Hydrogen Sulfide Suppresses Osteoclast Differentiation via Nrf2/GPX4 Pathway in RAW264.7 Cells

Excessive osteoclast activity is a primary driver of bone-resorptive disorders like osteoporosis, leading to bone loss and fragility. Current therapeutic strategies often have limitations, including side effects or incomplete efficacy. Oxidative stress is recognized as a crucial regulator of osteoclast differentiation and function, presenting a potential therapeutic target. Hydrogen sulfide (H2S), an endogenous gasotransmitter, possesses potent antioxidant properties, suggesting its potential to modulate osteoclastogenesis. However, the precise molecular mechanisms by which H2S influences this process, particularly concerning redox signaling, have remained largely undefined, representing a significant knowledge gap this study aims to address.

Researchers investigated the effects of hydrogen sulfide on osteoclast differentiation using RAW264.7 cells, a common macrophage cell line that differentiates into osteoclasts. Osteoclastogenesis was induced by RANKL (50 ng/mL). The H2S donor, sodium hydrosulfide (NaHS), was co-administered at concentrations ranging from 50-200 µM. Primary endpoints included evaluating osteoclast formation via TRAP staining and assessing the expression of key osteoclast markers like NFATc1 and Cathepsin K. The study also quantified oxidative stress markers, lipid peroxidation, mitochondrial function, and ferroptosis-related parameters. Mechanistic insights were sought by examining Nrf2 nuclear translocation and the expression of GPX4, HO-1, and NQO1 through western blot and qPCR analyses, with specific inhibitors used to confirm pathway involvement.

The administration of the H2S donor, NaHS, significantly suppressed RANKL-induced osteoclast differentiation in RAW264.7 cells across the tested dose range. This inhibitory effect was accompanied by a marked alleviation of oxidative stress and a restoration of mitochondrial function, both critical factors in osteoclastogenesis. Mechanistic investigations revealed that NaHS promoted the nuclear translocation of Nrf2, a master regulator of antioxidant responses. This activation led to the upregulation of key antioxidant enzymes, including GPX4, HO-1, and NQO1. Crucially, when Nrf2 or GPX4 were inhibited, the suppressive effects of NaHS on osteoclast differentiation were partially reversed, confirming the involvement of this specific redox axis. This indicates a direct link between H2S, the Nrf2/GPX4 pathway, and the regulation of bone-resorbing cells.

NaHS significantly suppressed RANKL-induced osteoclast differentiation, alleviated oxidative stress, and restored mitochondrial function, primarily by activating the Nrf2/GPX4 pathway.