The Peotective Study Of Melatonin On Retinal Pigment Epithelial Cells Encountered Oxidative Damage

OBJECTIVE: To explore the protective effect of melatonin retinal pigment epithelial cells encountered oxidative damage and to investiagate the protective mechanism.METHODS: Use hydrogen dioxide(H₂O₂) with the concentration of 600μmol·l^-1 to produce the model of oxidative damage on retinal pigment epithelial(RPE) cells cultured in vitro. The action time of the H₂O₂ was 1 hour.The cells cultured in vitro divided into normol control group, group treated with H₂O₂, group treated with H₂O₂ and melatonin(MEL) in the concentration of 1×10-7mol·l^-1,group treated with H₂O₂ and MEL in the concentration of 1×10-6mol·l^-1,group treated with H₂O₂ and MEL in the concentration of 1×10-5mol·l^-1,and group with treated with H₂O₂ and MEL in the concentration of 1×10-4mol·l^-1. We through methyl thiazolyl tetrazolium(MTT) mothed to detect the cytoactive of the retinal pigment epithelial cells;through mesuring the superoxide dismutase(SOD) and maleic dialdehyde(MDA) to detect the level of the oxidative damage with the cells;though DNA Ladders electrophoretic mothod to detect the cells apoptosis qualitatively,and Annexin V-FITC/PI double staining flow cytometry to detect the cells apoptosis quantitatively.RESULTS:1.Compared with normol control group, the results of A490 of the group treated with H₂O₂ , and other four groups treated with H₂O₂ and MEL in different concentration was decreased significantly, P<0.01,and the difference had statistical significance.It indicated that the oxidative damage of the H₂O₂ degraded the cytoactive of RPE cells. Compared with the group treated with H₂O₂, the results of A490 of all groups which had be treated with H₂O₂ and MEL in different concentration was significantly increased, and it increased gradually during the concentration increased, P<0.01,and the difference had statistical significance. 2. Compared with normol control group, not only the SOD of the group treated with H₂O₂,and other four groups treated with H₂O₂ and MEL in different concentration was decreased significantly,but also the MDA of those groups was increased significantly, P<0.01, and the difference had statistical significance.Compared with the group treated with H₂O₂, the SOD of all groups which had be treated with H₂O₂ and MEL in different concentration was significantly increased, and it increased gradually during the concentration increased. Correspondingly,the MDA of those groups treated with MEL was decreased significantly, and it decreased gradually during the concentration increased.To statistical analysis,all of the results P<0.01, and the difference had statistical significance.3. Observation the apoptosis of RPE:3.1 Though DNA Ladders electrophoretic mothod to detect the cells apoptosis qualitatively. In this experiment,we found the following phenomenon.In normol control group,there was scarce small fragments wichich could manifest the phenomenon of apoptosis. But in the group treated with H₂O₂ , there was larger differrent fragments with the length of 180-200bp or it several times, wichich just manifested the phenomenon of apoptosis.In all of the groups which had be treated with H₂O₂ and MEL in different concentration, there was some fragments also,but the number of those fragments was reduced comparing with the group treated with H₂O₂,and the fragments of the group treated with H₂O₂ and MEL in the concentration of 1×10-6mol·l^-1 was reduced most greatly.3.2 Annexin V-FITC/PI double staining flow cytometry to detect the cells apoptosis quantitatively. Using flow cytometry,we gained the cells apoptosis concretely. Compared with normol control group, the rate of apoptosis in the group treated with H₂O₂ increased cons图uously, however, Compared with the group treated with H₂O₂,the rate of apoptosis In all of the groups which had be treated with H₂O₂ and MEL in different concentration was down. On the whole, the rate of apoptosis was decreasing when the concentration of MEL was increased. The rate of apoptosis of the six groups was 4%,51%,45%,32%,20%,and 22% respectively.CONCLUSION:This empirical study discovered that: H2O2 can bring oxidative damage for RPE cells cultured in vitro.This oxidative damage embodied in details that it impaired the cytoactive of RPE cells,weakened the ability of oxidoreduction,and accelerated the apoptosis. Nevertheless,MEL can inhibit the oxidative damage.It can reinforce the cytoactive of RPE cells, strengthen the ability of oxidoreduction,and step down the progression of apoptosis.This study es表lished tiny theoretical fundament for the application of MEL in curing Retinal disease related to oxidative damage in the future.