Thymoquinone-Loaded Chitosan Nanoparticles Combat Testicular Aging and Oxidative Stress Through SIRT1/FOXO3a Activation: An In Vivo and In Vitro Study

Abstract

<b>Background:</b> Aging is a complex biological process characterized by the accumulation of molecular and cellular damage over time, often driven by oxidative stress. This oxidative stress is particularly detrimental to the testes, where it causes degeneration, reduced testosterone levels, and compromised fertility. D-galactose (D-gal) is commonly used to model aging as it induces oxidative stress, mimicking age-related cellular and molecular damage. Testicular aging is of significant concern due to its implications for reproductive health and hormonal balance. This research examines the protection by thymoquinone (TQ) or thymoquinone-loaded chitosan nanoparticles (NCPs) against D-galactose (D-gal)-induced aging in rat testes, focusing on biochemical, histological, and molecular changes. Aging, which is driven largely by oxidative stress, leads to significant testicular degeneration, reducing fertility. D-gal is widely used to model aging due to its ability to induce oxidative stress and mimic age-related damage. TQ, a bioactive ingredient of <i>Nigella sativa</i>, has earned a reputation for its anti-inflammatory, anti-apoptotic, and antioxidant characteristics, but its therapeutic application is limited by its poor bioavailability. <b>Methods</b>: Thymoquinone was loaded into chitosan nanoparticles (NCPs) to enhance its efficacy, and this was hypothesized to improve its stability and bioavailability. Four groups of male Wistar rats participated in the study: one for the control, one for D-gal, one for D-gal + TQ, and the last one for D-gal + NCP. <b>Results</b>: The results exhibited that D-gal substantially increased oxidative injury, reduced testosterone levels, and caused testicular damage. Treatment with TQ and NCPs significantly reduced oxidative stress, improved antioxidant enzyme levels, and restored testosterone levels, with NCPs showing a stronger protective effect than TQ alone. A histological analysis confirmed that NCPs better preserved testicular structure and function. Additionally, the NCP treatment upregulated the expression of key genes of oxidative stress resistance, mitochondrial function, and reproductive health, including SIRT1, FOXO3a, and TERT. <b>Conclusions:</b> The findings suggest that NCPs offer enhanced protection against aging-related testicular damage compared with TQ alone, which is likely due to the improved bioavailability and stability provided by the nanoparticle delivery system. This research emphasizes the potential of NCPs as a more effective therapeutic strategy for mitigating oxidative stress and age-related reproductive dysfunction. Future research should further explore the mechanisms underlying these protective effects.