Abstract
Individually, hypoxia and protein kinase R (PKR) induce retinal ganglion cells (RGCs) damage by aggravating reactive oxygen species (ROS), oxidative stress, inflammation, and apoptosis. However, it is still not established in hypoxia mediates such damaging effect by modulating PKR. This study investigated the expression and activation of PKR in hypoxic RGCs and tested if suppression of PKR by C16 is protective.
Isolated RGCs were under normoxic or hypoxic conditions for 12 h. In some cases, hypoxic cells were pre-treated with C16, a PKR inhibitor, or n-acetyl cysteine (NAC) a glutathione (GSH) precursor for 1 h and then exposed to hypoxia for the next 12 h.
Hypoxia increased cell death, lactate dehydrogenase (LDH) levels, and levels of single-stranded DNA (ssDNA). It also increased levels of ROS, the activity of the nuclear factor-kappa beta (NF-κB), JNK, and p38 MAPK, expression of Bax, p53, and cleaved caspase-3, levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and cytoplasmic levels of cytochrome-c. It concomitantly suppressed levels of GSH and Bcl-2. All these events were associated with increased phosphorylation (activation) of PKR and its target eukaryotic initiation factor 2 (eIF2). Pre-incubating the cells with NAC completely prevented all these effects in hypoxic cells. Similar protective effects without affecting levels of ROS and GSH levels were also seen in hypoxic cells pre-treated with C16.
Hypoxia induces oxidative stress, inflammation, and apoptosis in the RGCs mainly by ROS induced activation of PKR, whereas scavenging ROS by NAC or suppressing PKR by C16 is a novel protective mechanism.