Longhu Xingnao Granules Ameliorate Cerebral Ischemia-Reperfusion Injury by Modulating NUDT1-Associated Ferroptosis Signaling

Cerebral ischemia-reperfusion injury (CIRI) significantly exacerbates neuronal damage during ischemic stroke. Longhu Xingnao Granules (LHXNG) has been used clinically for stroke-related disorders and has shown neuroprotective potential against CIRI. However, the pharmacological basis and underlying molecular mechanisms remain to be clarified. This study aimed to explore the neuroprotective effects and potential mechanisms of LHXNG against CIRI. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to characterize representative chemical constituents of LHXNG. Neuroprotective efficacy was assessed in a rat middle cerebral artery occlusion/reperfusion (MCAO/R) model. An in vitro erastin-induced ferroptosis model in PC12 cells, combined with small interfering RNA (siRNA)-mediated NUDT1 knockdown, was established. UPLC-Q-TOF/MS identified gastrodin, chlorogenic acid, paeoniflorin, ferulic acid, and astragaloside IV as representative constituents. In vivo, pretreatment with LHXNG improved neurological function, reduced cerebral infarct volume, and alleviated histopathological injury. Biochemically, LHXNG decreased ferrous iron (Fe²⁺), malondialdehyde (MDA), reactive oxygen species (ROS), and lipid peroxidation (LPO) levels while restoring glutathione (GSH) content, indicating the suppression of ferroptosis-related oxidative damage after CIRI. At the molecular level, LHXNG treatment was associated with increased nudix hydrolase 1 (NUDT1) and glutathione peroxidase 4 (GPX4) expression and decreased acyl-CoA synthetase long-chain family member 4 (ACSL4) expression, together with changes in ferroptosis-associated injury markers. Importantly, NUDT1 knockdown weakened the anti-ferroptotic effects of LHXNG-medicated serum in PC12 cells, suggesting a possible contribution of NUDT1 to this process. In conclusion, LHXNG pretreatment attenuates experimental CIRI in association with reduced ferroptosis-related oxidative injury and changes in NUDT1, GPX4, and ACSL4 expression. The potential contribution of NUDT1 warrants further investigation.