Hypoxic postconditioning attenuates apoptosis via inactivation of adenosine A_2a receptor through NDRG3-Raf-ERK...

In recent years, many studies have demonstrated that endogenous adenosine induced by ischemia postconditioning reduces apoptosis in animal and cell models, but no study has clearly elucidated the effects of hypoxia postconditioning (HPC) in human dermal microvascular endothelial cells (HDMECs) of flaps, and the subtype of adenosine receptors involved remains unknown. In our study, we sought to identify the roles of adenosine A_2a receptor, NDRG3 (N-myc downstream-regulated gene 3) and Raf-ERK pathway in the anti-apoptotic effects of hypoxia postconditioning.

Human dermal microvascular endothelial cells were put into a hypoxic incubator (94% N₂ + 5% CO₂ + 1% O₂) for 8 h (hypoxia), and followed 24 h of normoxic culture with 95% air and 5% CO₂ (reoxygenation). Hypoxia postconditioning model of HDMECs was achieved as follows: Before HDMECs were put into a normoxic incubator, HDMECs were treated by three cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia. Opening level of mitochondrial permeability transition pore and change of mitochondrial membrane potential were detected with related Kit. The protein expressions of mitochondrion apoptosis, adenosine A_2a receptor and NDRG3-Raf-ERK pathway were measured by western blot.

Hypoxia/reoxygenation (H/R) resulted in injury in HDMRCs as evidenced by an increase in apoptosis percentage, mitochondrial membrane permeability and an increase in expression of pro-apoptosis proteins (Bax, c-caspase-3 and cytochrom C), meanwhile, hypoxia/reoxygenation increased expression of A_2a receptor, NDRG3, p-c-Raf, p-ERK, which was significantly attenuated by hypoxia postconditioning treatment. Moreover, Hypoxia/reoxygenation (H/R) resulted in a decrease in expression of anti-apoptotic protein (Bcl-2). However, the protective effect of hypoxia postconditioning treatment could be inhibited by adding CGS21680, a selective adenosine A_2a receptor agonist (all P values < 0.05).