Study On The VEGF Released By The Muller Cells Controlled By ERK1/2 Signal Pathyway In Diabetic Rat

Part one:The expressiong of oxidative stress, changes of ERK1/2 signal pathway and VEGF secreted in retina of early diabetic rats.Objective:To investigate the oxidative stress in early diabetic rat. Investigate whether the ERK1/2 signaling pathway activated in the retina. To study whether oxidative stress induced activating the ERK1/2 pathway. And study the dependability between oxidative stress and ERK1/2 activation. To study whether ERK1/2 activated in the retina Muller cell.Methods:Diabetic rats were induced with streptozotocin injection. At 7d,14d,21d, 1m,2m,3m we undertake the experiments. First, we observated the index of oxidative stress that include SOD, GSH, MDA. And observed the mitochondrial in several types cell by the electron microscope. Second, we used retina to investigate the phosphorylation of ERK1/2 by Western-blot. And study the AP-1 through the EMSA. We also investigated the VEGF protein by Western-blot and mRNA by Real-time PCR. Third, we observed phosphorylated ERK1/2 site in retina by immunohistochemical method. At last we interfered in oxidative stress by drug Tocopherol to investigate the variation of phosphorylated ERK1/2.Results:At 7d after induced diabetes, the index of oxidative stress showed that retina had been injured by oxidative stress and mitochondrial structural abnormalities in Muller cell and ganglion cell. Western-blot results demonstrated that ERK1/2 phosphorylatedly activated at DM7d. And phosphorylation of ERKs waved during the prolonging of inducing diabetes time. EMSA demonstrated that AP-1 DNA binding activity heightened in diabetic retina and were waved like phosphorylation of ERK1/2. Western-blot and Real-time PCR evaluated VEGF protein and mRNA risen in diabetic retina. The tendency of fluctuation of ERK1/2, AP-1 and VEGF are similar. Interfered the oxidative stress could down regulated the phosphorylation of ERK1/2. The expression of phosphorylated ERK1/2 is at inner nuclear layer and mainly appeared in polygon Muller cell nuclear.Conclusion:Oxidative stress injured retina in early diabetic retina and first occurred in Muller cell and ganglion cells. The ERK1/2 pathway was activated by oxidative stress corresponding to AP-1 DNA binding activity and variation of VEGF. The ERK1/2 activated possibly in Muller cell mainly.Part Two:in vitro to study the oxidative stress, ERKl/2 activity and VEGF secreted of Muller cell stimulated by high glucose.Objective:To investigate 1) oxidative stress occured in Muller cell stimulated by high glucose; 2) phosphorylatedly activated ERK1/2 with high glucose in Muller cell; 3) the DNA binding activity of nuclear factor AP-1 with high glucose in Muller cell; 4) the change of VEGF secreted by Muller cell.Methods:Cultured Muller cell was stimulated with 30mM high glucose. At 8h,12h, 24h,36h,48h after stimulation, the production of ROS in Muller cell were overviewed by flow cytometry. Membrane potential of mitochondria was observed by confocal microscopy with JC-1. and mitochondria ultrastructure was studied by electron microscope. ERKl/2 activity was detected by Western-blto and immunofluorescence. EMS A was used to detect the AP-1 DNA binding activity. c-Jun and c-Fos mRNAwere respectively deteced by Real-time PCR. VEGF mRNA and density were checked by Real-time PCR and ELISA.Results:the productions of ROS gradually increased during the stimulated time of high glucose prolonging and at 24h show suddenly changed. The membrane potential and ultrastructure of mitochondria were abnormal. ERK1/2 phosphorylated after stimulated by high glucose. EMS A show AP-1 DNA binding activated and Real-time PCR show the elements(c-Fos and c-Jun) of were increased. Real-time PCR and ELISA results show that VEGF excreted After glucose stimulation and achieved peak at 24h.Conclution:High glucose induced oxidative stress in Muller cell. ERK1/2 signal pathway activated at the same time. And VEGF secreted increasedly after stimulation.Part Three:the contribution of antioxidant agent and inhibtion ERKl/2 to the VEGF secreted.Objective:1)Study the changes of ERK1/2 pathway and VEGF after intervention of antioxidant agent.2)study the changes of ERK1/2 pathway and VEGF after intervention of ERK1/2 inhibitor.Methods:1)Interfered with NAC to inhibit oxidative stress. Western-blot, EMSA, Real time PCR, and ELISA respectively used to observed the changes of production of ROS, ERK1/2 phosphorylation, AP-1 DNA binding, VEGF mRNA and VEGF density.2)Interfered with U0126 to inhibit ERK1/2 activation. Western-blot, EMSA, Real time PCR, and ELISA respectively used to observed the changes of ERK1/2 phosphorylation, AP-1 DNA binding, VEGF mRNA and VEGF density.Results:1)The production of ROS decreased after NAC interfering. ERK1/2 activity and VEGF mRNA and density decreased.2)the ERK1/2 phoshphorylation were down regulated after U0126 used and VEGF mRNA and densith was inhibited partly.Conclution:1) Anti-oxidative stress partly down regulated the phosphorylation of ERK1/2 and decreased VEGF secreting.2)inhibting ERK1/2 pathway partly down regulated the phosphorylation of ERK1/2 and decreased VEGF secreting.3)oxidative stress induced ERK1/2 activation probably take part in the VEGF secre in Muller cell of diabetes.