ABSTRACT
Objective:
To evaluate the
role of
reactive oxygen species (ROS) in
hypoxia postconditioning-induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/
antioxidant response element (ARE) signaling pathway in
rat cardiomyocytes.
Methods:
Primary
cardiomyocytes of
adult rats were isolated and cultured and divided into 4 groups ( n=20 each) using a random number table
method:
normal group (group N),
hypoxia-reoxygenation group (group HR),
hypoxia postconditioning group (group HPO) and
hypoxia postconditioning plus an ROS scavenger N-(2-Amidinopropionyl)-
glycine (MPG) group (group HPO+ MPG).
Cells were exposed to
hypoxia for 45 min followed by 60 min reoxygenation to develop the
cardiomyocyte hypoxia-reoxygenation
injury model.In HPO group,
cells were subjected to 3 cycles of 5-min
hypoxia/5-min reoxygenation after 45 min
hypoxia, followed by reoxygenation for 60 min.In HPO+ MPG group, MPG (final concentration 2 mmol/L) was added at 35 min of
hypoxia,
cells were subjected to
hypoxia for 10 min, and the other
treatments were
similar to those previously described in group HPO.At the end of reoxygenation, the intracellular
calcium level and Nrf2 activity were measured, the
ultrastructure of
cardiomyocytes was observed, and the Flameng score of
mitochondria was assessed, and the expression of Nrf2,
NAD(P)H
quinine oxidoreductase 1 (NQO1),
superoxide dismutase 1 (SOD1) and
heme oxygenase-1 (HO-1)
protein and
mRNA was detected using
real-time polymerase chain reaction and
Western blot.
Results:
Compared with group N, the intracellular free Ca 2+ level, Nrf2 activity and Flameng score were significantly increased, and the expression of Nrf2, NQO1, SOD1 and HO-1
protein and
mRNA was down-regulated in group HR ( P<0.05). Compared with group HR, the intracellular free Ca 2+ level and Flameng score were significantly decreased, the Nrf2 activity was increased, and the expression of Nrf2, NQO1, SOD1 and HO-1
protein and
mRNA was up-regulated in group HPO ( P<0.05). Compared with group HPO, the intracellular free Ca 2+ level and Flameng score were significantly increased, the Nrf2 activity was decreased, and the expression of Nrf2, NQO1, SOD1 and HO-1
protein and
mRNA was down-regulated in group HPO+ MPG ( P<0.05).
Conclusions:
The mechanism by which
hypoxia postconditioning activates the Nrf2/ARE signaling pathway in
rat cardiomyocytes may be related to ROS.
