Pharmacological Activation of GPR68 Attenuates Ferroptosis in Spinal Cord Ischemia/Reperfusion Injury Through PI3K/Akt-Mediated Nrf2 Antioxidant Pathway

Spinal cord ischemia-reperfusion injury (SCIRI) is a devastating condition with limited therapeutic options. This study unveils a novel role of G protein-coupled receptor 68 (GPR68), a pH-sensing G protein-coupled receptor (GPCR), in mitigating ferroptosis-a lipid peroxidation-driven cell death-through the Phosphoinositide 3-Kinase/ Protein Kinase B/ Nuclear Factor Erythroid 2-Related Factor 2 (PI3K/Akt/Nrf2) antioxidant axis. Using in vitro (Oxygen-Glucose Deprivation/Reperfusion (OGD/R)-treated Pheochromocytoma Cell Line 12 (PC12) cells ) and in vivo (rat spinal cord ischemia-reperfusion (I/R) ) models, we demonstrate that GPR68 downregulation exacerbates Ferroptosis, evidenced by elevated Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4), Malondialdehyde (MDA), and Oxidized Glutathione/ Total Glutathione (GSSG/T-GSH) levels, alongside reduced Solute Carrier Family 7 Member 11 (SLC7A11) and Glutathione Peroxidase 4 (GPX4). Pharmacological activation of GPR68 with MS48107 or the clinically approved benzodiazepine Lorazepam robustly reversed Ferroptosis by enhancing Akt phosphorylation and Nrf2 nuclear translocation. Mechanistically, GPR68 siRNA or PI3K/Akt inhibition abolished these protective effects. Crucially, Lorazepam rescued neuronal viability and suppressed Ferroptosis in spinal I/R rats, effects fully negated by the GPR68 antagonist Ogremorphin (OGM). Our findings establish GPR68 as a key Ferroptosis regulator and propose repurposing Lorazepam as a therapeutic strategy for SCIRI.

Ferroptosis; GPR68; Ischemia-reperfusion; PI3K/Akt-Nrf2 axis; Spinal cord injury.