Nicotinamide adenine dinucleotide attenuates neuronal apoptosis in spinal cord ischemia-reperfusion injury via the activation of PI3K/AKT pathway

Spinal cord ischemia-reperfusion injury (SCIRI) leads to acute neurological deficits primarily driven by oxidative stress and apoptotic cell death. The potential of exogenous nicotinamide adenine dinucleotide (NAD⁺) to confer acute neuroprotection, as well as the underlying mechanisms, remains poorly understood. Network pharmacology analysis implicated the PI3K/AKT signaling pathway as a potential mediator of NAD⁺ effects. To investigate this, we assessed NAD⁺ in both in vivo (rodent SCIRI model; 75 mg/kg, intraperitoneal administration at reperfusion) and in vitro (oxygen-glucose deprivation/reperfusion, OGD/R; 32 μM) settings. Functional recovery was evaluated using the BBB locomotor rating scale, while histopathological changes, apoptosis (via TUNEL staining), oxidative stress markers (SOD, MDA, ROS), and key apoptotic proteins (Bcl-2/Bax, cleaved caspase-3) were analyzed alongside PI3K/AKT pathway activation. Pharmacological inhibition with LY294002 was employed to validate pathway involvement. Our results demonstrate that NAD⁺ significantly improved motor function recovery, attenuated histological damage, and reduced both apoptosis and oxidative stress following SCIRI. Consistent cytoprotection was observed in the OGD/R model with 32 μM NAD⁺. Mechanistically, NAD⁺ restored phosphorylation levels of PI3K and AKT and favorably modulated apoptotic regulators-effects partially reversed by LY294002, confirming the critical role of the PI3K/AKT axis. These findings provide mechanistic evidence that exogenous NAD⁺ exerts acute neuroprotection during the early reperfusion phase, mediated through PI3K/AKT pathway activation and subsequent suppression of oxidative stress and apoptosis.