Abstract
This study investigates the potential of isolated compounds from Paeonia lactiflora to mitigate heat stress-induced male infertility in Drosophila melanogaster, with egg-hatching rates as a quantitative fertility indicator. Exposure to thermal stress (27.5 °C) significantly impaired male fertility, resulting in egg viability declining to 16.18-23.08%. Supplementation with 10 µM of paeoniflorin (1), benzoic acid (2), and albiflorin (4) significantly restored egg-hatching rates to 55.17-93.48%, demonstrating protective effects against heat stress-induced reproductive impairment. Immunofluorescence analysis of testis tissue revealed that these compounds maintained spermatogonia structural integrity under thermal stress conditions. Molecular docking analyses identified specific binding interactions between compounds 1, 2, and 4 with Vasa protein, characterized by distinct patterns of hydrogen bonding, van der Waals forces, and hydrophobic interactions. Paeoniflorin (1) exhibited the highest binding affinity (- 9.64 kcal/mol), followed by compound 4 (- 9.14 kcal/mol), while compound 2 demonstrated a lower binding affinity. Molecular dynamics simulations conducted over 200 ns confirmed the thermodynamic stability of these complexes, with root mean square deviation values converging around 0.2 nm for all compounds. Analyses of root mean square fluctuation, hydrogen bond numbers, and molecular contact surface area provided further evidence of complex stability. Moreover, the free energy landscape and MM/PBSA analyses revealed that van der Waals and electrostatic interactions make significant favorable contributions to the thermodynamics of the system. These findings elucidate the molecular mechanisms by which secondary metabolites from P. lactiflora protect against heat stress-induced male reproductive dysfunction, offering potential therapeutic strategies for mitigating heat-induced infertility.
