Influence Of Ultraviolet Irradiation On Skin Dermis And Protective Effects Of Tempol, One Of Nitroxides, Against Ultraviolet Irradiation

Background:Photoaging is due to chronic ultraviolet radiation (UV) which contributes to a premature ageing phenotype of skin at sun-exposed area and characterized by coarsely wrinkled, yellowish skin in clinical appearance and "solar elastosis" change of dermis in histological appearance. It is a complex pathological process with the major damage seen in the connective tissue of the dermis and completely different from intrinsical ageing as related to morphological phenotype and underlying mechanisms, so photoaging can be prevented. Considering the important role of reactive oxygen species in the pathogenesis of photoaging. supplement with exogenic antioxidants to strengthen the antioxidant system of the skin is always one of important strategies to protect against photoaging. Since nitroxide has been reported to have antioxidant and some photoprotective properties, it may be hopeful to import this agent to fight against photoaging.Objective:1. To study the influence of UV on the dermal structure and metabolism, including the structure and proliferation of dermal fibroblasts. the oxidative damage, the structure and expression of collagen I, collagen III. elastic fiber and the expression of matrix metalloproteinase (MMP)-1. MMP-3.2. To determine the protective effects of Tempol, one of nitroxides. against ultraviolet irradiation and to confirm its proper concentration.Methods:The research was dividednto in -vitro study and animal study.Human dermal fibroblasts were used in the in-vitro study. Fibroblasts were irradiated by a single exposure to UVAI (340-400nm) or UVB (280-320nm) and at the same time incubated with, or without. Tempol and detected twenty-four hours later. Cellproliferation rate was determined by MTT assay and cell cycling was analyzed by flow cytometric method. Superoxide dismutase (SOD) activity and lipid peroxidation, as shown by accumulation malonyldialdehyde (MDA). were detected by biochemical assay. Expressions of procollagen I. procollagen 111 (protein levels) and MMP-1, MMP-3 (mRNA level) were detected by ELISA and semi-quantitative reverse transcriptase-PCR separately.In animal study, the guinea pig photoaging model with chronic sun-simulating radiation (SSR) exposure was set up firstly. The protective effects of Tempol, topically used before each exposure at the concentration of 5 mg/ml or 0.5 mg/ml, were also assessed. Dermal structure was observed after Hematoxylin-eosin stain or by electron microscopy. SOD activity and MDA level in the skin were detected by biochemical assay. The structure and the expression of elastic fiber were analyzed after Weigert's stain and the structure of collagen 1 and collagen III was observed after Siriured's stain by polarized microscopy.Results:In in-vitro study, cell proliferation curve after UVA1 or UVB irradiation showed dose dependent decrement pattern. More cells stopped at the GO/GI phase and the proliferation index (PI) decreased after 15 J/cm2UVAl irradiation. 15 J/cm2UVAl or 40 mJ/cm2 UVB significantly inhibited SOD activity and procollagen I, procollagen III protein levels, while increased MDA level and stimulated MMP-1 and MMP-3 mRNA expression. Tempol, between 0.03 mM and 8 mM, reversed these effects caused by UVA1 or UVB in some degree or even completely and in proper concentration, the results were statistically significant compared with irradiated group. Tempol at concentration of 0.5 mM had strongest photoprotective effects for UVA1 or UVB exposed human dermal fibroblasts and was even more effective than traditional antioxidant vitamin C (0.1%).In animal study, after depilated by rosin and beeswax mixture, albino guinea pigs were subjected to suberythemal dose of SSR three times a week. Seventeen weeks after exposure, there was typical "solar elastosis" damage in the upper dermis. Dermal fibroblasts appeared metabolically hyperactive and mitochondria in the cells were damaged. Some cells even broke up. Mature elastic fibers and collagen were severely degraded and there was large amount of elastotic material accumulated in the subepidermal dermis. SSR irradiation also provoked an infiltration into the dermis of...