The Effect And Mechanism Of Nrf2/HO-1 Pathway In Oxidative Stress-Induced Vascular Calcification In End-Stage Renal Disease

Objective:To investigate the effect and mechanism of Nrf2/HO-1 pathway in oxidative stress(OS)-induced vascular calcification in end-stage renal disease(ESRD),and explore a new target for the intervention of vascular calcification in ESRD.Methods:1. Grouping Logarithmic growth phase RASMCs(n=4) and cultured for 48h: the group of complete medium cultured; the group of calcification medium cultured; the group of complete medium cultured for 42 h then the medium containing 5μM SFN cultured for 6h; The group of complete medium cultured for 46 h then medium containing 0.01 m M hydrogen peroxide cultured for 2h. Analyze the expression of nuclear Nrf2 of each group with Western blot.2. Grouping logarithmic growth phase RASMCs(n=4): The group of calcification medium cultured alone until the RASMCs were harvested; Complete medium containing 0.01 m M hydrogen peroxide pretreated for 2h, and then calcification medium cultured until the RASMCs were harvested; Complete medium containing SFN 5μM pretreated for 6h, and then calcification medium cultured until the RASMCs were harvested; Complete medium containing SFN 5μM pretreated for 6h,complete medium containing 0.01 m M hydrogen peroxide pretreated for 2h once again, and then calcification medium cultured until the RASMCs were harvested. Detect the level of reactive oxygen species(ROS) of each groups by using DCFH-DA probe, and the expression of Nrf2, Heme oxygenase(HO-1), Runt-related transcription factor 2(Runx2) of total cellular protein of each groups by western blot.Results:1. SFN, hydrogen peroxide enhanced the Nrf2 expression of RASMCs nuclear. Nrf2 expression of RASMCs nuclear was not induced by complete medium and calcification medium.2. Expression of Nrf2,Runx2 and intracellular ROS induced by calcification medium of RASMCs enhanced in a time-dependent manner, but the expression of HO-1 did not have any effect; The expression of intracellular ROS of RASMCs were significantly reduced after Hydrogen peroxide, SFN, SFN + hydrogen peroxide pretreated, and the expression of intracellular ROS-induced calcification medium of RASMCs decreased gradually with incubation time(P<0.05); The expression of Nrf2, HO-1 enhanced after SFN pretreated,and then Runx2 over expression-induced calcification medium was inhibited; The expression of Nrf2, HO-1, Runx2 enhanced could be induced by exogenous hydrogen peroxide, and Runx2 over expression induced by exogenous hydrogen peroxide could not be inhibited by activated Nrf2,HO-1; The expression of Runx2-induced calcification medium decreased gradually within incubation time after Hydrogen peroxide, SFN + hydrogen peroxide pretreated.Conclusion:1. RASMCs calcification is associated with the overexpression of endogenous ROS.2. As an important endogenous antioxidant pathway, activated Nrf2/HO-1 pathway is no antagonism on RASMCs calcification which cause by Runx2 over expression-induced exogenous ROS(hydrogen peroxide).3. Activated Nrf2/HO-1 pathway may inhibit/attenuate RASMCs calcification caused by the over expression of Runx2-induced endogenous ROS.