Nano‐Rosmarinic Acid Protects Against Chlorfenapyr‐Induced Testicular Toxicity Through Modulation of NRF2/HO‐1 and NF‐κB/NLRP3 Signaling Pathways

Chlorfenapyr (CFP) is a widely used pesticide associated with significant toxicological effects, including reproductive toxicity. Rosmarinic acid (RA) possesses potent antioxidant and anti-inflammatory properties; however, its therapeutic application may be limited by poor physicochemical characteristics. This study investigated the molecular mechanisms underlying CFP-induced testicular toxicity and evaluated whether nano-encapsulation of RA within Chitosan nanoparticles (RA-CHNPs) improves its protective efficacy against CFP-induced reproductive damage. A total of 60 adult male Wistar rats were allocated into six groups (n = 10/group): control, RA, RA-CHNPs, CFP, CFP + RA, and CFP + RA-CHNPs. Animals received oral CFP (180 mg/kg/day) and/or RA (75 mg/kg/day, crude or nano-encapsulated) for 30 consecutive days. CFP exposure significantly reduced testicular weight, sperm count, sperm motility, luteinizing hormone, follicle-stimulating hormone, and testosterone levels. These alterations were accompanied by oxidative stress, increased pro-inflammatory cytokine production, apoptosis, and activation of NF-κB/NLRP3 signaling. Immunohistochemical analysis revealed marked NF-κB expression in spermatogenic and Leydig cells, together with strong NLRP3 expression within seminiferous tubules. Co-administration of RA-CHNPs was associated with a marked attenuation of these adverse effects, denoting greater protective efficacy than crude RA. RA-CHNPs reduced NF-κB and NLRP3 expression, which was accompanied by improved testicular architecture, restored spermatogenic activity, and normalized reproductive hormone levels. These protective effects were associated with activation of the NRF2/HO-1 antioxidant pathway, suppression of inflammatory responses, and regulation of apoptosis-related genes. Overall, RA-loaded Chitosan nanoparticles provided superior protection against CFP-induced testicular toxicity compared with crude RA, highlighting the potential of nanoformulation strategies to enhance RA's biological efficacy.