Effects Of CPDT On Cultured Müller Cells Under The High Glucose Condition

Objective : To study the effects of CPDT on cultured Müller cells under the high glucose condition, and to study the cells’ activity and mitochondrial reactive oxygen species (ROS) production and the level of Nrf2 and HO-1 protein of the Müller cells which CPDT has effected under the high glucose, and provide the foundation theory for the research of diabetic retinopathy. Methods: Experiment was divided into 4 parts: 1.Determined the appropriate high sugar concentration: Rat Müller cells which offered by Rochen Pharma Co., Ltd were randomly divided into 7 groups to add different levels of glucose: 15mmol/L, 25mmol/L, 35 mmol/L, 45mmol/L, 55mmol/L, 65mmol/L and 75mmol/L, preliminary experimented by CCK-8 test and observed the cell morphology, growth status. 65mmol/L was selected for the appropriate model of high glucose concentration tendency. 2. Screened of drug concentration of CPDT: rat Müller cells were divided into 7 groups randomly: 65mmol/L glucose group and High glucose group added with (5 、15、30、45、60 and 70μmol/L CPDT) and cultured them 72h. WST-8 was used to testify the activity of cells using different dose of CPDT under the high glucose.60μmol/L was selected for the appropriate model of CPDT concentration tendency. 3.Used FCM (flow cytometry) to see the differential of the Müller cells active oxygen and the apoptosis rate under different groups which contained normal group、65mmol/L group and 65mmol/L glucose+ 60μmol/L CPDT. 4.Meanwhile the test of Western blotting were used to measure the expression of Nrf2、HO-1 at protein levels in normal group、65mmol/L group and 65mmol/L glucose + 60μmol/L CPDT group.Results: 1.Through the CCK8 test: Compared with 25mmol/L normal group, 15mmol/L、65mmol/L、75mmol/L glucose group the activity of Müller cells decreased, and the difference was statistically significant (P < 0.05) and other groups (35mmol/L、45mmol/L and 55mmol/L) have no obvious statistical significance (P>0.05).The wst-8 showed that the activity of Müller cells apparently decreased under 65mmol/L.2.Through the CCK-8 test: Compared with the 65mmol/L glucose group the CPDT in 5、15、30、45μmol/L had no obvious change(P>0.05), while the activity of cells were recovered CPDT in 60μmol/L and in 70μmol/L were significantly different (P < 0.05). 3.Compared with the normal group, the FCM showed that the mitochondrial ROS levels were high in 65mmol/L high glucose group(t=-30.985, P=0.001), compared with the high glucose group, the mitochondrial ROS levels were distinctly decreased in 60umol/L CPDT group (t=27.676, P=0.001). 4. Compared with the normal group, the protein expression of NrfZ、HO-1 increased. However, compared with high glucose group, the protein expression of Nrf2、HO-1 decreased(t=-11.030,P=0.008; t=-63.170,P=0.000)Conclusions: 1. Under the high glucose, the rat’s Müller cells mitochondrial oxidative stress pathway is activated, active oxygen generation increases, cell activity is decreases and cell apoptosis increases. 2. The CPDT can inhibit oxidative stress reaction and decrease the apoptosis rate and the expression of protein levels. It also protects the Müller cells from damages.