Hamadan University of Medical Sciences Avicenna Journal of Medical Biochemistry 2345-4113 13 2 2025 12 31 Western Blotting of Salivary Interleukin-1β and Tumor Necrosis Factor-α for Non-Invasive Diagnosis of Orthodontically Induced Root Resorption 89 96 10.34172/ajmb.2676 EN Homa Farhadifard https://orcid.org/0000-0002-5564-7587 Parnia Yari https://orcid.org/0009-0001-9178-1687 Amir Taherkhani https://orcid.org/0000-0002-6546-8785 Vahid Siavashi https://orcid.org/0000-0003-1297-8295 Parzhin Moradi https://orcid.org/0009-0009-3035-1251 Journal Article 10.34172/ajmb.2676 2025 06 14 2025 11 07 Introduction: Orthodontically induced inflammatory root resorption (OIIRR) is an unpredictable side effect of orthodontic treatment, typically identified only after irreversible damage. This study sought to assess whether the salivary concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) could function as dependable diagnostic indicators for OIIRR. It was hypothesized that OIIRR patients would demonstrate higher salivary TNF-α and IL-1β than controls, and that these cytokines would be accurate diagnostic biomarkers. Methods: A case-control study was conducted involving 24 orthodontic patients, 12 with radiographically verified OIIRR and 12 controls free of root resorption, comparable in age and gender. Whole saliva samples were obtained, and protein expression of IL-1β and TNF-α was quantified via Western blotting using β-actin as a reference. Statistical methods included independent-samples t-tests, Cohen’s d for effect size estimation, and receiver operating characteristic (ROC) curve analysis. Results: Salivary IL-1β was significantly higher in cases (1.36±0.33) than in controls (0.68±0.16; t(22)=6.32, P<0.0001; Cohen’s d=2.58). Likewise, TNF-α was notably elevated in cases (1.38±0.29) versus controls (0.80±0.15; t(22)=6.27, P<0.0001; Cohen’s d=2.56). Moreover, ROC analysis revealed outstanding diagnostic performance for both markers, with AUC values of 0.986 for IL-1β and 0.951 for TNF-α. Optimal cutoffs were also 0.875 and 1.1 for IL-1β and TNF-α, respectively. Conclusion: Our findings indicated that both cytokines represent valuable non-invasive salivary biomarkers for the early diagnosis and monitoring of OIIRR, potentially supporting safer orthodontic care. Nonetheless, larger prospective studies are needed for confirmation.