot.Results: (1) RCM1 effectively inhibited IL-13-induced GCM, reduced the mucous oversecretion and decreased the expression of Fxoml, Spdef, Foxa3, Muc5ac and immunostaning for FOXM1, SPDEF and MUC5AC. RCM1 did not change the gene transcription and protein expression of Foxa2. RCM1 also did not alter the IL-13-induced upregulation of Agr2 and downregulation of Scgblal. (2) RCM1 relieved IL-13-induced airway inflammation and reduced the expression of chemokines, including Ccl2, CCl11, Ccl24 and cytokines, including IL-4, IL-5, IL-13,IL-25, IL-33. RCM1 also decreased the level of IL-4, IL-5, but not IL-33 in BALF.IL-13 reduced the expression of Ifng but RCM1 did not change it. Application of IL-13 and RCM1 did not change the expression of Ccl20, Ccr3, Ccr6, Cx3cll, Cx3cr1,Ifng, IL-12p35. Acta2, Ptgs2. (3) Either IL-13 or RCM-1 did not influence the number of inflammatory cells in BALF. (4) RCM-1 decreased airway resistance and increased the compliance in response to IL-13 exposure. (5) RCM-1 decreased the IL-13-induced expression of FOXM1 in mouse lung and NHBE cells. RCM1 also reduced the phosphorylation and total abundance of ERK1/2 in both IL-13-treated and control lungs and in NHBE cells independently of IL-13. RCM-1 did not alter the phosphorylation or total abundance of AKT or the total abundance of ERK5 and 14-3-3 in mouse lung tissue and in NHBE cells. (6) There was no evidence of systemic toxicity as assessed by intestinal morphology and serological tests of total protein, albumin. AST, ALT. ALK, BUN and CPK.Conclusions: RCM-1 suppressed goblet cell metaplasia, reduced airway inflammation and decreased airway resistance through inhibiting the FOXM1 expression and then blocking IL-13/STAT6 signaling pathway. These results of our studies strongly suggest that RCM1 is a nontoxic small molecular compound that has potential to be used for asthmatic patients. |
PDF Full Text Request