Does ginsenoside Rg1 promote intervertebral disc repair? An experimental study insights into ferroptosis mechanism

Abstract Background Intervertebral disc degeneration (IVDD) is a complex and multifactorial condition characterized by the progressive deterioration of the intervertebral discs. Ginsenoside Rg1, a bioactive compound isolated from Panax ginseng C.A.Mey., that has demonstrated promising therapeutic potential in the treatment of IVDD. Methods This study employed a multi-faceted approach to investigate the therapeutic effects of ginsenoside Rg1 on IVDD. Initially, histopathology, magnetic resonance imaging (MRI) were performed in clinical IVDD patients. Subsequently, histopathology, safranin green staining, X-ray, and MRI were utilized to evaluate the efficacy of ginsenoside Rg1 in alleviating in a rat model in vivo. Transcriptomics and gene set enrichment analysis (GESA) were conducted, and a network pharmacology visualization of ginsenoside Rg1-ferroptosis key targets-pathways-IVDD was constructed, along with molecular docking of ginsenoside Rg1 and targets, to identify the signaling pathways and proteins associated with the therapeutic effects of ginsenoside Rg1 on alleviating IVDD. Additionally, an Hydrogen peroxide (H2O2)-induced degeneration model of nucleus pulposus cells (NP cells) was used to evaluate the efficacy of ginsenoside Rg1 in alleviating IVDD in vitro. Methods including lipid-reactive oxygen species (ROS) detection, enzyme-linked immunosorbent assay (ELISA), FerroOrange staining, and transmission electron microscopy were employed to validate the effect and mechanism of ginsenoside Rg1 on alleviating IVDD in vivo and in vitro. ML385, a nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor, was used to reverse the effect of ginsenoside Rg1 in mitophagy and ferroptosis, respectively. The expression of proteins was assessed on immunochemical, immunofluorescence, and western blotting techniques. Results Significant ferroptosis was observed in the NP tissue of IVDD patients, with more effects in patients with higher imaging grades. Ginsenoside Rg1 significantly mitigated IVDD in rats and promoted intervertebral disc repair. Network pharmacology and transcriptomics analyses indicated the key targets of ginsenoside Rg1 for the treatment of IVDD, including NRF2, glutathione peroxidase 4 (GPX4), solute carrier family 7a member 11 (SLC7A11), and ferritin light chain 1 (FTL1). Molecular docking exhibited that ginsenoside Rg1 had good binding ability between ginsenoside Rg1 and these ferroptosis key targets. Ginsenoside Rg1 reduced the expression of ROS and malondialdehyde (MDA), decreased Fe2+ content, increased the expression of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and upregulated the expression of ferroptosis key proteins NRF2, GPX4, FTL1, and SLC7A11 in intervertebral disc tissues and NP cells. Treatment with ML385 attenuated the ginsenoside Rg1-induced upregulation of these proteins in NP cells, thereby promoting ferroptosis and reversing the protective effects of ginsenoside Rg1. Conclusions Ginsenoside Rg1 can mitigate IVDD by inhibiting ferroptosis in NP cells. The NRF2/GPX4 pathway was validated as the key ferroptosis pathway through which ginsenoside Rg1 exerts its therapeutic effects on IVDD. Graphical Abstract