The Effects And Study Involving Nrf-2 To Prevent Vascular Calcification In ESRD By Inhibition Of Oxidative Stress

Objective: Oxidative stress could induce cellular damage to vascular smooth muscle cells and lead to the transforming from vascular smooth muscle cells to osteoblasts in vascular calcification of ESRD.This study aims to investigate the role of Nrf-2 in vascular calcification about the transforming from vascular smooth muscle cells to osteoblasts,then further to explore the underlying mechanism involving oxidative stress.Methods:1.We used the rat vascular smooth muscle cells(RASMCs)model of ?-glycerophosphate induced calcification resembling vascular calcification in ESRD.2.In vitro,before the RASMCs were treated with calcification medium for 72 h,we divided RASMCs,which in logarithmic growth phase,into different groups.The groups are as below: ctrl,calcification,Nrf 2 siRNA,the inhibitor of Nrf-2(Retinoic Acid/Vitamine A Acid,VA,1 ?M or 5?M),the activator of Nrf-2(Sulforaphane,SFN,1 ?M or 5 ?M),the inhibitor of ROS(N-Acetylcysteine,NAC,5mM or 10 mM).3.We measured the calcification deposition in RASMCs of different groups by Von Kossa staining,then detected the calcium concentration in RASMCs of different groups.4.RT-PCR tested the mRNA level of Nrf-2?FGF-23?Klotho in RASMCs of each group.5.Detected the protein expression of Nrf-2?Keap-1?OPN?Runx-2 in RASMCs of each group by Western Bloting.6.Confocal observed mitochondria damage in RASMCs of each group after stained these cells by Mito-tracker Red FM.7.We also measured the ROS production and the mitochondria potential in RASMCs of each group.Reults:1.Nrf-2 siRNA transfection or Nrf-2 inhibitor Retinoic Acid could not reduce calcium deposition and calcium concentration in RASMCs following calcification.However,Nrf-2 agonist SFN or ROS inhibitor NAC could reduce calcium deposition and calcium concentration in RASMCs following calcification.2.Nrf-2 knockdown or Nrf-2 inhibitor Retinoic Acid could reduce the mRNA level of Nrf-2 and Klotho,then increase the mRNA level of FGF-23 in RASMCs following calcification.However,Nrf-2 agonist SFN or ROS inhibitor NAC could increase the mRNA level of Nrf-2 and Klotho,then decrease the mRNA level of FGF-23 in RASMCs following calcification.3.Nrf-2 silence or Nrf-2 inhibitor Retinoic Acid could reduce the protein expression of Nrf-2 and increase the protein expression of Keap-1,OPN and Runx-2 in RASMCs following calcification.However,Nrf-2 agonist SFN or ROS inhibitor NAC could increase the protein expression of Nrf-2 and reduce the protein expression of Keap-1,OPN and Runx-2 in RASMCs following calcification.4.Knockdown of Nrf-2 by Retinoic Acid could impair the red fluorescent intensity indicated mitochondria injury in calcific RASMCs.But overexpression of Nrf-2 by SFN or DMF or inhibition of ROS by NAC could increase the red fluorescent intensity indicated mitochondria injury in calcific RASMCs,overexpression of Nrf-2 by SFN or DMF could inhibit ROS production,increase mitochondria potential.Conclusion:Nrf-2 could regulate ROS production then inhibit oxidative stress.It also could ameliorate mitochondria injury by oxidative stress.Further Nrf-2 could inhibit the transforming from RASMCs to osteoblasts.Finally,Nrf-2 could prevents vascular calcification in ESRD.This study might indicate an anti-oxidant role of Nrf-2 following vascular calcification and set a novel stage for treatment to vascular calcification upon ESRD.