Tissue and Salivary NMR Metabolomics in Reticular-Type Oral Lichen Planus

Abstract

Oral lichen planus (OLP) is a chronic T-cell-mediated autoimmune disease, with low potential for malignant transformation. Its etiology remains unclear, necessitating immunohistochemical and molecular-level studies to enhance diagnosis and management. Thirteen patients diagnosed with OLP and 13 healthy controls (HCs) were enrolled from three centers. Mucosal tissue samples collected during diagnostic biopsies and unstimulated whole saliva samples were analysed. A comprehensive approach was taken, with high-resolution magic angle spinning (HR-MAS) ¹H-NMR spectroscopy performed on biopsies and liquid ¹H-NMR spectroscopy on saliva samples to identify potential biomarkers correlated with OLP. Multivariate analyses effectively distinguish OLP patients from HC based on metabolic profiles, with key metabolites contributing to the separation. In tissue, triglycerides were significantly elevated in OLP biopsies, whereas amino acids such as glutamate, glutamine, taurine, glycine and alanine were significantly decreased in OLP tissues compared with controls (p < 0.05). Salivary analysis revealed significant alterations in compounds of bacterial origin-such as isobutyrate, isocaproate, isovalerate and agmatine-suggesting dysbiosis in OLP patients. The metabolic alterations identified highlight the roles of oxidative stress and lipid metabolism in OLP pathogenesis and suggest potential biomarkers for OLP diagnosis. These findings provide new insights into the molecular mechanisms of OLP, which may have important clinical implications for future diagnostic and therapeutic strategies.

Keywords: metabolomics; nuclear magnetic resonance; oral lichen planus; oxidative stress; saliva; tissue.

FIGURE 1

¹H HR‐MAS CPMG NMR spectra from alveolar mucosa of a healthy control (HC) subject (black) and from buccal mucosa of an oral lichen planus (OLP) subject (blue). A: adenosine, Ac: acetate, Ade: adenine, Ala: alanine, Asc: ascorbate, Cho: choline, CHα: signals of CHα from alanine, Cr: creatine, For: formate, Glc: glucose, Gln: glutamine, Glu: glutamate, Gly: glycine, GPC: glycerophosphocholine, GSH, glutamine and glutamate, GSH: glutathione, Ile: isoleucine, Lac: lactate, Leu: leucine, MeOH: methanol, PC: phosphocholine, Phe: phenylalanine, Ser: serine, Tau: taurine, TG: triglyceride, Tyr: tyrosine, U: uridine, Val: valine. *Articaine signals (exogenous anaesthetic).

FIGURE 2

Principal component analysis (PCA) scores plot (a) and aromatic (b) and aliphatic (c) regions of PC loadings obtained from ¹H HR‐MAS CPMG NMR spectra of HC (healthy control, red), P (perilesional, green) and OLP (oral lichen planus, blue) tissue samples. A: adenosine, Ac: acetate, Ade: adenine, Ala: alanine, Asc: ascorbate, Cho: choline, CHα: signals of CHα from alanine, glutamine and glutamate, Cr: creatine, For: formate, Glc: glucose, Gln: glutamine, Glu: glutamate, Gly: glycine, GPC: glycerophosphocholine, Lac: lactate, PC: phosphocholine, Phe: phenylalanine, Tau: taurine, TG: triglyceride, Tyr: tyrosine, Val: valine.

FIGURE 3

PLS‐DA scores plot (a) and variable importance projection (VIP) scores along LV1 of important tissue metabolites for HC vs. OLP discrimination (b), obtained on deconvoluted ¹H HR‐MAS CPMG NMR signals of HC (red) and OLP (green) samples. Cross‐validation details are reported in Figure S2.

FIGURE 4

PLS‐DA of salivary metabolic profiles of OLP vs. HC. PLS‐DA 2D score plot (a). PLS‐DA VIP scores (b) depict the 15 most significant metabolites contributing to the OLP and HC separation. The red and blue boxes on the right side of the VIP score plot indicate the relative metabolite abundance in each cluster of the PLS‐DA. 3PhP: 3‐phenylpropionate, 4OHPhA: 4‐hydroxyphenylacetate, Agm: agmatine, Ala: alanine, Crn: creatinine, EA: ethanolamine, For: formate, Gln: glutamine, His: histidine, iBu: isobutyrate, iCa: isocaproate, iVa: isovalerate, Pyr: pyroglutamate, Tyr: tyrosine, Ur: uracil. Cross‐validation details are reported in Figure S3.

FIGURE 5

(a) Volcano plot analysis of the differential salivary metabolites of OLP vs. HC. Each point on the volcano plot is based on specific p and fold‐change (FC) values. The points that satisfy the condition p ≤ 0.05 and |FC| ≥ 1.5 were considered significant and appear in red or blue if the concentration of the corresponding metabolite is higher or lower in OLP than in HC. (b) Box plots of the normalized concentrations of the significant metabolites identified in (a) for both sets of samples; red is for OLP patients, and green is for HC. Agm: agmatine, Crn: creatinine, iBu: isobutyrate, iCa: isocaproate, iVa: isovalerate, Ur: uracil.

FIGURE 6

Hypothesis of a ROS‐mediated mechanism in OLP disease.