Titanium nanoparticles provoke ferritinophagy-mediated ferroptosis via inhibition of the Nrf2-HO-1 signaling pathway in osteocytes.

Wear particles-induced osteolysis is the primary cause of aseptic loosening and subsequent joint replacement failure. Osteocytes, the most abundant cells in bone, are in direct contact with implants; however, the interaction between osteocytes and wear particles remains largely unelucidated. In the present study, in vivo results showed that titanium (Ti) nanoparticles (with diameters of 40 nm and 800 nm) markedly triggered bone destruction and increased osteocytes death, and this effect was mitigated by the ferroptosis inhibitor ferrostatin-1 (Fer-1). Our in vitro experiments revealed that Ti nanoparticles significantly inhibited the viability of osteocytes MLO-Y4, and induced cell death. Meanwhile, Ti nanoparticles remarkably promoted ferroptosis in MLO-Y4 cells, as evidenced by iron overload, lipid peroxidation accumulation, downregulated protein expressions of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), and glutathione (GSH) depletion. Notably, this ferroptotic response was associated with enhanced ferritinophagy and degradation of ferritin heavy chain 1 (FTH1), since inhibition of ferritinophagy with 3-methyladenine (3-MA) alleviated Ti nanoparticles-induced ferroptosis. Additionally, Ti nanoparticles significantly suppressed the erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1(HO-1) pathway; the Nrf2-specific activator sulforaphane (SFN) reversed Ti nanoparticles-induced Fe²⁺ accumulation and lipid peroxidation, attenuated ferroptosis and ferritinophagy, and thereby restored the viability of MLO-Y4 cells. Collectively, these findings identify ferritinophagy-mediated ferroptosis as a novel mechanism underlying Ti nanoparticles-induced osteocytes death, and demonstrate that the Nrf2/HO-1 pathway is essential for regulating Ti nanoparticles-induced ferritinophagy and ferroptosis. These results further suggest that targeting the Nrf2/HO-1pathway may represent a promising therapeutic strategy for preventing or alleviating ferritinophagy-mediated ferroptosis in wear particles-related osteoclastogenesis and periprosthetic osteolysis.