Abstract:

Objective To investigate the role of Toll-like receptor 4 （TLR4） in the regulation of homocysteine （Hcy）-induced ferroptosis in macrophages. Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group， Hcy intervention group （Hcy group）， and Hcy plus ferroptosis inhibitor group （Hcy+Fer-1 group）. After transfection with interference fragments， macrophages were treated with Hcy， and then divided into control group， Hcy intervention group （Hcy group）， TLR4 interference negative control plus Hcy intervention group （si-NC+Hcy group）， and TLR4 interference plus Hcy intervention group （si-TLR4+Hcy group）. Macrophages were transfected with overexpression lentivirus and treated with Hcy， then were divided into control group， Hcy intervention group （Hcy group）， a TLR4 overexpression negative control plus Hcy intervention group （OE-NC+Hcy group）， and a TLR4 overexpression plus Hcy intervention group （OE-TLR4+Hcy group）. After 48 hours of intervention， real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy； western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4， GPX4， and FTH1 in macrophages， and ferrous ion assay kit to detect the concentration of Fe²⁺ in macrophages； reactive oxygen species （ROS） assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species. Results Compared with those in the control group， the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group （P < 0.05）， while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased （P < 0.05）； the concentration of Fe²⁺ was increased （P < 0.05）， and the content of ROS was increased. Meanwhile， the protein and mRNA expression levels of TLR4 were both increased in the Hcy group （P < 0.05）. After macrophages were transfected with TLR4 interference fragments， compared with those in the si-NC+Hcy group， the expression levels of GPX4 and FTH1 were increased （P < 0.05）； the expression level of ACSL4 was decreased （P < 0.05）； the concentration of Fe²⁺ was decreased （P < 0.05）， and the content of ROS was reduced in the si-TLR4+Hcy group. After macrophages were transfected with TLR4 overexpression lentivirus， compared with those in the OE-NC+Hcy group， the expression levels of GPX4 and FTH1 were decreased （P < 0.05）， and the expression level of ACSL4 was increased （P < 0.05） in the OE-TLR4+Hcy group. Conclusion Hcy induces the occurrence of ferroptosis in macrophages， and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Key words: TLR4, macrophages, homocysteine, ferroptosis