Abstract: BACKGROUND: Ferroptosis is an iron-dependent form of programmed cell death closely associated with periodontal and peri-implant inflammatory diseases. Cytidine/uridine monophosphate kinase 2 is a kinase localized in mitochondria that participates in regulating tissue cell activation and inflammatory responses. The regulatory mechanisms of both in peri-implantitis remain unclear.
OBJECTIVE: To construct an in vitro cell model of peri-implantitis using lipopolysaccharide and explore the regulatory mechanism of cytidine/uridine monophosphate kinase 2 on lipopolysaccharide-induced ferroptosis in human gingival fibroblasts.
METHODS: (1) Human gingival fibroblasts were cultured using the tissue explant culture method and treated with 0 μg/mL (normal control group) or 10 μg/mL lipopolysaccharide for 12 hours. The mRNA and protein expression levels of ferroptosis-related markers glutathione peroxidase 4, solute carrier family 7 member 11, and acyl-CoA synthetase long-chain family member 4, as well as cytidine/uridine monophosphate kinase 2, were detected by RT-qPCR and western blot assay. Lipid peroxidation levels were detected by reactive oxygen species staining and C11-Bodipy staining. Intracellular malondialdehyde, superoxide dismutase levels, and the reduced glutathione/oxidized glutathione ratio were detected by colorimetric methods. (2) Cytidine/uridine monophosphate kinase 2 was silenced using siRNA, and then cells were stimulated with 10 μg/mL lipopolysaccharide for 12 hours. The changes in the above indicators were then detected.
RESULTS AND CONCLUSION: (1) After stimulating human gingival fibroblasts with 10 μg/mL lipopolysaccharide for 12 hours, the expression of the ferroptosis marker acyl-CoA synthetase long-chain family member 4 was upregulated, while the expression of the negative ferroptosis regulators glutathione peroxidase 4 and solute carrier family 7 member 11 was downregulated. Reactive oxygen species release increased, malondialdehyde levels increased, superoxide dismutase activity and the reduced glutathione/oxidized glutathione ratio decreased, and cytidine/uridine monophosphate kinase 2 expression was upregulated. (2) After silencing cytidine/uridine monophosphate kinase 2 with siRNA, and then stimulating the cells with 10 μg/mL lipopolysaccharide for 12 hours, acyl-CoA synthetase long-chain family member 4 expression was downregulated, glutathione peroxidase 4 and solute carrier family 7 member 11 expression were upregulated, reactive oxygen species release decreased, malondialdehyde levels decreased, and superoxide dismutase activity and the reduced glutathione/oxidized glutathione ratio increased. These results indicate that cytidine/uridine monophosphate kinase 2 is a key regulatory gene for ferroptosis in gingival fibroblasts during peri-implantitis, and silencing cytidine/uridine monophosphate kinase 2 can inhibit lipopolysaccharide-induced ferroptosis in human gingival fibroblasts.

Key words: cytidine/uridine monophosphate kinase 2, ferroptosis, lipopolysaccharide, human gingival fibroblasts, peri-implantitis, glutathione peroxidase 4, reactive oxygen species, superoxide dismutase