Differential Expression Analysis of Key Inflammatory Mediators in Irreversible Pulpitis for Diagnostic Biomarkers

Introduction and aims: The diagnosis of pulpal inflammatory conditions poses significant challenges in clinical endodontics, requiring precise differentiation between reversible and irreversible states. This study investigated the differential expression patterns of 10 inflammatory genes: LCP2, PTPRC, CXCL8, TNFα, IL6, CCL2, MMP9, NOD2, ICAM1, and TLR8 in irreversible pulpitis as potential molecular diagnostic biomarkers. Methods: In this prospective cross-sectional analytical investigation, pulp tissue samples were collected from 30 subjects with irreversible pulpitis and 10 control subjects with healthy pulp tissue from orthodontically indicated extractions. Clinical diagnosis followed standardized American Association of Endodontists guidelines by two calibrated endodontists (Cohen's kappa >0.85). RNA isolation utilized TRIzol methodology, followed by quantitative Real-Time PCR analysis with GAPDH normalization and >2-fold change threshold for differential expression. Results: Analysis revealed varying frequencies of differential gene expression, with CCL2 demonstrating the highest frequency (63.3%, P = .001587), followed by MMP9 (60%, P = .001453), TNFα and IL6 (56.7% each), CXCL8 and NOD2 (53.3% each), TLR8 (46.7%), ICAM1 and PTPRC (43.3% each), and LCP2 (36.7%). Concurrent differential expression of all 10 markers occurred in 3.3% of cases. Significant correlations were identified between gene expression and clinical parameters, particularly CXCL8 with thermal hyper-response (r = 0.641, P < .001), TLR8 with thermal hyper-response (r = 0.758, P < .001) and pain type (r = 0.379, P = .039), and TNFα with visual analogue scale pain intensity and thermal hyper-response. Conclusion: Six genes (MMP9, CXCL8, TNFα, IL6, CCL2, NOD2) demonstrated statistically significant upregulation with notable correlations between specific genes and clinical manifestations. These preliminary findings suggest potential utility as diagnostic biomarkers, though clinical implementation requires validation through larger cohort studies. Clinical relevance: This molecular profiling approach addresses critical limitations in current endodontic diagnostics with 30% to 40% diagnostic inaccuracies. The identified inflammatory gene signatures provide quantifiable molecular evidence that could enhance clinical decision-making in determining vital pulp therapy vs conventional root canal treatment. Implementation through point-of-care diagnostic platforms may reduce diagnostic uncertainty and facilitate evidence-based endodontic therapy.