Integrative LC-MS/MS, Bioinformatics, and Molecular Dynamics Reveal Multitarget Antioxidant Mechanisms of Bushen...

Intervertebral disc degeneration (IVDD) is a major cause of low back pain and disability. Excessive reactive oxygen species (ROS) disrupt disc homeostasis. Bushen Huoxue (BSHX) decoction, a traditional Chinese medicine, shows clinical efficacy in IVDD, but its antioxidant mechanisms are not well understood. Public IVDD transcriptomic datasets were analyzed to identify ROS-related gene signatures, followed by immune infiltration assessment. LC-MS/MS was used to characterize the chemical profile of BSHX decoction. Network pharmacology, machine learning, and bioinformatics analyses were integrated to identify potential compound-target interactions. Molecular docking and molecular dynamics simulations were applied to validate binding stability between active components and key targets. Six ROS-related hub genes (NCF1C, SLC11A1, MPO, GSTA4, SLC7A11, AKR1C3) were identified, with immune profiling revealing increased neutrophils, NK cells, and pro-inflammatory macrophages in IVDD. LC-MS/MS identified 77 active compounds in BSHX decoction, including flavonoids and phenolic acids. Network analysis and docking suggested strong interactions with oxidative stress targets, notably MPO. Molecular dynamics simulations confirmed the stability of key compound-target complexes. BSHX decoction shows promise as a multitarget antioxidant therapy for IVDD, offering mechanistic insight into its traditional efficacy. These findings support its further development and experimental validation.