The Effect Of NF-κB Pathway On Proliferation And Apoptosis Of Human Umbilical Vein Endothelial Cells Induced By Intermittent High Glucose

Objective: To observe the effect of NF-κB pathway on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by intermittent high glucose using the NF-κB p65-targeting RNAi recombinant adenovirus vector. Also, we detected the change of bcl-2 expression which was related to cell apoptosis.Methods: Homologous recombination and cloning techniques were used to construct RNAi recombinant adenovirul expressive vectors specific to NF-κB p65 gene. Then, the adenovirus plasmids was transfected into HEK293A cells to produce adenovirus and amplify the adenovirul stock. Plaque forming assay was used to titer the adenovirul stock. Third, we transducted the RNAi adenovirus to HUVECs induced by intermittent high glucose. The p65 expression was detected by Western blot. We measured proliferation of HUVECs in the indicated conditions by AlamarBlue assay, detected bcl-2 expression by Western blot.Results: 1) The RNAi adenovirus exppression vectors targeting to p65 gene were successfully constructed; 2) The results of western blot showed the expression of p65 protein in HUVECs could be inhibited efficiently by NF-κB/p65-targeting RNAi adenovirus; 3) The decrease of p65 expression in HUVECs exposed to intermittent high glucose was also observed after infection with RNAi adenovirus, and the decrease of p65 expression was enhanced along with the continuation of infection time, and at least lasted for 6 days; 4) P65 protein of nuclear extracts was significantly increased in intermittent high glucose culture in the control group, but only slightly increased in the NF-κB-specific knockdown group, which maintained at basal state; 5) In AlamarBlue assay, knockdown of NF-κB p65 decrease(P<0.01)the proliferative damage of HUVECs in intermittent high glucose (30.5mmol/L/5.5mmol/L)(P<0.01),but the proliferative is lower than the control level (5.5mmol/L) (P<0.01); 6)Knockdown of NF-κB p65 up-regulated the bcl-2 expression when HUVECs were cultured in intermittent high glucose, thus may reduce cell apoptosis. Conclusion: The NF-κB p65-targeting RNAi adenovirus is an important tool which can efficiently inhibit the expression of p65 gene in HUVECs. Intermittent high glucose mediated proliferation inhibition and apoptosis in HUVECs. Knockdown of NF-κB p65 partly protected HUVECs from proliferation inhibition and may reduce apoptosis by preventing intermittent high glucose-induced NF-κB nuclear translocation.