Hepcidin regulates neuronal ferroptosis: A mechanism for postoperative cognitive dysfunction

Abstract

Toll-like receptor 4 (TLR4) is a signaling molecule responsible for the expression of hepcidin (Hepc), while myeloid differentiation protein 2 (MD2) is one major accessory protein of TLR4. This study focuses on exploring the neurocyte ferroptosis mediated through the regulation of Hepc expression by MD2, which is also one of the mechanisms for postoperative cognitive dysfunction (POCD). An experimental study was carried out using aged wild-type (Wt) and MD2 transgenic (Tg) mice. The neurocyte ferroptosis and POCD in the mice were assessed following splenectomy. Morris water maze was utilized to assess the neurocognitive abilities, hematoxylin and eosin (H&E) assay was performed to examine histopathology, and Nissl staining was used to evaluate the neurocyte damage. The Fe²⁺ , superoxide dismutase(SOD), malondialdehyde (MDA), glutathione(GSH), and glutathione peroxidase 4 (GPX4) levels were determined with kits. The expressions of transferrin receptor (TFR), Hepc, and MD2 were measured by Western blotting, while the expressions of TFR and GPX4 were measured by immunohistochemical staining. In Tg mice, we observed neurocyte ferroptosis and POCD following treatment with an MD2 inhibitor. PC12 cells were used as a neurocyte model. Ferroptosis was induced after treatment with an MD2 inhibitor, and the cell viability was assayed by Cell Counting Kit-8. Immunofluorescent staining was used to measure the TFR and GPX4 expressions. Meanwhile, the intracellular levels of Fe²⁺ , SOD, MDA, GSH, GPX4, and Hepc were also measured. POCD occurred among aged Wt and Tg mice. The Tg-POCD mice had more apparent POCD than the Wt-POCD mice. Nissl and H&E staining revealed neurocyte damage in brain tissues. Besides this, the Fe²⁺ and MDA expressions were upregulated, while the SOD, GSH, and GPX4 expressions were downregulated. Elevations in tissue levels of TFR, Hepc, and MD2 were observed, which were higher than those of Wt-POCD mice. After treatment with an MD2 inhibitor, the POCD could be prominently ameliorated in Tg-POCD mice, the Fe²⁺ and MDA levels could be reduced, while the SOD, GSH, and GPX4 levels could be elevated. In the PC12 model, ferroptosis could be suppressed by inhibiting the expression of MD2. MD2 is capable of regulating neurocyte ferroptosis by promoting Hepc expression, which has great potential as a novel target for POCD therapy.

Keywords: POCD; ferroptosis; hepcidin; myeloid differentiation protein; neurocyte.