Downregulation of Glycogen Synthase Kinase-3 Beta by Urtica dioica L. Contributes to Neuroprotection Against Aluminum Chloride-Mediated Neurotoxicity in Zebrafish Larvae.

Urtica dioica L. (U. dioica; Family Urticaceae) has been utilized since ancient times, both as a nutritional source and for its therapeutic properties in addressing multiple disease conditions. The present study aimed to assess the neuroprotective effect of a hydroalcoholic U. dioica leaf extract (Ud) in a zebrafish larva model of aluminum-induced neurotoxicity. The phytochemical quantification of Ud was performed using a high-performance liquid chromatography (HPLC) photodiode array detection method, and the molecular interactions of the identified compounds were studied using in silico docking simulations. Zebrafish embryos were exposed to aluminum chloride (AlCl) with or without Ud. Whole-mount in situ imaging using dichlorofluorescein diacetate and NeuN was performed in the grown larvae to study oxidative stress and neuronal loss, respectively. Quantitative real-time PCR and western blot analyses measured the expression of selected genes and proteins. Neurochemical estimation was performed by the HPLC electrochemical detection method. HPLC-based analysis quantified 12 major flavonoids and phenolic compounds in Ud. The benchmark dose lower confidence limit of the Ud was found to be 127.63 µg/mL. The extract treatment alleviated Al-induced locomotor deficit in larvae and reduced oxidative stress and neuronal damage in the cephalic region of the larvae. Modulation of gene expression related to oxidative stress, inflammation, apoptosis, and catenin beta was observed in larvae treated with Ud. The extract increased the brain expression of β-catenin, p-Gsk-3β(Ser), and p-CREB(Ser) proteins and also restored the brain levels of gamma-aminobutyric acid and glutamate. The results concluded that Ud protects against AlCl-induced neurotoxicity in zebrafish larvae.