Psoraleae Fructus combined with Walnut kernels improves postmenopausal osteoporosis by inhibiting ferroptosis through the Nrf2/GPX4/SLC7A11 pathway

Background: Postmenopausal osteoporosis (PMOP) is a bone metabolic disorder caused by estrogen (E2) deficiency. The traditional Chinese medicine Psoraleae Fructus (P) is often used in combination with walnut kernels (J, Juglans regia L.) to treat osteoporosis. However, whether the combination of walnut kernels and Psoraleae Fructus (PJ) is more effective than Psoraleae Fructus alone remains unclear, and the material basis and mechanism of the synergistic effects of this combination are not fully understood.

Purpose: This study aimed to elucidate the synergistic mechanisms of PJ in the treatment of PMOP and to identify the active components and their targets.

Methods: The compounds in PJ were analyzed using UPLC-MS/MS. Bilateral ovariectomy (OVX) was performed to establish a rat model of PMOP. Femoral pathological changes were evaluated by serum ELISA, micro-CT, H&E and TRAP staining. UPLC-MS/MS analysis was also performed to screen for active components in the serum, focusing on key monomers. Transcriptome Sequencing was conducted to identify core pathways. An MC3T3-E1 Ferroptosis model was established by erastin treatment. A combination index was used to evaluate the synergistic effects of the monomers. In addition to measuring cell viability, the effects of the key monomers on Ferroptosis and osteoblastic differentiation were verified using cryogenic transmission electron microscopy, confocal laser scanning microscopy, and flow cytometry. Molecular docking, molecular dynamics (MD) simulations, microscale thermophoresis (MST), immunofluorescence, and western blotting were performed to validate the targets of this activity.

Results: In vivo experimental results indicated that PJ significantly improved OVX-induced osteoporosis more prominently than P alone. The UPLC-MS/MS results showed that the serum concentrations of eight potentially active components increased significantly after compatibility with J. Transcriptome analysis revealed significant enrichment of Ferroptosis in the OVX group, whereas in vivo experiments demonstrated that PJ inhibited Ferroptosis by regulating the Nrf2/GPX4/SLC7A11 pathway. In vitro experiments identified norbakuchinic acid (NA) and α-linolenic acid (ALA) as the main pharmacodynamic components of PJ and confirmed that both components synergistically inhibited Ferroptosis while promoting osteogenic mineralization. This effect was dependent on Nrf2/GPX4/SLC7A11 pathway activation. MST and MD simulations revealed that both NA and ALA were directly bind to Keap1, thereby promoting Nrf2 nuclear translocation and triggering downstream biological responses. Furthermore, when Nrf2 expression was inhibited in vitro by ML385, the inhibitory effects of NA and ALA on Ferroptosis were suppressed.

Conclusion: This study provided the first systematic evidence that walnut kernels can enhance the therapeutic efficacy of Psoraleae Fructus against PMOP by improving the bioavailability of active constituents and forming a synergistic network of NA and ALA. Furthermore, it has been determined that the Nrf2/GPX4/SLC7A11 pathway serves as the principal mechanism through which NA and ALA synergistically inhibit osteoblast Ferroptosis.

Ferroptosis; GPX4/SLC7A11; Nrf2; Postmenopausal osteoporosis; Psoraleae fructus; Walnut kernels.