Astragaloside IV Ameliorates Atrazine-Induced Male Reproductive Toxicity: An In vivo and In Silico Analysis

Srinivasa Rao Sirasanagandla¹Mohamed Al Mushaiqri¹Firas Al-Majrafi²Nadia Al- Abri³Selvaraj Jayaraman⁴Isehaq Saif Al Huseini^1*

• ¹Department of Anatomy, Sultan Qaboos University College of Medicine and Health Sciences, Seeb, Oman
• ²Sultan Qaboos University College of Medicine and Health Sciences, Seeb, Oman
• ³Department of Pathology, Sultan Qaboos University College of Medicine and Health Sciences, Seeb, Oman
• ⁴Centre of Molecular Medicine and Diagnostics, Department of Biochemistry, SIMATS Deemed University Saveetha Dental College, Chennai, India

Atrazine (ATZ) stands as the most widely utilized herbicide globally, and is known for its adverse impacts on the reproductive system. Although astragaloside IV (AS IV) is well known for possessing various health benefits, its protective effects against the ATZ-induced toxicity remain unexplored. This study aimed to investigate the ameliorative potential of AS IV against ATZ-induced male reproductive toxicity in mice. Eight-week-old CD-1 mice were allocated into four groups (n=10). The ATZ and AS IV were administered at a dose of 100 mg/kg/day and 40 mg/kg/day, respectively. Treatments were continued for 21 days, followed by sacrifice for plasma biochemical analyses and testes collection for histo-pathological examination. One-way ANOVA followed by Bonferroni’s multiple comparison test was used for data analysis. Molecular docking studies were performed to evaluate ATZ and AS IV interactions with oxidative stress and inflammation-related proteins, including GSH, GPx, SOD, and Nrf2, NF-β, IL-1β, IL-6, TNF-α, cullin-3, and keap-1. Biochemical analysis revealed significant reductions in the GSH levels (p<0.001), SOD (p<0.001), and GPx activity (p<0.05), alongside elevated malonaldehyde levels (p<0.01) following ATZ exposure. AS IV treatment in ATZ-exposed mice significantly improved these markers (p<0.05). ATZ exposure led to significant decreases in testosterone (p<0.001) and androgen-binding protein (ABP) levels (p<0.001) within the ATZ group, whereas AS IV supplementation significantly improved these markers (p<0.05). Histopathological examination revealed sloughed and collapsed seminiferous epithelia with vacuoles and poorly formed spermatids in ATZ-exposed mice, which were mitigated by AS IV treatment. The docking study revealed ATZ's moderate interactions with key oxidative stress and inflammation-related proteins (binding energies: -4.7 to -5.5 kcal/mol), with glutathione (-5.5 kcal/mol) showing the strongest binding. Notable stabilizations include SOD (3 hydrogen bonds) and modulation of antioxidants (SOD, Nrf2) and anti-inflammatory (IL-1β, TNF-α) pathways. Moreover, AS IV demonstrated significant binding affinities between glutathione (-9.2 kcal/mol), cullin-3 (-9.1 kcal/mol), and keap-1 (-8.9 kcal/mol). MD simulations showed strong stability for GPx and IL-1β target against ATZ, and AS IV exhibited strong stability for GSH and cullin-3. AS IV appears to be a promising natural compound for preventing ATZ-induced male toxicity. Further investigations to elucidate the molecular mechanisms behind such positive effects are warranted.