The Effect Of The Resveratrol On Proliferation In Human Retinal Capillary Endothelial Cells Induced By High Glucose And Its Possible Mechanism

Objective:DM is a disease of metabolic dysregulation that results in reduced life expectancy due to disease specific microvascular(retinopathy, nephropathy, neuropathy, impaired wound healing) and macrovascular(heart disease and stroke) complications.Diabetic retinopathy(DR) is one of the common and seriousdiabetic microvascular complications, is a major cause of blindness in adult, blindness is caused by DR in 85% patients. DR is characterized by the formation of new blood vessels, pathogenesis is due to retinal microvascular system damagecaused by many factors, is the interaction results, mainly including the pericyte loss,optic nerve retinal abnormalities and neovascularization. Retinal capillary endothelial cell proliferation is a prerequisite for the formation of new blood.Red wine contains a variety of polyphenolic compounds in abundance,The major actions on the eye include: anti-oxidant,anti-apoptotic, anti-tumourogenic, anti-inflammatory, anti-angiogenic and vasorelaxant. As the new angiogenesis is one of the pathogenesis of diabetic eye diseases,this study discovered the effect of the Res to the human retinal capillary endothelial cells(HRCRCs) proliferation and cell cycle, and its molecular mechanisms are discussed in this paper, for the Res provides a new experimental basis in diabetic eye disease treatment.Methods:1 CCK-8 method to detect the proliferation activity of the HRCECsHRCRCs cells were plated in a 96-well plate, and then treated with 25 mmol/L Mannitol(group M), 5.5 mmol/L glucose(group N), 25 mmol/L glucose(group H) and H+Res(group R1, R2, R3 and R4, combined 25 mmol/L glucose with 25, 50, 100, 200 μmol/L Rec, respectively) for 24 h. The cck-8 assay was preformed to detect the cell proliferation.2 Flow cytometry was used to detect the effect of Res to the cell cycle of the HRCECs. HRCECs were divided into group M, group N, group H and group R(group R2, 25 mmol/L glucose plus 50μmol/L Rec, and group R3, 25 mmol/L glucose plus 100 μmol/L Rec). After 24 h of treatment, cell cycle distribution was then analyzed by flow cytometry.3 Western blot and real-time PCR were performed to detect the expressions of the cell cycle related proteins and its upstream m RNA.Results:1 The results of the cck-8 showed that the cell proliferative viability was significantly increased in group H compared with group N(P<0.05), and treatment of mannitol had no remarkable effect on proliferation in HCRECs; Res(25, 50, 100, 200 μmol/L) suppressed the proliferation of HRCECs cells by 4%、11%、16、47% respectively at the 24 th hour compared with the group H, displayed in a dose-dependent manner. Significant differences were observed in group R2, R3 and R4 when compared with group H respectively(P<0.05).The cell count was much higher when HCRECs exposed to high glucose compared with treating with normal glucose(P<0.05), whereas there was no significant difference when exposed to mannitol; Res(25, 50, 100, 200 μmol/L) can decreased the cell count of HCRECs by high glucose treatment in a dose-dependent manner. When compared with group H, the cell count of group R1, R2, R3 and R4 decreased by 17 %, 41 %, 58 %, 75% respectively(P<0.05).2 Flow cytometry results showed that high glucose treatment can increase the cell numbers in S phase(P<0.05), but the cell cycle was not altered after mannitol culture. After the treatment with the Res(50,100, μmol/L), the number of cells distributed in S phase increased dramatically, indicating the inhibitory role of Res in the cell cycle of HRCECs induced by high glucose.3 Western blot analysis and Real-time PCR results demonstrated that there were no changes of cyclin A1,CDK2, PCNA and P21CIP1 m RNA and protein levels between group N and group M. High glucose significantly increased cyclin A1, CDK2 and PCNA protein levels, decreased P21CIP1 levels in HRCECs cells. Treatment of Res(50, 100 μmol/L) downregulated the expressions of cyclin A1, CDK2 and PCNA and up-regulated P21CIP1 in HRCECs exposed to high glucose in a does-dependent manner(P<0.05).Conclusions:1 High glucose increased the proliferative viability of HRCECs by affecting cell cycle progression, upregulating the expressions of cyclin A1、CDK2、PCNA and downregulating P21CIP1 expression;.2 Res inhibited the proliferation of HRCRCs exposed to high glucose in a does-dependent manner possibly via blocking the cell cycle in the S phase, suppressing CDK2, PCNA and cyclin A1 expressions and promoting P21CIP1 expression.