Betulinic Acid Modulates Redox Imbalance and Dysregulated Metabolisms, While Ameliorating Purinergic and Cholinergic Activities in Iron-Induced Neurotoxicity

The neuroprotective properties of betulinic acid were investigated in oxidative-mediated neurotoxicity induced with Fe²⁺ in isolated rats’ brains. Oxidative neurotoxicity was induced ex vivo by incubating brain tissue homogenates with 0.1 mM FeSO₄ for 30 min at 37 °C in a 5% CO₂ incubator in the presence and/or absence of different concentrations of betulinic acid. Induction of oxidative neurotoxicity led to suppressed levels of glutathione, superoxide dismutase, catalase, and ectonucleotidase activities, with concomitant exacerbation of malondialdehyde and nitric oxide levels, ATPase, acetylcholinesterase, and α-chymotrypsin activities. These activities and levels were significantly reversed following the treatment with betulinic acid. Liquid chromatography coupled to mass spectrometry analysis revealed treatment with betulinic acid restored oxidative-depleted 8-hydroxythioguanine, adenylylselenate, adenosine tetraphosphate, and phosphatidylinositol-3,4,5-trisphosphate, while generating cysteic acid, adenosine 3′,5′-diphosphate, cytidine triphosphate, and deoxycytidine triphosphate. Pathway analysis revealed inactivation of seleno-compound metabolism, phosphatidylinositol signaling system, and inositol phosphate metabolism pathways on induction of oxidative neurotoxicity. These pathways were reactivated following treatment with betulinic acid, while concomitant activating taurine and hypotaurine metabolism, mannose type O-glycan biosynthesis, pantothenate, and CoA biosynthesis, as well as cysteine and methionine metabolism pathways. MTT assay revealed betulinic acid was not cytotoxic against HT22 cells. Molecular docking of betulinic acid showed a strong binding affinity for ATPase, with a binding energy −9.7 kcal/mol. These results suggest that betulinic acid confers neuroprotective effect against iron-mediated neurotoxicity as indicated by its ability to mitigate redox imbalance and cholinergic and proteolytic enzyme activities, with concomitant modulation of purinergic enzyme activities and dysregulated metabolic pathways in isolated rat brain tissue. Graphical Abstract: [Figure not available: see fulltext.]