RESUMEN
Cell penetrating peptide derived from human eosinophil cationic protein (CPPecp) is a 10-amino-acid peptide containing a core heparan sulfate (HS)-binding motif of human eosinophil cationic protein (ECP). It binds and penetrates bronchial epithelial cells without cytotoxic effects. Here we investigated airway-protective effects of CPPecp in BEAS-2B cell line and mite-induced airway allergic inflammation in BALB/c mice. In BEAS-2B cell, CPPecp decreases ECP-induced eotaxin mRNA expression. CPPecp also decreases eotaxin secretion and p-STAT6 activation induced by ECP, as well as by IL-4. In vivo studies showed CPPecp decreased mite-induced airway inflammation in terms of eosinophil and neutrophil count in broncho-alveolar lavage fluid, peri-bronchiolar and alveolar pathology scores, cytokine production in lung protein extract including interleukin (IL)-5, IL-13, IL-17A/F, eotaxin; and pause enhancement from methacholine stimulation. CPPecp treated groups also showed lower serum mite-specific IgE level. In this study, we have demonstrated the in vitro and in vivo anti-asthma effects of CPPecp.
Asunto(s)
Antiasmáticos/farmacología , Asma/tratamiento farmacológico , Péptidos de Penetración Celular/farmacología , Proteína Catiónica del Eosinófilo/química , Mucosa Respiratoria/efectos de los fármacos , Alérgenos/inmunología , Animales , Antiasmáticos/uso terapéutico , Antígenos Dermatofagoides/inmunología , Asma/inmunología , Asma/patología , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/inmunología , Línea Celular , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/uso terapéutico , Citocinas/inmunología , Citocinas/metabolismo , Evaluación Preclínica de Medicamentos , Eosinófilos/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: We evaluated the effects of T helper cell differentiation in a mite-allergic animal model treated with inhaled heparins of different molecular weight. METHOD: BALB/c mice were divided into four groups: 1. Control, 2. Mite intratracheal (mIT), 3. Inhaled heparin (hIN), 4. Inhaled low-molecular-weight heparin (lmwhIN). Groups 2, 3, and 4 were sensitized twice with Der p allergen subcutaneously on day 1 and day 8. Der p allergen was administered intratracheally on day 15. Groups 3 and 4 were treated with heparin or low-molecular-weight (lmw) heparin intranasally from day 1 to 22. Splenocytes from sacrificed mice stimulated with 16 µg/ml of Der p were cultured for 72 hours. Supernatants of splenocyte were collected to analyze the effect of Interleukin (IL)17-A/F, Interferon(IFN)-γ, IL-4, IL-13, and IL-10. Serum was also collected for Der P-specific IgE level on day 23. Total RNA was extracted from spleen tissue for mRNA expression. Gene expression of Foxp3, IL-10 IFN-γ, GATA3, IL-5, and RORγt were analyzed. RESULTS: Both hIN and lmwhIN groups had lower serum IgE level than that of the mIT group (both p<0.0001). Both hIN and lmwhIN groups showed significantly decreased transcripts of GATA-3, IFN-γ, IL-5, and RORγt mRNA in their spleen. Regarding the supernatant of splenocyte culture stimulated with Der p, compared with the mIT group, there were significant decreases in IL-17A/F, IFN-γ, IL-4, IL-13, and IL-10 secretion in inhaled hIN and lmwhIN groups. CONCLUSIONS: From this balb/c mice study, the analyses of mRNA and cytokines revealed that both intranasal heparin and lmw heparin treatment decreased the expression of Th1, Th2, and Th17 in spleen. The underlying mechanism(s) warrant further studies.