Effects And Mechanism Of Curcumin On Oxidative Damage And Fibrosis Of Retinal Pigment Epithelial Cells Induced With High Glucose

ObjectiveTo study the protective effect of curcumin on oxidative damage and the inhibitory effect of curcumin on fibrosis of retinal pigment epithelial cells induced by high glucose,and to explore its mechanism.MethodsHuman retinal pigment epithelial cell line(hRPE)cells were cultured and divided into:(1).normal culture group(complete medium),(2).high glucose culture group(glucose 25mmol/L+complete medium),(3)-(6)on the basis of the high glucose culturing,adding of curcumin with the concentrations: 5?g/ml,10?g/ml,20?g/ml,40?g/ml.1.MTT assay was used to detect the proliferation of cells in each group.2.Reactive oxygen species(Reactive oxygen species,ROS)kit was used to detect the expression of ROS in cells.3.Immunofluorescence method was used to detect the location and expression of NF–?B.4.ELISA kit was used to detect the expression of Tumor Necrosis Factor alpha(Tumor Necrosis Factor alpha,TNF alpha)and Interleukin-1 beta(Interleukin-1 beta,IL-1 beta).5.Cell scratch experiment was conducted to detect the changes in cell morphology and migration in each group.6.Western-blot tests are used to detect the expression of the Nuclear transcription factors-?B-p65(NF-?B-p65),Intercellular Adhesion factor-1(ICAM-1),Vascular Endothelial Growth Factor(VEGF),Transforming Growth Factor-beta 1(TGF-beta 1),alpha-Smooth Muscle Actin(alpha-SMA),and Matrix Metalloproteinase-2(MMP-2).Results1.Compared with the normal cell group,the cell proliferation activity of the high-glucose group decreased,while the cell activity of the high-glucose group increased with the increase of drug concentration,and the results were statistically significant(F=25.468,P<0.05).2.Compared with the normal group,the expression level of ROS in cells in the hyperglycemia group was significantly increased.Compared with the hyperglycemic group,the ROS expression level of cells in the hyperglycemic group decreased significantly with the increase of curcumin concentration.3.NF-?B rarely expresses in normal group of cells in the nucleus,the expression of NF-?B content in high sugar group cells' nucleus increased significantly.Comparing with the high sugar dosing group of cells,with the increase of drug concentration,the content of the nucleus of NF-?B becomes significantly lower compared with the high sugar group and has a certain dose dependent.4.Only a small amount of TNF-? and IL-1? were expressed in the supernatant of normal cells,while the contents of TNF-? and IL-1? were significantly increased in the supernatant of the high-glucose group,which was statistically significant(P < 0.05).In the hyperglycemic group,the contents of TNF-? and IL-1? in the supernatant decreased with the increase of drug concentration,which was statistically significant(TNF-?,F=28.143,P <0.05;IL-1?,F = 17.585,P<0.05).5.High glucose can cause fibrosis of retinal pigment epithelial cells and enhance the migration ability of cells;With the increase of drug concentration,the ability of cell fibrosis and migration was gradually inhibited.6.The results of western-blot showed that the protein contents of NF-?B-p65,ICAM-1,VEGF,TGF-?1,?-SMA,and MMP-2 of the hyperglycemia group cells were significantly higher than those in the normal group,and the results were statistically significant(P < 0.05).With the continuous increase of drug concentration,the expression of various proteins in the high-sugar group decreased,with statistical significance(P<0.05).Conclusions1.High glucose can induce oxidative damage to retinal pigment epithelial cells.2.High glucose can induce fibrosis of retinal pigment epithelial cells.3.Curcumin can inhibit oxidative damage and fibrosis of retinal pigment epithelial cells induced by high glucose by inhibiting over-activation of NF-kappa B in a dose-dependent manner.