Molecular Assessment of Nano-Hesperetin in Restoring Testosterone Biosynthesis and Antioxidant Balance in Testicular Toxicity

Abstract

Chemotherapeutic drugs still represent a major clinical challenge for testicular toxicity mainly due to the persistent oxidative insult that disrupt steroidogenesis and spermatogenic function. Hesperetin, an aglycone citrus flavonoid, is widely known for its antioxidant action, but its medicinal utility is hampered by its poor water solubility and modest oral bioavailability. In the present study, hesperetin nanoparticles (Nano-Hesperetin) were synthesised by an antisolvent precipitation-ultrasonication method and characterised by FTIR, XRD and FESEM. Forty adult male Wistar rats were randomly allocated into four groups: Control, Cyclophosphamide (CP, 150 mg/kg), CP + free Hesperetin (50 mg/kg/day) and CP + Nano-Hesperetin (25 mg/kg/day). Serum testosterone, LH and FSH were tested by ELISA and testicular SOD, CAT, GSH and MDA were determined spectrophotometrically. The mRNA expression of StAR, CYP17A1 and Nrf2 was determined by quantitative RT-PCR. FESEM results showed the presence of almost spherical particles with an average particle size of around 81 nm. The crystalline nature of hesperetin was confirmed by the XRD. CP significantly reduced serum levels of testosterone and gonadotropins, depleted enzymatic antioxidants, increased MDA and downregulated the expression of steroidogenic and Nrf2 genes. When administered together, nano-hesperetin restored the hormonal balance, refilled the antioxidant pool and reactivated the StAR/CYP17A1 axis to near control levels, but free hesperetin only partially restored these parameters. The nano-formulation provided much better protection than the bulk flavonoid suggesting that reduction in particle size leads to improved testicular protection by jointly restoring steroidogenic gene expression and redox equilibrium.