Ursolic Acid and Caprylic Acid Cocktail Approach Against Pentylenetetrazole-induced Seizures-like Behavior in Adult Zebrafish: Preclinical Study

Epilepsy is a neurological disorder characterized by recurrent seizures. Pentylenetetrazole (PTZ) is a chemoconvulsant that impairs GABAergic and glutamatergic neurotransmission, promoting excitotoxicity and seizures. Ursolic acid (UA) and caprylic acid (CA) have anti-inflammatory, anti-oxidant, and neuroprotective properties. The objective of the present study was to investigate the combined effect of UA and CA on seizures, neuronal damage, and inflammation induced by PTZ in adult zebrafish. Adult zebrafish (~6-8 months old; 470-530 mg, n=20) were randomly assigned to 10 different groups namely- I) vehicle: 10% DMSO (20µl/kg, i.p), II) Diazepam 1.25 mg/kg per se (DZP, i.p), III) UA 150 mg/kg per se (i.p), IV) CA 60 mg/kg per se (i.p), V) PTZ 170 mg/kg (i.p), VI) DZP 1.25 + PTZ 170 (30 min after DZP 1.25), VII) UA 50 + PTZ 170 (30 min after UA 50), VIII) UA 150 + PTZ 170 (30 min after UA 150), IX) CA 60 + PTZ 170 (30 min after CA 60), and X) UA 50 and CA 60 + PTZ 170 (30 min after UA 50 and CA 60) administration followed by seizure scoring, neurobehavioral (Novel tank test and open field test), biochemical [lipid peroxidase (LPO), acetylcholinesterase (AChEs), superoxide dismutase catalase (SOD), and glutathione-s-transferase (GSH)], molecular (TNF-α, IL-10, Nrf-2 and IL-1β), mitochondrial (complex I, II, IV), cell viability assay (MTT assay) and histopathological analysis. UA of both doses and CA decreased mean seizure score, and mean seizure time. Importantly, combination of UA 50 mg/kg and CA 60 mg/kg attenuated seizure-like behavioural scores, decreased mean seizure time, mean seizure score and reduced the frequency of clonic-like seizures (score 4) in PTZ-treated zebrafish. A combination of UA 50 mg/kg and CA 60 mg/kg also prevented oxidative stress in PTZ-challenged fish by decreasing lipid peroxidation, AChEs activity and increasing catalase, GSH and SOD levels. Additionally, the combination therapy prevented inflammatory response by declining TNF-α and IL-1β levels and raising IL-10 and Nrf-2 levels. Moreover, combination of UA 50 mg/kg and CA 60 mg/kg significantly improved mitochondrial complex I, II and IV activities as well as increase mitochondrial viability in MTT assay. Furthermore, morphology of neuronal cell was prevented in combination of UA 50 mg/kg and CA 60 mg/kg as seen in H & E staining. The protective effect of UA 50 mg/kg and CA 60 mg/kg cocktail is comparable with standard drug Diazepam. Together, we demonstrate that UA 50 mg/kg and CA 60 mg/kg cocktail significantly attenuated PTZ-induced seizure-like behaviours, brain oxidative stress, mitochondrial dysfunction and morphological damage of neuronal cell in zebrafish, suggesting the involvement of its strong anti-inflammatory and antioxidant mechanisms in neuroprotection.