Ursolic acid ameliorates 3-Nitropropionic acid-induced Huntington’s disease-like symptoms in adult zebrafish: in-silico, in-vitro and in-vivo evidences

Background 3-Nitropropionic acid (3-NP), a natural mycotoxin, induces Huntington's Disease (HD)-like pathology and motor abnormalities. Ursolic acid (UA), a triterpenoid, possesses antioxidant and anti-inflammatory properties. The study investigated the therapeutic potential of UA against HD using network pharmacology and molecular docking to identify its possible molecular targets and mechanisms. Its efficacy was further supported through in-silico, in-vitro and in-vivo studies. In vitro, UA showed protective effect against 3-NP-induced toxicity in the SHSY-5Y cell line, while in vivo it demonstrated protection against 3-NP-induced neurotoxicity in adult zebrafish. Methodology The investigation commenced with in-silico analyses for the lead chemical using SwissTargetPrediction, where UA targets were forecasted, and HD-related genes were discovered via UniProt and GeneCards. A protein interaction network was established using STRING and displayed with Cytoscape. Docking investigations encompassed UA, 3-NP, TBZ, and KEAP1 together with IKB kinase. In vitro, SH-SY5Y cell lines were utilized, while in vivo investigations involved adult zebrafish, which were randomly divided into seven groups and pre-treated with UA (50 and 100 mg/kg; i.p.) and tetrabenazine (TBZ; 3 mg/kg; i.p.) for three days, followed by administration of 3-NP (15 mg/kg; i.p.) for five days. Thorough evaluations included behavioral, biochemical, molecular, mitochondrial, and histopathological investigations. Results In-silico analyses revealed that UA exhibits stronger binding affinity as compared to 3-NP. In vitro, SH-SY5Y cell lines treated with UA alleviated 3-NP-induced neuronal damage by stabilizing mitochondrial potential, diminishing oxidative stress, and inhibiting neuroinflammation through the NF-κB/NLRP3 and Nrf2 pathways. In vivo findings further demonstrated that UA pre-treatment ameliorated behavioural deficits (NTT and OFT), reduced oxidative stress markers (LPO, SOD and GSH), quelled neuroinflammation TNF-α and IL-1, and enhanced mitochondrial function compared to 3-NP-treated zebrafish. Additionally, UA downregulated GSK-3β, MAPK14 expression while upregulating AMPK levels and improvements in mitochondrial structure. Further, pre-treatment with Plumbagin significantly improved mitochondrial morphology (as observed through TEM analysis), and neuronal morphology (assessed via Haematoxylin and Eosin Staining an Nissl staining) as compared to 3-NP-treated zebrafish. Conclusion UA demonstrates potential in mitigating neurotoxicity and offering neuroprotection, potentially via the control of the Nrf2 pathway, which may aid in reducing symptoms of HD. Our research indicates that ursolic acid has the potential to lower neurotoxicity and may provide neuroprotective benefits; yet additional investigation is required to fully elucidate the underlying mechanisms.