Calycosin-7-O-β-D-glucoside modulates copper homeostasis through SLC31A1 to mitigate cuproptosis in cerebral ischemia/reperfusion injury.

Cuproptosis is a type of copper-dependent cell death, yet whether it participates in neuronal death during cerebral ischemia/reperfusion injury (CI/RI) has not been thoroughly investigated. As one of the characteristic components of Astragali radix, calycosin-7-O-β-D-glucoside (CG) exhibits potential pharmacological benefits on CI/RI, but its specific regulatory mechanisms are still insufficiently understood. This study was intended to illustrate the incidence of cuproptosis in CI/RI and to explore the mechanism of CG in mitigating CI/RI. CI/RI was simulated in vitro and in vivo by establishing the oxygen-glucose deprivation/reoxygenation (OGD/R) model of SH-SY5Y cells and the middle cerebral artery occlusion/reperfusion (MCAO/R) model of Sprague-Dawley rats. The results demonstrated that CI/RI induced the onset of cuproptosis accompanied by the expression changes of cuproptosis-associated proteins (FDX1, DLAT, LIAS, and ACO2), intracellular copper enrichment, and depletion of the endogenous copper chelator GSH. The cuproptosis inhibitor ATTM ameliorated cuproptosis, and similarly, CG inhibited cuproptosis, as evidenced that CG significantly boosted OGD/R cell survival, promoted neuronal cell growth, attenuated nerve damage in MCAO/R rats, and reversed the abnormal changes in cuproptosis proteins. Further studies showed that the inhibition of cuproptosis by CG was achieved through inhibition of the GSK-3β/ATF3/SLC31A1 pathway. Either CG or GSK-3β inhibitor LiCl upregulated p-GSK-3β protein expression, inducing a decrease in ATF3 protein expression, which in turn led to a downregulation of SLC31A1 protein expression, a decrease in copper accumulation, and an inhibition of the occurrence of cuproptosis. Investigation of cuproptosis offered therapeutic strategies for CI/RI and other diseases associated with copper disorders.