Geniposide activates the transcription of Ndufs8 and enhances PD-L1 blockade for inhibiting growth of osteosarcoma

Geniposide, a key bioactive component of Gardenia jasminoides, is an iridoid glycoside known for its therapeutic potential in various diseases characterized by oxidative stress or inflammation, including atherosclerosis, brain disorders, and diabetes. However, its effects on osteosarcoma and the underlying mechanisms are unknown. Therefore, we explored the impact of geniposide on osteosarcoma growth through proteomic analysis. After subcutaneous tumorigenesis of osteosarcoma cells proteomic alterations were analyzed. GO and KEGG pathway analyses were performed to identify enriched pathways and critical proteins. Immunohistochemistry and Western blotting were conducted to measure the protein expression of AMPK, SIRT1, OVOL3, and Ndufs8. Flow cytometry, CCK-8, and colony formation assays were employed to assess osteosarcoma growth. ELISA assay was utilized to measure alterations in pyruvate, lactate, and ATP following geniposide treatment. ChIP were performed to confirm OVOL3 transcriptionally regulates Ndfus8. The results demonstrated that geniposide inhibited osteosarcoma growth by 45–65 % in tumor weight, with proteomic data revealing oxidative phosphorylation as the critical pathway. Geniposide reduced Ki-67 and PCNA expression by 30–55 %, while increasing apoptosis rates by 2.5-fold. Mechanistically, Ndufs8 was upregulated 1.8–2.3-fold by geniposide, enhancing ROS levels by 40–60 % and suppressing osteosarcoma cell viability by 50–70 %. Additionally, geniposide suppressed PD-L1 expression by 40–50 % and synergized with PD-L1 blockade. These findings reveal that geniposide inhibits osteosarcoma through dual mechanisms: Ndufs8-mediated oxidative phosphorylation activation and PD-L1/PD-L1 axis suppression, offering a novel therapeutic strategy. These findings suggest that geniposide is a promising therapeutic agent for osteosarcoma and may enhance the efficacy of immunotherapy combined with PD-L1 blockade.