Salidroside derivative SHPL-49 attenuates glutamate excitotoxicity in acute ischemic stroke via promoting NR2A-CAMKIIα-Akt /CREB pathway

Background: Ischemic stroke is a significant cause of death and disability with a limited treatment time window. The reduction of early glutamate excitotoxicity using neuroprotective agents targeting N-methyl-D-aspartic acid (NMDA) receptors have attracted recent research attention. SHPL-49, a structurally modified derivative of salidroside, was synthesized by our team. Previous studies have confirmed the neuroprotective efficacy of SHPL-49 in rats with ischemic stroke. However, the underlying mechanisms need to be clarified. Methods: We conducted in vivo experiments using the permanent middle cerebral artery occlusion rat model to investigate the role of SHPL-49 in glutamate release at different time points and treatment durations. Glutamate transporters and receptor proteins and neural survival proteins in the brain were also examined at the same time points. In vitro, primary neurons and the coculture system of primary neurons-astrocytes were subjected to oxygen-glucose deprivation and glutamate injury. Proteomics and parallel reaction monitoring analyses were performed to identify potential therapeutic targets of SHPL-49, which were further confirmed through in vitro experiments on the inhibition and mutation of the target. Results: SHPL-49 significantly reduced glutamate release caused by hypoxia-ischemia. One therapeutic pathway of SHPL-49 was promoting the expression of glutamate transporter-1 to increase glutamate reuptake and further reduce the occurrence of subsequent neurotoxicity. In addition, we explored the therapeutic targets of SHPL-49 and its regulatory effects on glutamate receptors for the first time. SHPL-49 enhanced neuroprotection by activating the NMDA subunit NR2A, which upregulated the cyclic-AMP response binding protein (CREB) neural survival pathway and Akt phosphorylation. Since calcium/calmodulin-dependent kinase II alpha (CaMKII alpha) is necessary for synaptic transmission of NMDA receptors, we explored the interaction between CaMKII alpha and SHPL49, which protected CaMKII alpha from hypoxia-ischemia-induced autophosphorylation damage. Conclusion: Overall, SHPL-49 enhanced neuronal survival and attenuated acute ischemic stroke by promoting the NR2A-CAMKII alpha-Akt/CREB pathway. Our study provides the first evidence demonstrating that the neuroprotective effect of SHPL-49 is achieved by promoting the NR2A subunit to extend the treatment time window, making it a promising drug for ischemic stroke.