The Effect And Mechanism Of PFKFB3 On Human Umbilical Vein Endothelial Cells In Hyperglycemic Environment

BACKGROUND: As an important mark of diabetic retinopathy(DR),neovascularization is a key event in the development of DR.Numerous studies have found that endothelial cells rely on anaerobic glycolysis to generate energy to maintain cell function,and form “branches” to build neovascularization.However the mechanism of action of endothelial cell glycolysis on neovascularization in hyperglycemic environment remains unclear.Our paper mainly studies the possible effect of PFKFB3 on angiogenesis in hyperglycemic environment.METHODS: This study was divided into five parts.The first part was cultured with normal glucose(5 mmol/L,NG)and high glucose(30 mmol/L,HG)Western Blot(WB)and real-time polymerase chain reaction(RT-PCR)were used to detect the expression of PFKFB3 under normal and high glucose conditions.The second part used normal glucose(5 mmol/L,NG),normal glucose transfected with SiRNA-1(NG+SiRNA-1),normal glucose with Si RNA-2(NG+SiRNA-2),normal glucose with SiRNA-3(NG+Si RNA-3)to culture for 24 h.WB was used to detect the expression of PFKFB3.The third part of the experiment was carried out with normal glucose(NG),normal glucose with SiRNA(NG+SiRNA),high glucose(HG)and high glucose with SiRNA(HG+SiRNA),then used tube formation assay to analyze.In the fourth part,the expressions of pAkt and Akt were detected by WB using normal glucose(NG),normal glucose with SiRNA(NG+SiRNA)respectively.In the fifth part,the expression of pAkt and Akt was detected by WB after using normal glucose(NG),high glucose(HG),high glucose with SiRNA(HG+SiRNA)respectively.RESULTS: In the first part,the expression of PFKFB3 protein in HUVECs cultured in high glucose medium was significantly higher than that in normal glucose environment(t=5.457,P=0.00,P<0.05),and the expression of PFKFB3 mRNA in HUVECs cultured in high glucose medium was much higher(t=4.466,P=0.00,P<0.05).The inhibitory effect of SiRNA-1 on PFKFB3 in the second part was the most significant higher(P=0.000,P<0.01).The third part of the tube formation ability of HUVECs cultured in high glucose medium was significantly lower than that of the normal control group(P=0.005,P<0.01).The tube formation capacity of SiRNA addition to normal glucose was also significantly higher than that of the control group(P=0.031,P<0.05).The addition of SiRNA to high glucose medium formation capacity was significantly increased compared with that of high glucose medium(P=0.041,P<0.05).There was no significant difference between normal control group and high glucose medium with SiRNA addition(P=0.173,P>0.05).In the fourth part,the expression of pAkt in HUVECs after adding SiRNA to PFKFB3 in normal glucose medium was significantly lower than that in normal glucose medium(t=16.91,P=0.00,P<0.05).The pAkt expression in the normal glucose medium group in the fifth part was significantly higher than that in the high glucose medium group(P=0.004,P<0.01).The pAkt expression in the high glucose medium added SiRNA group was significantly higher than that in the high glucose medium group(P=0.000,P<0.01).The expression of pAkt in high glucose medium added SiRNA group was significantly higher than that in normal glucose medium group(P=0.008,P<0.05).CONCLUSIONS: The results of this study indicate that the inhibition of PFKFB3 can significantly alleviate the pathological neovascularization induced by high glucose,and the inhibition of PFKFB3 can significantly improve the expression of pAkt,a protective factor that is decline in high glucose environment,thereby protecting the cells.PFKFB3 can be used as a new target for the treatment of neovascularization in high glucose environment.