Abstract:

Objective To investigate the effect of lipopolysaccharide （LPS） of Porphyromonas gingivalis （P.g） on the expression levels of ferroptosis-related factors in the macrophages， and to clarify the influence of ferroptosis-related factors in the pathogenesis of periodontitis. Methods The mouse mononuclear/macrophage line RAW264.7 cells were cultured and divided into experimental group and control group. The cells in experimental group were treated with 10 mg·L^－1P.g-LPS for 6， 12， and 24 h， respectively； the cells in control group were not treated. Real-time fluorescence quantitative PCR （RT-qPCR） method was performed to detect the expression levels of long chain acyl-CoA synthetase 4 （ACSL4）， glutathione peroxidase 4 （GPX4）， and transferrin receptor 1 （TfR1） mRNA in the RAW264.7 cells in two groups. The levels of reactive oxygen species （ROS） in the RAW264.7 cells in two groups were detected by 2'，7'-dichlorodihydrofluorescein diacetate（DCFH-DA） fluorescence probe method. The levels of malondialdehyde （MDA） in the RAW264.7 cells in two groups were detected by thiobarbituric acid （TBA） method and the levels of ferrous ion （Fe²⁺） in the RAW264.7 cells in two groups were detected by Ferrousion fluorescent probe （FeRhonox-1） method. The expression levels of ACSL4， GPX4， and TfR1 proteins in the RAW264.7 cells in two groups were detected by Western blotting method. Results Compared with control group， the expression levels of GPX4 mRNA in the RAW264.7 cells in experimental group after treated for 6， 12， and 24 h were decreased （P<0.05）， the expression levels of ACSL4 and TfR1 mRNA in the RAW264.7 cells in experimental group after treated for 12 and 24 h were increased （P<0.05）； the expression levels of ACSL4 and TfR1 proteins in the RAW264.7 cells in experimental group after treated for 12 and 24 h were increased （P<0.05）；the expression levels of GPX4 protein in the RAW264.7 cells in experimental group after treated for 12 and 24 h were decreased （P<0.05）.Compared with control group，the levels of ROS， MDA， and Fe²⁺ in the RAW264.7 cells in experimental group were significantly increased （P<0.05）， and showed an increasing trend with time prolongation. Conclusion The expression levels of ferroptosis-related factors ACSL4 and TfR1 are increased and the expression level of GPX4 is decreased in a time-dependent manner induced by P.g-LPS， suggesting that the changes of ferroptosis-related factors may be related to the pathogenesis of periodontitis.

Key words: Macrophages, Porphyromonas gingivalis, Lipopolysaccharide, Ferroptosis, Reactive oxygen species