Based on network pharmacology and experimental validation of β-sitosterol alleviates cisplatin-induced hearing loss via phosphorylated MAPK3.

PURPOSE: This research aimed to investigate the protective role as well as molecular mechanisms of Rehmanniae Radix Praeparata and its bioactive compound β-sitosterol in preventing CIHL. METHODS: Network pharmacology and bioinformatic analyses were used to identify the pharmacologically active constituents of Rehmanniae Radix Praeparata and predict their potential molecular targets. The influence of β-sitosterol on hair cell damage, apoptosis, reactive oxygen species (ROS) levels, and target protein expression were evaluated in vitro as well as in vivo. RESULTS: The primary active constituents of Rehmanniae Radix Praeparata were β-sitosterol and stigmasterol. They shared 50 potential targets with CIHL, with MAPK3, SRC, and ESR1 as the top three. Molecular docking combined with dynamics simulations demonstrated strong binding and stable complex formation between β-sitosterol and MAPK3. In vitro, β-sitosterol mitigated cisplatin-induced hair cell death, reduced ROS levels, and restored phosphorylated MAPK3. In vivo, it enhanced hearing, decreased hair cell loss, and partially restored phosphorylated MAPK3 in the cochleae of cisplatin-treated mice. CONCLUSION: Rehmanniae Radix Praeparata and its active component β-sitosterol protected cochlear hair cells from cisplatin-induced damage by restoring MAPK3 phosphorylation signaling pathway, providing new molecular targets and strategies for CIHL treatment.