| Background:Periodontitis is a chronic inflammatory disease of the tooth support tissue that occurs with plaque as the initial factor,which is characterized by the chronic progressive destruction of the alveolar bone and can lead to tooth loss in adults.Many studies have shown that Porphyromonas gingivalis(P.g)is the most dominant bacteria in the active lesion area of chronic periodontitis,which is closely related to the occurrence and development of periodontal disease.Periodontal immune cells are activated under the stimulation of P.g and its virulence factor lipopolysaccharide(LPS),then excessive reactive oxygen species(ROS)are released through "respiratory burst" to kill bacteria.However,excessive secretion of ROS will cause damage to lipid,DNA and protein in itself and its surrounding tissues and cells.At present,the treatment drugs of periodontitis mainly include nitroimidazole and tetracycline antibacterial drugs,but long-term use of antibiotics has the disadvantage of causing adverse reactions and producing drug-resistant strains leading to dysbacteriosis.Traditional Chinese medicine curcumin can not only play a bacteriostatic effect,but also has a variety of biological activities such as regulating inflammation,improving oxidative stress and inhibiting apoptosis,and is considered to be an effective natural compound for the treatment of periodontitis.At present,curcumin has attracted much attention by inhibiting ferroptosis to ameliorate tissue damage,and this pharmacological effect may be related to its antioxidant effect.Ferroptosis is a novel type of cell death caused by the destruction of cell membrane injured by lipid ROS,and its main characteristics include mitochondrial shrinkage,glutathione(GSH)depletion,and abnormal accumulation of lipid peroxidation products.The abnormal accumulation of lipid peroxides is not only a direct cause of ferroptosis,but also closely related to periodontitis.Studies have shown that plasma and saliva of periodontitis patients contain high levels of lipid peroxide.The changes of ferroptosisrelated genes in the gingival tissue of mice with periodontitis were found,and the changes could be reversed by the ferroptosis inhibitor Fer-1.The regulation of ferroptosis during the occurrence and development of periodontitis may provide a new therapeutic strategy for improving the inflammatory response of periodontitis.This experiment intends to establish ligature-induced periodontitis in vivo and P.gLPS stimulated RAW264.7 in vitro,using molecular biology,immunohistochemistry,and other methods to detect whether curcumin can reduce the inflammatory response of periodontitis by inhibiting ferroptosis.Method:(1)The state of systemic oxidative stress was evaluated by detecting the enzyme activity of superoxide dismutase(SOD)in mice;Paraffin sections of gingival tissue were made for HE staining to compare the degree of inflammatory infiltration of periodontal tissue;RNA and proteins of gingival and alveolar bone tissue were extracted,Solute carrier family 7 member 11(SLC7A11)and GPX4 were detected from both genes and proteins,and immunohistochemical staining was performed on gingival tissue sections to verify the expression level of SLC7A11 and GPX4.Gingival alveolar bone tissue homogenates were prepared according to different methods to detect the contents of GSH and malondialdehyde(MDA).(2)Mitochondrial morphology,mitochondrial membrane potential and mitochondrial ROS level were observed to prove that curcumin can maintain the stability of mitochondrial morphology and function;DCFH-DA probe was used to detect ROS level to demenstrate the clearance effect of curcumin on intracellular ROS;Lactate dehydrogenase(LDH)release,Calcein/ PI cytotoxicity detection and Annexin V-FITC apoptosis detection experiment suggested that curcumin has the effect of protecting cells;The expression levels of SLC7A11 and GPX4 were detected to illustrate that curcumin could inhibit ferroptosis by regulating the SLC7A11/GPX4 axis.By measuring the level of antioxidant GSH and the accumulation of lipid peroxide MDA to prove that curcumin promotes the clearance of lipid peroxidation and improves the resistance of cells to ferroptosis.Results:(1)Results in vivo showed that different concentrations of curcumin could increase serum SOD enzyme activity,improve inflammatory infiltration of gingival tissue,upregulate SLC7A11 and GPX4 expression,increase GSH content in gingival and alveolar bone,and reduce MDA accumulation.It was confirmed that curcumin can inhibit ferroptosis by regulating SLC7A11/GPX4 axis,increase the content of antioxidants GSH in periodontal tissues,reduce MDA accumulation,and improve the inflammatory response of periodontitis.(2)Results in vitro showed that different concentrations of curcumin could improve mitochondrial morphology,increase mitochondrial membrane potential,reduce mitochondrial ROS and cellular ROS levels,reduce cell death rate,increase the expression of SLC7A11 and GPX4,increase the content of GSH in inflammatory cells,and reduce the abnormal accumulation of intracellular MDA.It was shown that curcumin could inhibit ferroptosis of RWA264.7 under P.g-LPS stimulation by regulating the SLC7A11/GPX4 axis.Conclusion:(1)Curcumin has a regulatory effect on the inflammation of periodontitis in experimental mice,which can reduce the local inflammatory infiltration of periodontal tissue and protect periodontal tissue.(2)Curcumin inhibits the accumulation of lipid peroxide in periodontitis by regulating the SLC7A11/GPX4 pathway,thereby protecting gingival tissue and alveolar bone tissue cells.(3)Curcumin can maintain the stable morphology and normal function of the mitochondria in RAW264.7 cells under inflammation,and play a protective role on cells.(4)Curcumin affects the expression of ferroptosis-related genes SLC7A11 and GPX4 in macrophages,and it is inferred that the mechanism of curcumin reducing periodontitis inflammation is achieved by regulating the SLC7A11/GPX4 axis to inhibit the phenomenon of cellular ferroptosis. |
