Mechanism of Formononetin in Improving Energy Metabolism and Alleviating Neuronal Injury in <scp>CIRI</scp> Based on Nontargeted Metabolomics Research

Cerebral ischaemia-reperfusion injury (CIRI), resulting from thrombolytic therapy for ischaemic stroke, presents a considerable challenge during postoperative recovery. Formononetin (FMN) has shown promise in the prevention and treatment of neurological diseases. However, its specific mechanism in ameliorating CIRI remains uncertain. Initially, we established a CIRI rat model to evaluate FMN's therapeutic potential by assessing neurological function, infarct area and pathological changes. Subsequently, we employed metabolomics technology to investigate FMN's impact on metabolite levels in the ischaemic brain tissue of CIRI rats. Based on the metabolomics findings, we validated FMN's effects on nicotinate and nicotinamide metabolism, as well as alanine, aspartate and glutamate metabolism, along with its influence on neuronal injury and repair. Our investigation unveiled that FMN intervention significantly diminished the Longa score and asymmetry score in CIRI rats, constricted the infarct area and ameliorated pathological alterations in the ischaemic brain tissue, including reduced DCI index and augmented Nissl body count. Metabolomics analysis indicated that FMN exerted regulatory effects on nicotinate and nicotinamide metabolism, as well as alanine, aspartate and glutamate metabolism. Following FMN intervention, there was a notable increase in the levels of related metabolites such as nicotinamide (NAM), L-aspartic acid (L-Asp), fumaric acid (FA), gamma-aminobutyric acid (GABA) and L-glutamic acid (L-Glu). RT-qPCR and Western blot outcomes demonstrated that FMN upregulated the gene and protein expression of key enzymes adenylosuccinate lyase (ADSL) and glutamic acid decarboxylase (GAD) involved in alanine, aspartate and glutamate metabolism. Moreover, FMN intervention bolstered SOD activity, diminished MDA and ROS levels and reduced TUNEL-positive expression. Furthermore, FMN intervention elevated ATP levels and markedly increased Ki67-positive expression. FMN exhibits the potential to alleviate oxidative stress injury in CIRI rats by enhancing nicotinate and nicotinamide metabolism along with alanine, aspartate and glutamate metabolism, consequently reinstating energy metabolism and conferring neuroprotective effects to ameliorate CIRI.