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

Abstract

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.