Abstract: BACKGROUND: Through analyzing the expression of cuproptosis-related genes in periodontitis tissues, this study attempted to explore the significance of cuproptosis-related biomarkers in the diagnosis and treatment of periodontitis during the development of periodontitis. 
OBJECTIVE: To explore the expression of cuproptosis-related genes in periodontitis and their potential mechanisms through bioinformatics analysis. 
METHODS: Periodontitis transcriptome datasets GSE10334 and GSE156993 were obtained from the Gene Expression Omnibus (GEO) database. After eliminating batch effects, they were integrated into a meta-cohort. Hub genes were screened and identified using the Least Absolute Shrinkage and Selection Operator, Support Vector Machine-Recursive Feature Elimination, and Venn analysis. Subsequently, a comprehensive analysis of the distribution and role of these hub genes in periodontitis was performed using receiver operating characteristic curves, functional enrichment analysis, immune cell infiltration analysis, single-cell analysis (dataset GSE171213), gene-drug network analysis, nomogram model construction, and consensus clustering. Further validation was conducted in a Sprague-Dawley rat periodontitis model.

RESULTS AND CONCLUSION: (1) Three hub cuproptosis signature genes (NOD-like receptor pyrin domain-containing protein 3, dihydrolipoamide succinyltransferase and glutamine synthetase) in periodontitis were identified using machine learning algorithms. These genes were significantly upregulated in periodontal tissues, with NOD-like receptor pyrin domain-containing protein 3 (the area under the curve=0.828) and glutamine synthetase (the area under the curve=0.830) showing good diagnostic value, suggesting their potential as diagnostic biomarkers for periodontitis. (2) Dihydrolipoamide succinyltransferase induced mitochondrial dysfunction by regulating the tricarboxylic acid cycle and lipoic acid metabolism; NOD-like receptor pyrin domain-containing protein 3 amplified inflammatory responses through inflammasome activation; and glutamine synthetase supported immune cell function by enhancing glutamine metabolism. These three genes synergistically regulated cuproptosis to drive the progression of periodontitis. (3) The expression levels of NOD-like receptor pyrin domain-containing protein 3, dihydrolipoamide succinyltransferase and glutamine synthetase were upregulated in the Sprague-Dawley rat model of periodontitis and positively correlated with the amount of alveolar bone resorption, providing in vivo evidence for the proposed mechanistic hypothesis. These findings indicate that NOD-like receptor pyrin domain-containing protein 3, dihydrolipoamide succinyltransferase and glutamine synthetase, as hub genes of cuproptosis, play important roles in the occurrence and development of periodontitis and possess potential diagnostic value.

Key words: cuproptosis, periodontitis, bioinformatics, NOD-like receptor pyrin domain-containing protein 3 (NLRP3), glutamine synthetase (GLS), dihydrolipoamide succinyltransferase (DLST)