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Background <p>Osteoarthritis (OA) is a progressive joint disorder characterized by cartilage degradation and increased oxidative stress. Recently, OA pathogenesis has been related to ferroptosis, an iron-dependent programmed cell death associated with oxidative damage. However, its regulatory mechanisms remain unclear. We sought to clarify the involvement of Targeting Protein for Xklp2 (TPX2) in ferroptosis regulation and its interaction with the nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) pathway in OA.</p> Methods <p>To mimic OA conditions in vitro, C28/I2 chondrocytes were incubated with 5&#xa0;µg/mL Lipopolysaccharide (LPS) for a 24-hour period. Intracellular iron levels (Fe²⁺, Fe³⁺, total iron) were measured using colorimetric assays. Glutathione peroxidase (GSH-Px) activity and glutathione (GSH/GSSG) levels were determined. Mitochondrial morphology was examined by transmission electron microscopy. Key ferroptosis markers (<i>Solute Carrier Family 7 Member 11</i>, <i>SLC7A11</i>; <i>Glutathione Peroxidase 4</i>, <i>GPX4</i>) were analyzed at the mRNA and protein levels. Cellular responses were evaluated utilizing cell counting kit-8 (CCK-8) viability assays, 5-Ethynyl-2’-deoxyuridineb (EdU) proliferation staining, and flow cytometry.</p> Results <p>LPS treatment significantly increased intracellular iron accumulation while decreasing GSH-Px activity and GSH/GSSG levels, accompanied by characteristic mitochondrial damage. <i>TPX2</i> expression was significantly elevated in LPS-induced chondrocytes, whereas <i>TPX2</i> knockdown reversed ferroptosis-related biochemical alterations and restored <i>GPX4</i> and <i>SLC7A11</i> expression via inhibition of NF-κB p65 phosphorylation. Moreover, NF-κB p65 overexpression attenuated these protective effects of <i>TPX2</i> silencing. Ferroptosis activation by erastin abolished the protective effects conferred by <i>TPX2</i> knockdown.</p> Conclusion <p>Our in vitro findings suggest that TPX2 may promote ferroptosis in LPS-induced C28/I2 chondrocytes via NF-κB p65 signaling, reducing antioxidant defenses. Further in vivo and clinical studies are needed to clarify its role and therapeutic potential in OA.</p>

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TPX2 promotes ferroptosis in LPS-induced C28/I2 chondrocytes via NF-κB p65-mediated downregulation of GPX4 and SLC7A11

  • 期刊论文

  • 发表日期：

    2025年12月10日

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  • Biao Chen,
  • Huashun Liu,
  • Yaming Xie,
  • Jingtao Yu

摘要

Background

Osteoarthritis (OA) is a progressive joint disorder characterized by cartilage degradation and increased oxidative stress. Recently, OA pathogenesis has been related to ferroptosis, an iron-dependent programmed cell death associated with oxidative damage. However, its regulatory mechanisms remain unclear. We sought to clarify the involvement of Targeting Protein for Xklp2 (TPX2) in ferroptosis regulation and its interaction with the nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) pathway in OA.

Methods

To mimic OA conditions in vitro, C28/I2 chondrocytes were incubated with 5 µg/mL Lipopolysaccharide (LPS) for a 24-hour period. Intracellular iron levels (Fe²⁺, Fe³⁺, total iron) were measured using colorimetric assays. Glutathione peroxidase (GSH-Px) activity and glutathione (GSH/GSSG) levels were determined. Mitochondrial morphology was examined by transmission electron microscopy. Key ferroptosis markers (Solute Carrier Family 7 Member 11, SLC7A11; Glutathione Peroxidase 4, GPX4) were analyzed at the mRNA and protein levels. Cellular responses were evaluated utilizing cell counting kit-8 (CCK-8) viability assays, 5-Ethynyl-2’-deoxyuridineb (EdU) proliferation staining, and flow cytometry.

Results

LPS treatment significantly increased intracellular iron accumulation while decreasing GSH-Px activity and GSH/GSSG levels, accompanied by characteristic mitochondrial damage. TPX2 expression was significantly elevated in LPS-induced chondrocytes, whereas TPX2 knockdown reversed ferroptosis-related biochemical alterations and restored GPX4 and SLC7A11 expression via inhibition of NF-κB p65 phosphorylation. Moreover, NF-κB p65 overexpression attenuated these protective effects of TPX2 silencing. Ferroptosis activation by erastin abolished the protective effects conferred by TPX2 knockdown.

Conclusion

Our in vitro findings suggest that TPX2 may promote ferroptosis in LPS-induced C28/I2 chondrocytes via NF-κB p65 signaling, reducing antioxidant defenses. Further in vivo and clinical studies are needed to clarify its role and therapeutic potential in OA.