The Damage Mechanism Of UVA On Human Fibroblast And Keratinocyte And The Photo-protection Role Of Resveratrol From UVA Induced Damage

Part One Effects of UVA Irradiation on Cell Proliferation, iNOS Expression and NO Production in Human FibroblastsObjectiveTo investigate the effects of UVA irradiation on cell proliferation, iNOS expression and NO production in cultured human skin fibroblasts in vitro and further explore the damage mechanism of UVA on human skin fibroblasts.MethodsFibroblasts from normal skin cultured in vitro were irradiated with 1, 5,10J/cm~2 of UVA respectively,then,the cell survival rate,the expression of iNOS mRNA/protein and NO production of skin fibroblasts at different time point(24h,48h,72h)after irradiation of various dosages of UVA were measured by MTT assay,RT-PCR,Western-blot and Griess Reaction.ResultsThe survival rate of normal fibroblasts increased gradually as time extends during 72h,the expression of iNOS mRNA/protein wasn't detected,and only low level of NO production detected in normal human skin fibroblasts(control groups).But at 24h,48h,72h after the irradiation of various dosages of UVA(5,10J/cm~2),the decrease of cell survival rate, the increase of iNOS mRNA/protein expression and NO production became most significant following the increase of UVA dosage and more than that in control group at the same time point,there were significant differences with statistical analysis(P＜0.001).The decrease of cell survival rate,the increase of iNOS mRNA/protein expression and NO production were most significant at 24h after irradiation of various dosages of UVA,there was significant difference compared to that at 24h and 48h niter the same UVA dosage(P＜0.05 or 0.01).ConclusionUVA can injure the proliferation activity of human skin fibroblasts, it might be related to the up-regulation of iNOS gene expression and the over-secretion of NO induced by UVA.Part Two Effects of UVA Irradiation on Cell Proliferation, iNOS Expression and NO production in Human KeratinocytesObjectiveTo investigate the effects of UVA irradiation on cell proliferation, iNOS expression and NO production in human keratinocytes(HaCaT cell line). MethodsHaCaT cells were cultured and irradiated with UVA of 1,5,10J/cm~2 dosages respectively.The survival rate,iNOS mRNA/protein expression and NO production at different time point after UVA irradiation of the aboved various dosages were measured by MTT assay,RT-PCR, Western-blot and Griess Reaction,respectively.ResultsThe survival rate increased gradually in normal HaCaT cells as time extends,iNOS mRNA/protein expression wasn't detected and low level of NO also detected.At 48h,72h after the irradiation of 1J/cm~2 UVA and At 24h,48h,72h after the irradiation of 5,10J/cm~2 UVA,the survival rate of UVA-irradiated HaCaT cells was lower than that in control group at the same time point,but the iNOS mRNA/protein expression and NO production significantly increased at 24h and 48h after irradiation of three different dosage of UVA compared to that in the normal control group at the same time point,and iNOS mRNA/protein expression reach the peak at the 48h after UVA irradiation,then disappeared at 72h after UVA irradiation,and NO level decreased significantly compared to that at 24h and 48h after irradiation of the same UVA dosage(P＜0.001).ConclusionUVA induced expression of iNOS mRNA/protein and promote NO secretion,it may be implicated in the damage of UVA on the proliferation of HaCaT keratinocytes. Part Three The Effect of Resveratrol on Inducible Nitric Oxide Synthase Expression in Human Fibroblasts and Keratinocytes Irradiated by UVAObjectiveTo investigate the photo-protective mechanism of resveratrol from UVA-induced damage on cultured human fibroblasts and keratinocytes (HaCaT cell line).MethodsFibroblasts and human immortalized keratinocyte(HaCaT cell line) were treated with 0.01,0.1mmol/L resveratrol after irradiation with UVA in 5J/cm~2 dosage,meanwhile,normal control group(without UVA irradiation and resveratrol treatment)and only UVA irradiation group were set as control groups.The proliferation activity of fibroblasts and HaCaT cells was detected with MTT assay,and the expression of iNOS mRNA/protein in irradiated cells was quantified with RT-PCR and Western-blot analysis techniques,respectively.ResultsThe decrease of proliferation activity and the increase of iNOS mRNA/protein expression were more significant in both fibroblasts and HaCaT cells at 24h after exposure to 5J/cm~2 of UVA than that in UVA non-irradiated groups(control groups),there were significant differences with statistical analysis(P＜0.01 or 0.001).In UVA plus resveratrol-treated groups,the survival rate was higher and the level of iNOS mRNA/protein expression decreased than that in only UVA irradiation groups in both two kinds of cells,there was significant differences between two groups(P＜0.01)ConclusionResveratrol can repair the damage degree of UVA on both fibrobalsts and HaCaT cells through down-regulating iNOS expression and NO secretion induced by UVA.It may be play a repair role in UVA-induced skin damage.