A luz intensa pulsada é amplamente utilizada para o tratamento do fotoenvelhecimento e de lesões vasculares congênitas e adquiridas, porém, seus efeitos biológicos ainda não são completamente esclarecidos. Neste estudo, foram avaliadas as respostas proliferativas e de síntese em fibroblastos e células endoteliais humanas após a irradiação com a luz intensa pulsada. As culturas de fibroblastos e células endoteliais foram irradiadas com luz intensa pulsada com variação de comprimento de onda entre 420 e 1110 nm e fluências de 10, 16 e 20 J/cm2. Os experimentos foram realizados após 24 e 48 horas da irradiação e analisados os seguintes parâmetros: 1-alterações morfológicas; 2- determinação da capacidade proliferativa e citotóxica; 3- produção de radicais livres; 4- fases do ciclo celular; 5- expressão dos marcadores do ciclo celular e apoptose; 6- avaliação do potencial elétrico da membrana mitocondrial; 7- análise da atividade mitocondrial por microscopia confocal; 8- biossíntese de colágeno e 9- microscopia eletrônica de varredura dos fibroblastos e matriz extracelular. Os resultados demonstraram que, nos fibroblastos, a luz intensa pulsada promoveu aumento da densidade celular e da capacidade proliferativa; aumento na produção de radicais livres diretamente proporcional à intensidade de energia; estímulo na fase de síntese; estímulo na síntese de colágeno mais significativo em 10 e 16 J/cm²; induziu apoptose pela via extrínseca, dependente da intensidade de energia; a microscopia eletrônica de varredura demonstrou detalhes da organização e síntese da matriz extracelular, bem como o colágeno recém sintetizado. Nas células endoteliais a luz intensa pulsada não provocou mudanças morfológicas; promoveu aumento na produção de radicais livres, induziu citotoxicidade e apoptose pela via extrínseca e diminuiu a capacidade proliferativa, proporcional à intensidade de energia. Este estudo permitiu a determinação in vitro dos mecanismos biológicos envolvidos após a interação da luz intensa pulsada com os fibroblastos e células endoteliais humanas / Intense pulsed light is widespread used in the treatment of intrinsic and extrinsic photo damaged skin and congenital or acquired vascular diseases but its biological effects have not yet been clearly demonstrated. This study assessed the proliferative and synthesis responses of fibroblasts and endothelial cells. The fibroblasts and endothelial cells cultures were irradiated with intense pulsed light, with wavelength ranging from 420 to 1110 nm, fluencies of 10, 16 e 20 J/cm2 and pulse delay 10 ms. Experiments were performed 24 and 48 hours after irradiation and the following parameters were analyzed: 1- morphological changes; 2- measurement of proliferative capacity and citotoxicity; 3- free radical production; 4- cell cycle phases; 5- expression of cell cycle and apoptosis markers; 6- electrical potential of mitochondrial membrane; 7- mitochondrial activity by confocal microscopy; 8- collagen synthesis; 9- Scanning electron microscopy of fibroblasts and extracellular matrix. The results evidenced that intense pulsed light on fibroblasts increased cell density and proliferative capacity; stimulus on synthesis phase and collagen synthesis more significant at fluences of in 10 and 16 J/cm²; increase of free radical formation, citotoxicity and apoptosis through the extrinsic pathway proportional to the energy intensity. The scanning electron microscopy evidenced the organization details of extracellular matrix and new collagen fibers. Regarding the endothelial cells, intense pulsed light did not cause morphological changes; increased free radicals formation, induced citotoxicity and apoptosis through the extrinsic pathway and decreased the proliferative capacity proportionally to the intensity of energy. This study allowed the in vitro determination of the biological mechanisms involved after the interaction of intense pulsed light and human fibroblasts and endothelial cells

Le photo-vieillissement cutané est en partie dû aux effets néfastes des rayonnements UV qui induisent un stress oxydant. Celui-ci joue un rôle promoteur dans l’installation d’un statut micro-inflammatoire lié à la production de médiateurs tels que des cytokines pro-inflammatoires, des métalloprotéinases de la matrice extracellulaire (MMPs) et des prostaglandines E2. Mes travaux ont été effectués sur le kératinocyte, cellule majeure de l’épiderme et acteur du processus de vieillissement cutané. Dans une première partie, nous décrivons différentes expériences menées pour évaluer l’association de plusieurs molécules anti-oxydantes connues afin d’en potentialiser l’activité. Cette étude a mis en évidence que dans certains cas, un effet pro-oxydant pouvait être observé. Dans une seconde partie, grâce à un système de multiplexing, nous avons pu identifier 39 cytokines et 4 MMPs sécrétées par les kératinocytes suites à une irradiation par des UVB. Nous avons également pu mettre en évidence une différence entre donneurs jeunes et âgés, avec une augmentation des cytokines pro-inflammatoires et une diminution des cytokines anti-inflammatoires pour ces derniers. Dans une dernière partie, l’activité d’un extrait de fleurs de Butea monosperma a été étudiée. L’extrait présente une activité anti-oxydante en piégeant les EORs intracellulaires, anti-inflammatoire en diminuant la sécrétion de cytokines pro-inflammatoires (IL-1; IL-6 et IL-8) et protectrice du derme en inhibant la production de MMP-1 ; 2 ; 9 et 10. Cette étude multifactorielle a permis d’expliquer les utilisations traditionnelles de cette plante comme anti-inflammatoire. En conclusion, afin de comprendre et de limiter les effets des radiations UVB sur le vieillissement cutané, il est nécessaire d’avoir une approche multifactorielle. Comme nous l’avons observé, le stress oxydant n’est pas le seul responsable du statut micro-inflammatoire cutané mais en est le promoteur. Il faut donc agir en amont en limitant la production d’EORs et en aval en contrôlant la sécrétion de cytokines pro-inflammatoires et de MMPs. / Early signs of skin aging are related amongst others by UV irradiation that induces an oxidative stress. This one is associated with a skin micro-inflammatory status which is the consequence of mediator productions as pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and prostaglandin E2. Studies reported in this thesis were mainly conducted with human keratinocyte, one of the cells involved in the skin aging process. In a first part, we describe various experiments led to evaluate the association of several antioxydant molecules in the aim to increase their activities. This study has shown that in certain cases, a pro-oxidizing effect could be observed. In a second part, through a multiplexing system, we have identified 39 cytokines and 4 extracellular MMPs that are secreted upon UVB irradiation. We have also found a difference between the cytokines secreted by keratinocytes from young and old donors; the latter has an increase of pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines. Finally, in a last part, an extract of flowers of Butea monosperma has been studied. The extract present an anti-oxidant activity by scavenging intracellular ROS, an anti-inflammatory effect by reducing the secretion of pro-inflammatory cytokines (IL-1 , IL-6 and IL-8) and a protective effect of the dermis by inhibiting the production of extracellular MMP-1, 2, 9 and 10. These results explain the strong protective anti-inflammatory activity of this plant which is widely used. In conclusion, in order to understand and limit the impact UVB radiations on skin aging, it is necessary to have a multifactorial approach. As we have observed, oxidative stress is not solely responsible for the micro-inflammatory status associated with photo-aging, but is the proponent. We must act upstream by limiting production of ROS and downstream by controlling the secretion of pro-inflammatory cytokines and MMPs.

A luz intensa pulsada é amplamente utilizada para o tratamento do fotoenvelhecimento e de lesões vasculares congênitas e adquiridas, porém, seus efeitos biológicos ainda não são completamente esclarecidos. Neste estudo, foram avaliadas as respostas proliferativas e de síntese em fibroblastos e células endoteliais humanas após a irradiação com a luz intensa pulsada. As culturas de fibroblastos e células endoteliais foram irradiadas com luz intensa pulsada com variação de comprimento de onda entre 420 e 1110 nm e fluências de 10, 16 e 20 J/cm2. Os experimentos foram realizados após 24 e 48 horas da irradiação e analisados os seguintes parâmetros: 1-alterações morfológicas; 2- determinação da capacidade proliferativa e citotóxica; 3- produção de radicais livres; 4- fases do ciclo celular; 5- expressão dos marcadores do ciclo celular e apoptose; 6- avaliação do potencial elétrico da membrana mitocondrial; 7- análise da atividade mitocondrial por microscopia confocal; 8- biossíntese de colágeno e 9- microscopia eletrônica de varredura dos fibroblastos e matriz extracelular. Os resultados demonstraram que, nos fibroblastos, a luz intensa pulsada promoveu aumento da densidade celular e da capacidade proliferativa; aumento na produção de radicais livres diretamente proporcional à intensidade de energia; estímulo na fase de síntese; estímulo na síntese de colágeno mais significativo em 10 e 16 J/cm²; induziu apoptose pela via extrínseca, dependente da intensidade de energia; a microscopia eletrônica de varredura demonstrou detalhes da organização e síntese da matriz extracelular, bem como o colágeno recém sintetizado. Nas células endoteliais a luz intensa pulsada não provocou mudanças morfológicas; promoveu aumento na produção de radicais livres, induziu citotoxicidade e apoptose pela via extrínseca e diminuiu a capacidade proliferativa, proporcional à intensidade de energia. Este estudo permitiu a determinação in vitro dos mecanismos biológicos envolvidos após a interação da luz intensa pulsada com os fibroblastos e células endoteliais humanas / Intense pulsed light is widespread used in the treatment of intrinsic and extrinsic photo damaged skin and congenital or acquired vascular diseases but its biological effects have not yet been clearly demonstrated. This study assessed the proliferative and synthesis responses of fibroblasts and endothelial cells. The fibroblasts and endothelial cells cultures were irradiated with intense pulsed light, with wavelength ranging from 420 to 1110 nm, fluencies of 10, 16 e 20 J/cm2 and pulse delay 10 ms. Experiments were performed 24 and 48 hours after irradiation and the following parameters were analyzed: 1- morphological changes; 2- measurement of proliferative capacity and citotoxicity; 3- free radical production; 4- cell cycle phases; 5- expression of cell cycle and apoptosis markers; 6- electrical potential of mitochondrial membrane; 7- mitochondrial activity by confocal microscopy; 8- collagen synthesis; 9- Scanning electron microscopy of fibroblasts and extracellular matrix. The results evidenced that intense pulsed light on fibroblasts increased cell density and proliferative capacity; stimulus on synthesis phase and collagen synthesis more significant at fluences of in 10 and 16 J/cm²; increase of free radical formation, citotoxicity and apoptosis through the extrinsic pathway proportional to the energy intensity. The scanning electron microscopy evidenced the organization details of extracellular matrix and new collagen fibers. Regarding the endothelial cells, intense pulsed light did not cause morphological changes; increased free radicals formation, induced citotoxicity and apoptosis through the extrinsic pathway and decreased the proliferative capacity proportionally to the intensity of energy. This study allowed the in vitro determination of the biological mechanisms involved after the interaction of intense pulsed light and human fibroblasts and endothelial cells