The neuroprotective mechanisms of naringenin: Inhibition of apoptosis through the PI3K/AKT pathway after hypoxic-ischemic brain damage

Ethnopharmacological relevance: Naringenin (NGN) is a widely distributed flavonoid with potent antioxidant and neuroprotective properties. Neuroprotective agents play a crucial role in the treatment of hypoxic-ischemic encephalopathy (HIE). It has shown potential therapeutic effects for neurological disorders. However, its efficacy on HIE is yet to be investigated. Aim of the study: This study aims to investigate the potential neuroprotective effect of naringenin and its underlying molecular mechanisms in reducing oxidative stress, apoptosis, and improving brain outcomes following HIE. Additionally, the study aims to identify the potential targets, mechanisms, and functions of naringenin using network pharmacology analysis. Materials and methods: Neonatal mice were exposed to the hypoxic-ischemic brain damage (HIBD) model to determine brain water content, and brain tissue was subjected to hematoxylin and eosin (HE), immunohistochemistry (IHC), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and Nissl staining to investigate its neuroprotective effects. Furthermore, the neonatal mouse primary neuron oxygen-glucose deprivation (OGD) model to measure reactive oxygen species (ROS) production in vitro. The protein levels were characterized by Western Blot, and mRNA levels were evaluated by a real-time quantitative PCR detecting system (qPCR). Transmission electron microscopy (TEM) and mitochondrial fluorescent staining were used to observe mitochondrial morphology. Neuronal nuclei (NeuN) and microtubule-associated protein 2 (MAP2) were detected by Immunofluorescence (IF). Finally, network pharmacology was employed to determine the common target of naringenin and HIE. The core genes were obtained via protein-protein interaction networks (PPI) analysis and molecular docking was examined, and the mechanism of action was explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Additionally, small interfering RNA (siRNA) was constructed for verification. Results: Naringenin has a neuroprotective effect in HIBD by modulating Vegfa expression and activating the PI3K/AKT pathway to inhibit apoptosis. Furthermore, molecular docking results suggest that Vegfa is a potential binding target of naringenin, and silencing Vegfa partially reverses the pharmacological effects of NGN. Conclusion: Our findings suggest that naringenin demonstrates potential clinical application for treating HIE as a novel neuroprotective agent.