| Objectives The purpose of this study was to investigate the effects of ultraviolet (UV) irradiation on cultured keratinocytes in vitro, including: 1. proliferation, apoptosis and death of keratinocytes, the expression of apoptosis related genes bcl-2 and Fas. 2. UV-induced NF-kappa B (NF- k B) p65 expression and IL-6 secretion of human keratinocytes. 3. the effect of IL-6 on the production of matrix metalloproteinase-1 (MMP-1) and MMP-3 by human fibroblasts in vitro. 4. the protective effects of (-)-epigallocatechin - 3- gallate (EGCG) , the major polyphenolic constituent in green tea, on UV-induced keratinocytes damage. 5. the effects of antisense NF-kB p65 oligonucleotides and antisense EGF-R oligonucleotides on UV-induced IL-6 secretion of keratinocytes and c-jun activity in keratinocytes respectively. Our study will help elucidate the mechanisms of skin photoaging caused by UV irradiation on keratinocytes and its potential preventive and theraputic methods.Methods The proliferation of keratinocytes were observed by MTT method. Flow cytometry was applied to detect the number of apoptotic cells and dead cells and the level of bcl-2 protein. The IL-6, pro-MMP-1 and MMP-3 in cell-free supernants were measured byELISA. NF-kBp65 in keratinocytes was determined by immunohistochemical analysis and Western Blot. The mRNA expression was quantified by RT-PCR.Results 1. 21mJ/cm2UVB could inhibit proliferation of keratinocytes. 42mJ/cm2 UVB and 1.98J/cm2 UVA could inhibit proliferation both of keratinocytes and fibroblasts. Tea polyphenol (0.1ug/uL) protected keratinocytes from UVB and UVA irradiation. 2. The numbers of apoptotic cells and dead cells in all of the UVB irradiation groups were significantly higher than normal controls. Compared to 42mJ/cm2 UVB groups, there were fewer apoptotic cells in 126mJ/cm2 UVB groups, whereas there was an increase in the number of dead cells. However, bcl-2 and Fas mRNA levels had no difference between the two groups. In 42mJ/cm2 UVB irradiation and EGCG treatment groups, the numbers of apoptotic cells and dead cells, as well as Fas mRNA level decreased, wherease bcl-2 protein level increased. 3.IL-6 secretion and NF-kB expression of keratinocytes were induced by UVB 20. 30 mJ/cm2 and UVA 10, 20 J/cm2 significantly. 4. MMP-1 and MMP-3 production of keratinocytes could not be induced by UVB(10. 20, 30mJ/cm ). But the protein and mRNA expression of MMP-1 and MMP-3 of fibroblasts were increased by both UVB 30mJ/cm2 and various concentration of IL-6(8, 16, 24pg/mL). 5. In keratinocytes IL-6 secretion and NF-kappaB expression and in fibroblasts MMP production induced by UV could beinhibited by EGCG(0.15mM and 0.3mM). But EGCG had no effect on IL-6 induced MMP production in fibroblasts. 6. UV-induced NF- k B p65mRNA expression and IL-6 secretion were inhibited after the keratinocytes were transfected with p65 antisense oligonucleotides at 2, 4 and 8ug/mL concentrations. Similarly, UVB induced EGF-R mRNA expression and c-jun activity were inhibited after EGF-R antisense oligonucleotides transfection at 2, 4 and 8ug/mL concentrations.Conclusions Our study indicated that ultraviolet irradiation could severely damage keratinocytes. Lower dosage UVB irradiation could induce keratinocytes apoptosis, whereas higher dosage might result in cells death. After UV irradiation, NF-kappa B in keratinocytes was activated and translocated into nucleus to facilitate IL-6 secretion. MMP-1 and MMP-3 production in fibroblasts could be enhanced by IL-6 that was secreted by keratinocytes after UV-irradiation. EGCG, which is an antioxydant in nature, could reduce the damage caused by UV irradiation, which suggested that anti-oxidation may be an important pathway to prevent UV-induced skin photoaging. The results also indicated that Antisense oligonucleotides transfection mediated by lipofectin might be a useful technique that could be used to investigate UV-induced skin damage. It may have potential prevenive and theraputic significance to skin injury caused by ultraviolet.
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