Study On The Protective Effects Of Estradiol Against Injury Caused By Ultraviolet B To Rat's Skin Keratinocytes

Background: Keratinocytes (KC) are main components of epidermis which are subject to a vast array of environmental insults. As ultraviolet B (UVB) component of the solar spectrum exposure is one of the leading causes of skin cancer, it is imperative to try to understand how KC responds to UVB irradiation. A hallmark feature of UV exposure in skin are so-called sunburn cells, which represent apoptotic epidermal KC. Previously, the functional role of these UVB-damaged KC was obscure, but recent evidence suggests that sunburn cells are not only markers for severe sun damage, and also this apoptotic process is important for preventing skin cancer. UVB damage KC via similar intracellular responses, jointly termed "the stress response", including genotoxic stress which is induced by direct DNA damage, and oxidative stress which is induced by reactive oxygen species (ROS). The inability of KC to UVB-induced stress responses can lead to the development of skin cancers and skin photoaging. Lots of researches show that Estrogens have a profound influence on skin. Estrogen treatment in postmenopausal women has been repeatedly shown to increase collagen content, dermal thickness and elasticity, and data on the effect of estrogen on skin water content are also promising. but at present, the study on capability of Estrogen antagonize oxidative injury and apoptosis of skin cell is few .Object: To establish cellular damage model with UVB on rats'Keratinocyte from culture in vitro. Based on these models, the antioxidative and antiapoptotic effects of estrogen are investigated.Methods: Adopt postnatal clean Wistar ped-rats within 24 h, primary culture skin Keratinocyte and let Keratinocyte exaltation. Detect the effect of different dos of UVB to Keratinocyte survival by MTT essay to determine the dos of UVB injury. Detect the effect of different concentration of DMSO to Keratinocyte survival by MTT essay to determine the concentration of DMSO as excitatory of E2 .The third generation Keratinocyte in exponential phase of growth were divided 6 groups, cell concentration of each group is 1×106/ml: blank group, UVB group, prevention group, prevention control group, treatment group, treatment control group. Keratinocyte of Blank group were cultured in 36h commonly ; Keratinocyte of UVb group accepted radiation of UVB only once; Keratinocyte of prevention group, which were added DMSO and E2 at first, accepted radiation of UVB after 24h and continued to be cultured 24 h; the difference from prevention control group and prevention group is that prevention control group were only added DMSO without E2; treatment group accepted radiation of UVB at first, after 24 h culture, added DMSO and E2 and continued to culture 24 h, the difference from treatment group and treatment control group is that reatment control group were added DMSO without E2. at last, collected all Keratinocyte to detect oxidative damage and apoptosis.Detected the erythrocuprein (SOD), malonaldehyde (MDA), glutathione (GSH), glutathion peroxydase (GSH-Px), superoxide anion free radical (O2-·) and hydroxy radical (·OH) in the dealed Keratinocyte with kit. Detect apoptosis in the method of AO/EB.The results of the data were represented as mean±root-mean square deviation ( x_±s), applied analysis of variance of Q analysis to difference in each group. Results: (1) Survival of Keratinocyte were decrease significantly after the radiation of UVB in diffident time 3min, 5min, 7min, 10min (P<0.05) ; (2) there is no significant statistic difference from 10-1 mol/L DMSO group and blank group (P>0.05) ; (3) the value of SOD, GSH-Px and GSH of UVB group, prevention control group and treatment control group is significant lower than blank group (P<0.05) ; the value of SOD, GSH-Px and GSH of prevention control group, treatment control group, and UVB group is not different significantly (P>0.05); the value of SOD, GSH-Px and GSH of prevention group is higher than prevention control group significantly (P<0.05) ; the value of SOD, GSH-Px and GSH of treatment group is higher than treatment control group significantly (P<0.05) ; (4) the value of MDA,·OH and O2-·of UVB group, prevention control group and treatment control group is higher than blank group significantly (P<0.05); about MDA,·OH and O2-·, there is no significant statistic difference between treatment control group and prevention control group (P>0.05); the value of MDA,·OH and O2-·of prevention group is lower than prevention control group significantly (P< 0.05) ; the value of MDA,·OH and O2-·of treatment group is lower than treatment control group significantly (P<0.05) ; (5) after AO/EB dyeing, under the fluorescence microscope there are lots of apoptosis cell in UVB group, prevention control group and treatment control group, the Apoptosis rate of black group is significantly different from the rate of UVB group (P<0.05), and the rate of prevention group is lower than the rate of prevention control group significantly (P<0.05); the rate of treatment control group is lower than the rate of treatment control group significantly (P<0.05);Conclusion: The results indicated that 10-6mol/L E2 can resist UVB radiating cellular oxidation injury and apoptosis of Keratinocyte, and effect of prevention and treatment of E2 are not different fundamentally .