Transferrin receptor upregulation mediates homocysteine-induced cytotoxicity in cerebral endothelial cells.

Hyperhomocysteinemia (HHcy) is a recognized risk factor for cognitive impairment, including Alzheimer's disease, but the mechanisms linking homocysteine (Hcy) accumulation to cerebral dysfunction remain unclear. Brain microvascular endothelial cells (BMECs) are an important cellular component of the blood-brain barrier (BBB) and play a key role in maintaining central nervous system homeostasis. However, how Hcy affects BMECs remains incompletely understood. In this study, we used the human brain microvascular endothelial cell line hCMEC/D3 to determine the half-maximal inhibitory concentration (IC50) of Hcy by CCK-8 assay, and then performed transcriptomic analysis to identify Hcy-regulated genes associated with endothelial cell injury. Hcy reduced hCMEC/D3 cell viability in a dose-dependent manner, with an IC50 of approximately 12.25 mM; therefore, 10 mM Hcy was selected for subsequent experiments. Transcriptomic sequencing revealed significant upregulation of the transferrin receptor (Tfrc) gene, which we confirmed by quantitative real-time PCR and Western blot analysis. Functionally, TFRC overexpression mimicked the Hcy-induced reduction in cell viability, whereas TFRC knockdown partially rescued cells from Hcy-induced cytotoxicity. TFRC overexpression reduced hCMEC/D3 cell viability, whereas TFRC knockdown partially alleviated Hcy-induced cytotoxicity. These results suggest that TFRC is involved in Hcy-induced injury of brain microvascular endothelial cells. Taken together, our in vitro findings provide a possible clue for future in vivo studies on HHcy-associated BBB dysfunction.