Docosahexaenoic Acid Modulates Autophagy and Confers Neuronal Resilience under Hypoxia-Reoxygenation Stress.

Hypoxia dramatically worsens neuronal death after injuries and strokes, as neurons rely heavily on oxidative phosphorylation for energy. This study investigated the effects of docosahexaenoic acid (DHA) in reducing cell death in nerve cells. Neuron-like cells, nerve growth factor-differentiated PC12 (NGFDPC12) cell cultures, with or without DHA pretreatment, were exposed to hypoxic conditions (0.5% O₂) for 12 to 48 h, followed by reoxygenation. Results showed that hypoxia caused a significant increase in the stress-response gene HIF-1α, with mRNA levels rising 5.47- and 6-fold at 24 and 48 h, respectively, compared to normoxic controls. Similarly, the pro-apoptotic gene BNIP3 was elevated 3.37- and 2.9-fold at the same time points. After 24 h of hypoxia, cells showed significant reactive oxygen species (ROS) buildup and displayed apoptotic features. The expression of fatty acid-binding protein 5 (FABP5), which is involved in responding to oxidative stress, also increased under hypoxic conditions. Treatment with DHA or rapamycin significantly improved cell viability after hypoxic exposure. To determine whether DHA protects by activating autophagy, we measured the expression of critical autophagy-related genes Atg5, Atg7, and Atg12. Hypoxia suppressed Atg5 and Atg7 expression; however, DHA restored and markedly increased the levels of Atg5, Atg7, and Atg12 beyond those in normoxic conditions. Immunoblot analysis supported these findings, showing higher levels of phosphorylated Beclin-1 and conjugated LC3, two key autophagy markers. These findings support our prior research, suggesting that DHA’s neuroprotective effects during hypoxia may involve the activation of autophagy pathways, highlighting its potential as a therapeutic strategy.Impact StatementThis study reveals that docosahexaenoic acid (DHA), an omega-3 fatty acid abundant in the brain, enhances neuronal resilience to hypoxia–reoxygenation injury by restoring autophagy function and limiting oxidative stress. These findings uncover a previously underappreciated role for DHA as a modulator of redox-sensitive autophagy pathways and suggest potential therapeutic strategies for ischemic and metabolic neurodegenerative disorders.