RESUMO
During tissue regeneration and wound healing of the skin, migration, proliferation and differentiation of keratinocytes are important processes. Here we assessed the effect of a neuropeptide, bombesin, on keratinocytes during regeneration from scratch wounding. Bombesin purified from amphibian skin, is homologous of mammalian gastrin-releasing peptide and is active in mammals. Its pharmacological effects mediate various physiological activities: hypertensive action, stimulating action on gastric secretion, hyperglycemic effect or increased insulin secretion. In vitro it shows a hyperproliferative effect on different experimental models and is involved in skin repair. The aim of this study was to elucidate the effect of Bombesin in an in vitro experimental model on a mechanically injured human keratinocyte monolayer. We evaluated different mediators involved in wound repair such as IL-8, TGFbeta, IL-1, COX-2, VEGF and Toll-like receptors 2 and 4 (TLR2 and TLR4). We also studied the effects of bombesin on cell proliferation and motility and its direct effect on wound repair by observing the wound closure after mechanical injury. The involvement of the bombesin receptors neuromedin receptor (NMBR) and gastrin-releasing peptide receptor (GRP-R) was also evaluated. Our data suggest that bombesin may have an important role in skin repair by regulating the expression of healing markers. It enhanced the expression of IL-8, TGFbeta, COX-2 and VEGF. It also enhanced the expression of TLR2, while TLR4 was not expressed. Bombesin also increased cell growth and migration. In addition, we showed that NMBR was more involved in our experimental model compared to GRP-R.
Assuntos
Bombesina/farmacologia , Bombesina/fisiologia , Cicatrização/efeitos dos fármacos , Animais , Anuros , Bombesina/isolamento & purificação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Corantes/metabolismo , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Interleucina-18/metabolismo , Interleucina-8/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Receptores da Bombesina/análise , Receptores da Bombesina/metabolismo , Fatores de Tempo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Azul Tripano/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/fisiologiaRESUMO
BACKGROUND: Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. PT is from the V2 region of HIV-1 gp120, an exterior envelope glycoprotein that is a target for host immune responses. The anti-inflammatory mechanisms of PT are not well understood. OBJECTIVES: We studied the immunomodulatory effects of PT on the human keratinocyte cells. METHODS: Cultured human keratinocytes were treated with PT, proteins extracted and analysed by Western blotting and reverse transcriptase polymerase chain reaction. RESULTS: Our findings show reduced expression of intercellular adhesion molecule 1 and an increase in transforming growth factor (TGF)-beta and heat shock protein (HSP)-70 in human keratinocyte cells treated with PT. The HSP-70 increase is modulated by TGF-beta. In fact, we demonstrated that anti-TGF-beta antibodies reduce HSP-70 overexpression. In addition, we show a modulation of alphav integrins after 4 hours of treatment with PT. These receptors favour keratinocyte migration and epidermal regeneration. It has been reported that overexpression of HSP results in dramatic changes to intermediate filaments. These proteins act on keratin intermediate filaments and determine their retraction. The consequence is cell-cell contact detachment and inhibition of cellular hyperproliferation. CONCLUSIONS: Our results support the beneficial effect of PT found in vivo, suggesting, moreover, the primary role of keratinocytes upon which PT acts directly by stimulating the anti-inflammatory function and favouring the regeneration of tissue.
Assuntos
Queratinócitos/efeitos dos fármacos , Peptídeo T/farmacologia , Adulto , Western Blotting , Técnicas de Cultura de Células , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Cadeias alfa de Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta1RESUMO
The lipophilic yeast Malassezia furfur is a member of the cutaneous microbiota, also associated with several chronic diseases such as pityriasis versicolor, folliculitis, seborrhoeic dermatitis, and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. In this study we determined the immunomodulatory and invasive capacity of M. furfur in a human keratinocyte cell culture, HaCat. At a yeast cell to HaCat ratio of 30:1, M. furfur penetration was only 30% with poor phagolysosome fusion and with cytoskeleton modification. Transglutaminase I gene expression was also inhibited, supporting the hypothesis that M. furfur causes an initial break in the barrier function of the epidermis. Moreover, we demonstrated that M. furfur modulates proinflammatory and immunomodulatory cytokine synthesis by downregulating IL-1alpha and by inhibiting IL-6 and TNF-alpha and by upregulating IL-10 and TGF-beta1. The suppressed inflammatory response induced by M. furfur may play a role in chronic disease.