Ampelopsin preserves glutamate homeostasis against cerebral ischemia

The glutamate (Glu)–glutamine (Gln) cycle between astrocytes and neurons plays a crucial role in maintaining extracellular Glu levels during excitatory neurotoxicity, with glutamine synthetase (GS) and glutaminase (GLS) serving as the key enzymes. Ampelopsin (Amp) exhibits diverse biological and pharmacological activities, including antioxidant, anti-inflammatory, and antitumor effects. Notably, Amp regulates GLS, which is also the established target of CB-839, a potent and specific inhibitor known to suppress tumorigenesis. However, the influence of this effect on neuroprotective effects and Glu homeostasis remains unclear. This study aimed to construct a middle cerebral artery occlusion/reperfusion (MCAO/R) model to assess the effects of Amp and CB-839 on Glu-induced ischemic injury in vivo. Herein, Glu and Gln levels within the Glu–Gln metabolic cycle, and protein levels of GLS, GS, glutamate transporter-1 (GLT-1), and N-methyl- D -aspartate receptor (NMDAR) were analyzed. Immunofluorescence staining was performed to determine the distribution of GLS and GS in brain astrocytes and neurons. Notably, both Amp and CB-839 exerted neuroprotective effects in MCAO/R mice by reducing cerebral infarction area, alleviating brain edema, and improving neurological function. Furthermore, they attenuated neuronal necrosis and mitigated the ischemia-induced damage to neurons and Nissl bodies. In addition, both Amp and CB-839 enhanced GS-mediated Glu-to-Gln conversion in astrocytes by increasing GS activity in the ischemic brain and reducing Glu accumulation through GLT-1 upregulation, which facilitated Glu uptake. In neurons, both Amp and CB-839 inhibited GLS-mediated Gln hydrolysis to Glu by downregulating GLS expression, leading to elevated Gln and decreased Glu levels during cerebral ischemia. Simultaneously, NMDAR expression was reduced, preventing excitatory neurotoxicity driven by excessive glutamatergic signaling. Overall, this study highlights the potential of Amp in providing neuroprotection in mice subjected to transient focal cerebral ischemia by promoting the dynamic Glu homeostasis.