Orientador: Mary Luci de Souza Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T21:16:10Z (GMT). No. of bitstreams: 1 Dieamant_GustavodeCampos_D.pdf: 11544856 bytes, checksum: 05f884d1804a10bd91e58e71c4df4681 (MD5) Previous issue date: 2008 / Resumo: O envelhecimento cutâneo é resultado de dois processos independentes, clinica e biologicamente distintos, que afetam a pele simultaneamente. Ao primeiro, conhecido como "relógio biológico", damos o nome de envelhecimento intrínseco, o qual afeta a pele na mesma proporção em que órgãos internos são afetados, como por exemplo, a lenta e irreversível degeneração tecidual. O segundo, denominado fotoenvelhecimento, é resultado da exposição da pele a agentes agressores ambientais, primeiramente radiação ultravioleta (RUV). Sobre o impacto da RUV, especialmente RUVA e RUVB, a pele passa por inúmeras alterações, incluindo hiperpigmentação, fotodano, alterações histológicas e imunológicas. Tendo em vista a complexidade do fotoenvelhecimento, podemos dividir este trabalho em duas partes, onde avaliamos em uma primeira etapa os efeitos da RUV sobre a resposta neuroimuno-endocrinológica da pele através da mensuração de parâmetros específicos em culturas de fibroblastos e queratinocitos humanos. Após a realização dos experimentos propostos para a primeira parte deste trabalho, passamos a considerar para a segunda etapa, a possível ação in vitro e in vivo do extrato de Rhodiola rosea (ERR) em associação com o dipeptídeo Lcarnosina (LC) sobre a reversão ou controle dos efeitos deletérios da RUV sobre a pele. Nossos resultados demonstram que durante a foto-exposição, culturas celulares in vitro apresentaram importantes alterações nos parâmetros avaliados, especialmente após a exposição crônica, dentre elas: redução na produção de IL-1a, TNF-a e IFN-g, redução nos níveis de b-endorfina e encefalina, aumento na atividade de metaloproteinases (MMPs), inibição na atividade do inibidor tecidual de metaloproteinase 1 (TIMP-1), redução nos níveis de colágeno e elastina e redução na atividade das enzimas superóxido dismutase (SOD) e catalase (CAT). No perfil de resposta das células tratadas com ERR, pudemos observar uma importante função adaptógena do extrato, onde na maioria dos parâmetros, constatamos uma modulação da resposta celular de forma dose dependente. Os resultados obtidos com L-carnosina foram importantes, especialmente no que diz respeito aos níveis de colágeno, elastina e atividade das enzimas antioxidantes. Quando o perfil de resposta da associação entre ambos (ARRLC) é observado, pode-se perceber que, em alguns parâmetros, não houve uma potencialização dos efeitos. No entanto, dada a complexidade da resposta neuro-imuno-endocrinológica da pele relacionada ao fotoenvelhecimento, pode-se concluir que a utilização de compostos com mecanismos de ação distintos é imprescindível, uma vez que diferentes processos metabólicos levam ao dano tecidual e à aparência estética comprometida da pele fotoenvelhecida. Para a realização dos estudos clínicos, uma emulsão placebo ou emulsão contendo 1% de ARRLC foi utilizada pelas voluntárias em dois estudos independentes, sendo que o primeiro foi realizado durante 28 dias para avaliação dos parâmetros subjetivos - conforto da pele, redução da sensação de desconforto após Stinging-Test (ST) e melhora do ressecamento da pele. O segundo estudo foi realizado durante 56 dias, onde avaliou-se em D1, D28 e D56, a melhora do relevo cutâneo e a redução da perda de água transepidérmica. Os resultados obtidos demonstram que aproximadamente 90% das voluntárias reportaram uma melhora do conforto da pele e 75% reportaram redução do ressecamento. Além disso, a utilização da emulsão contendo 1% de ARRLC apresentou uma redução de aproximadamente 36% na sensação de desconforto após ST. Além disso, o tratamento com a emulsão contendo 1% de ARRLC resultou em redução considerável da perda de água transepidérmica e na melhora do relevo cutâneo. Estes resultados clínicos, associados aos resultados obtidos in vitro, nos encorajam a sugerir o uso de ARRLC como alternativa terapêutica no tratamento tópico do fotoenvelhecimento. / Abstract: Cutaneous ageing is a complex biological phenomenon consisting of two components; intrinsic ageing, which is largely genetically determined and extrinsic ageing caused by environmental exposure, primarily UV light. Under the impact of ultraviolet radiation (UVR), skin undergoes several changes, including enhanced pigmentation, photodamage and immunosuppression. Because photoaging culminates in important alterations in skin function and appearance, we can separate this work in two different phases. In the first phase, we evaluated the effects of ultraviolet radiation (UVR) on skin neuro-immuneendocrinological response through the measurement of specific parameters in fibroblasts and keratinocytes human cell cultures. After the conclusion of experiments proposed for the first phase of this work, the possible in vitro and in vivo effects of Rhodiola rosea extract (ERR) in combination to di-peptide Lcarnosine (LC) in the control of deleterious effects of UVR exposure on the skin were evaluated. Our results demonstrated that during the UVR, the cell cultures promoted crucial alterations in the parameters evaluated, mainly after chronic exposure. The alterations observed were: the reduction in the production of IL-1a, TNF-a, IFN-g, b-endorphin and enkephalin, reduction in the levels of matrix metalloproteinase tissue inhibitor-1, increase of metalloproteinase activity, reduction of collagen and elastin production and inhibition of the antioxidant enzymes superoxide dismutase and catalase. In the cell groups that received ERR, we observed an important adaptogenic function of the extract, because in the majority of the parameters, it was able to modulate the cell response in a dose-dependent manner. The results obtained in the LC treated groups, we could also observe important effects, mainly in collagen and elastin synthesis and in the antioxidant enzymes activities. When we consider the response obtained with the association of both ERR and LC (ARRLC), we could see that it is not able to enhance the effects obtained with ERR or LC separately. Therefore, due to the complexity of the neuro-immune-endocrinological response during the photoaging, we can conclude that the use of compounds with different mechanisms of action is crucial, because distinct metabolic process lead to tissue damage and terrible appearance of photodamaged skin. For the clinical trials, a placebo emulsion or a emulsion containing 1% of ARRLC were used by the volunteers in two independent studies, in which the first one was performed during 28 days for evaluation of subjective parameters, including skin comfort, reduction of skin dryness sensation and reduction of skin discomfort sensation after Stinging-Test (ST). The second study was performed during 56-days and the evaluations were carried out on D1, D28 and D56. The parameters evaluated were the improvement of cutaneous relief and the reduction of transepidermal water loss. The obtained results demonstrated that approximately 90% of the volunteers perceived an increase in skin comfort and 75% reported reduction of skin dryness sensation. In addition, the emulsion containing 1% of ARRLC was able to reduce in approximately 36% the sensation of discomfort after ST. These clinical results and the in vitro findings, encourage us to suggest the use of ARRLC as alternative ingredient in the topical treatment of skin photoaging. / Doutorado / Doutor em Farmacologia

In man the metabolic heat produced during physical exercise stresses the thermoregulatory system, particularly if hot, humid environmental conditions prevail. It has recently been postulated that endogenous opioids may play a role in regulating body temperature at rest and because it has also been shown that blood levels of these substances increase during exercise, the possibility exists that endogenous opioids may play a role in thermoregulation during exercise. A study was conducted in two parts to determine the thermoregulatory response during exercise with and without pharmacologic blockade of the opioid receptor. In Part I nine healthy male subjects performed 30 minutes cycling at 50 % maximal aerobic capacity in an environmentally controlled laboratory. The subjects received either placebo, 2mg or 10mg naloxone hydrochloride in a randomized double-blind crossover fashion prior to the exercise test. Rectal temperatures were recorded at one-minute intervals and cardiorespiratory parameters were measured during the test. Water loss was calculated from differences in nude body weight. In part II eight male subjects performed a graded maximal cycle ergometer test after receiving either placebo or 2mg naloxone in a randomized double-blind crossover fashion. Rectal and sublingual temperatures were recorded before and after the test and oesophageal temperature was recorded at one-minute intervals during the test. Cardiorespiratory parameters were recorded during the test. The results of Part I show that rises in rectal temperature as well as calculated water losses were similar for placebo and after the administration of both 2mg and 10mg naloxone. Similarly, during maximal exercise (Part II) the rise in rectal and oesophageal temperatures was equivalent for placebo and 2mg naloxone but sublingual temperature failed to rise during exercise following the 2mg naloxone dose. Cardiorespiratory responses did not differ between placebo and naloxone tests in both Part I and Part II of the study. These results indicate that naloxone-mediated blockade of opioid receptors does not affect rectal and oesophageal temperature responses to either submaximal or maximal exercise. Naloxone appears to selectively alter the sublingual temperature response to exercise possibly by altering local blood flow. It is concluded that insofar as naloxone induced opioid receptor blockade provides a measure of the function of the endogenous opioid system, this study suggests that the endogenous opioid system does not play a significant role in thermoregulation during exercise.

Summary: The effects of an alpha2 adrenoceptor blocker, yohimbine, and an alpha1 adrenoceptor blocker, phenoxybenzamine, and the central alpha2 adrenoceptor agonist, clonidine, on changes in arterial blood pressure and heart rate were studied during fatiguing muscular contractions to determine whether an adrenergic-opioidergic system might be involved in the mediation of cardiovascular function. Fatiguing contractions of the gastrocnemius and plantaris muscles of cats caused an increase in mean arterial blood pressure to 150-170 mmHg from resting values of 110-120 mmHg. Injection of clonidine into the cerebral aqueduct eliminated the increase in blood pressure; this effect was dose dependent. Naloxone antagonised the effects of the highest dose of clonidine (5 μg). Injections of yohimbine (1 μg) into the cerebral aqueduct had no significant effect on this pressor response. Yohimbine (1 μg) effectively counteracted the antipressor effects of clonidine when the two drugs were injected together until higher doses of clonidine (2-5 μg) were used. Phenoxybenzamine had no effect on the pressor response itself but unlike yohimbine was able to attenuate the effects of clonidine only when injected together. These data suggest that activation of muscle ergoreceptor afferent nerve fibres (group III and IV fibres) during muscular contractions may cause an increase in arterial blood pressure by interfering with an inhibitory adrenergic-endorphinergic pathway in the medullary region of the brainstem.

Five adult male volunteers were studied to investigate the effect of opiate receptor blockade on the physiological response to a maximum of 60 minutes of stationary cycling at 70% V02peak in a hot (33 0 C/65% RH) environment. Exercise bouts were conducted following the administration of naloxone (4mg IV) 5 minutes prior to exercise with a follow-up 4mg dose at 25 minutes of exercise. In the placebo trial, volume-matched doses of saline were administered at the same points. No significant drug effect was observed on rectal or mean skin temperature during exercise. Post-exercise skin temperature was significantly (P<.001) higher on naloxone versus saline. Forearm blood flow (FBF) was consistently higher from minute 25 of exercise until test termination, although only the minute 25 and minute 55 data points were significantly elevated (P<.05, P<.005, respectively) . The rectal temperature threshold at which FBF plateaued was higher on naloxone (P=.054), and the FBF: rectal temperature slope was higher on naloxone throughout the trial. No significant changes were observed in heart rate or estimated mean arterial pressures, although both were consistently lower on naloxone. Gross sweat response was not altered by the drug. Plasma Beta-Endorphin was significantly (P<.Ol) higher on naloxone versus saline, and Beta-Endorphin was significantly elevated in the naloxone trial only. The observation that FBF was significantly higher on naloxone without inducing compensatory heart rate or blood pressure changes suggests that the opioids may be involved in the blood volume shifts that occur during prolonged exercise in the heat. / Master of Science