Dietary supplementation of Lactobacillus casei alleviates permethrin exposure-induced zebrafish testis damage through modulation of TLR4/NF-κB and AKT/Nrf2 pathways: Oxidative stress, inflammation and ferroptosis

The widespread use of permethrin (Per) poses significant risks to aquatic ecosystems, particularly through reproductive toxicity. This study investigated the protective effects of Lactobacillus casei (L. casei) against Per-induced testicular damage in zebrafish (Danio rerio) and elucidated the underlying molecular mechanisms. Male zebrafish were exposed to environmentally relevant concentrations of Per (1 μg/L) and supplemented with different doses of L. casei (10⁷-10⁹ CFU/g) or heat-killed controls for 30 days. Results showed that Per exposure suppressed growth parameters, disrupted sex hormone homeostasis and induced histopathological testicular lesions, including spermatogenic tubule disorganisation and reduced sperm density. Mechanistically, Per induced oxidative stress and activated inflammatory cascades (IL-1β, IL-6, TNF-α upregulation) via TLR4/NF-κB signaling, while suppressing Akt/Nrf2-mediated antioxidant responses. Notably, Per exposure promoted Ferroptosis as evidenced by Fe²⁺ accumulation and GPX4/SLC7A11 downregulation. Dietary L. casei supplementation (10⁸ CFU/g optimal dose) dose-dependently reversed these effects, restoring redox balance, attenuating inflammation and reactivating Nrf2-driven antioxidant defences. Transcriptomic analysis revealed that L. casei modulated 3732 differentially expressed genes, predominantly in the TLR4/NF-κB and PI3K-AKT pathways. Pharmacological validation confirmed that L. casei protection was based on dual regulation: TLR4 inhibition (via TAK-242) abolished the anti-inflammatory effects, while Akt activation (via SC79) increased Nrf2 expression. Crucially, heat-killed L. casei failed to attenuate Per toxicity, highlighting the need for viable probiotics. This study provides novel evidence that L. casei alleviates Per-induced testicular injury through coordinated suppression of oxidative inflammation and Ferroptosis, mediated by TLR4/NF-κB inactivation and Akt/Nrf2 pathway activation. These findings advance strategies for probiotic-based interventions in aquatic toxicology.

AKT/Nrf2; Ferroptosis; Lactobacillus casei; Oxidative stress; Permethrin; TLR4/NF-κB; Testicular injury.