Astragalus Polysaccharides Protect against Di-n-butyl phthalate-induced testicular damage by modulating oxidative stress, apoptosis, and the PI3K/Akt/mTOR pathway in rats

Manal Bakeer¹seham samir soliman^2*Omaima Ahmed¹Fady Sayed¹Ghada E. Ali^1,3Nada Aljarba^4,5George Zouganelis⁶Maha M. Rashad³

• ¹Cairo University, Giza, Giza, Egypt
• ²Department of Animal Production, National Research Centre (Egypt), Giza, Egypt
• ³Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Cairo University, Giza, Beni Suef, Egypt
• ⁴Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
• ⁵Department of Biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia
• ⁶School of Electrical Engineering & Computer Science, College of Engineering & Mines, University of North Dakota, Grand Forks, North Dakota, United States

Di-n-butyl phthalate (DBP), a widely used plasticizer, is linked to oxidative stress and male reproductive toxicity. Astragalus polysaccharides (APS) possess antioxidative and antiinflammatory properties, but their role in male reproductive health remains unclear. This study investigates the protective effects of APS against DBP-induced testicular injury in rats. Twentyfour male rats were randomly assigned to four groups (n = 6 per group): control, DBP-only (500 mg/kg/day), APS-only (200 mg/kg/day), and APS+DBP (500 mg/kg/day DBP + 200 mg/kg/day APS). Treatments were administered via oral gavage for eight weeks. DBP exposure significantly reduced serum testosterone levels, catalase (CAT) activity, lactate dehydrogenase (LDH) activity, and sperm quality while increasing malondialdehyde (MDA) levels and apoptotic markers (Casp3, Casp9). APS co-treatment mitigated these effects by enhancing antioxidant enzyme activity, lowering MDA levels, and restoring testicular function. Histological analysis revealed reduced necrosis and degeneration in APS-treated rats compared to the DBP-only group. APS also modulated oxidative stress-related genes (Nrf2, SOD) and influenced the PI3K/AKT/mTOR signaling pathway. These findings suggest that APS effectively protects against DBP-induced oxidative stress and reproductive toxicity. APS may serve as a promising therapeutic agent to help prevent male infertility caused by environmental toxins.Keywords dibutyl phthalate, Astragalus polysaccharides, oxidative stress, reproductive toxicity, apoptosis, PI3K/AKT/mTOR pathway, male infertility. integrity (3-5). An excess of ROS disrupts this equilibrium, leading to oxidative damage.Additionally, DBP directly impairs Leydig cell function, resulting in decreased weights of the testis and accessory reproductive organs and reduced testosterone levels (6). Recent studies have suggested that antioxidants may mitigate DBP-induced reproductive dysfunction. For instance, coadministration of naringin, an antioxidant flavonoid, with DBP improved reproductive performance in animal models, highlighting oxidative stress as a central mechanism underlying DBP toxicity (7,8).Astragalus polysaccharides (APS), bioactive components derived from Astragalus membranaceus, exhibit potent antioxidant, anti-inflammatory, immune-modulating, and anticancer properties (9).Although APS have been reported to confer protection against oxidative damage in various tissues, their role in preventing DBP-induced testicular toxicity remains unexplored. This study aims to investigate the toxic effects of DBP on the testes and evaluate the protective efficacy of APS against DBP-induced reproductive toxicity. By analyzing physiological, biochemical, pathological, and molecular parameters,