Bu-Shen-Tian-Jing Formula alleviates oxidative-inflammatory stress in granulosa cells of polycystic ovary syndrome through AGEs-RAGE/NOX4/NF-κB pathway

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent reproductive endocrine disorder. The traditional Chinese medicine Bu-Shen-Tian-Jing Formula (BSTJF) has demonstrated efficacy in ameliorating PCOS-related pathologies, however its therapeutic mechanisms remain incompletely understood. This study aimed to investigate the pharmacological mechanisms by which BSTJF improves ovarian microenvironment in PCOS. METHODS: BSTJF-containing serum was applied to PCOS granulosa cells (GCs) in vitro for cellular functional assays and transcriptomic sequencing, combined with mass spectrometry-based identification of bioactive components. Network pharmacology and molecular docking were employed to predict multi-target mechanisms of BSTJF against PCOS. In vivo validation utilized an androgen-induced PCOS mouse model divided into five groups: control, PCOS, low-dose BSTJF, high-dose BSTJF, and FPS-ZM1 (RAGE inhibitor). The estrous cyclicity, glucose tolerance, reproductive hormones, ovarian morphology, and granulosa cell apoptosis of mice were detected. Serum inflammatory cytokines and biomarkers of oxidative stress in ovarian GCs were measured. Untargeted metabolomics was employed for comprehensive metabolic profiling in the serum of mice. Molecular analyses included AGEs-RAGE-NOX4 axis expression in GCs, paralleled by p38 MAPK phosphorylation kinetics and NF-κB p65 nuclear translocation dynamics. RESULTS: Transcriptomic analysis identified differentially expressed genes with significant enrichment in the AGEs-RAGE signaling pathway, revealing oxidative-inflammatory regulatory hubs (NOX4, SOD3, GPX2; TNF, TLR7, CCR2). Network pharmacology provided supports of BSTJF's multi-target engagement, demonstrating high-affinity interactions between its bioactive components and core targets. In vivo, BSTJF mirrored the RAGE inhibitor FPS-ZM1's efficacy by ameliorating PCOS phenotypes through reducing GC apoptosis, attenuating AGEs accumulation, inflammatory cytokines and state of oxidative stress, normalizing carbohydrate metabolism and lipid homeostasis, and inhibiting AGEs-RAGE-NOX4 axis activation and NF-κB nuclear translocation in ovarian GCs. CONCLUSION: Our study indicated that BSTJF could ameliorate oxidative-inflammatory stress in ovarian GCs of PCOS through AGEs-RAGE/NOX4/NF-κB pathway.