Salivary proteome of aphthous stomatitis reveals the participation of vitamin metabolism, nutrients, and bacteria

Abstract

There are currently no preventative options for recurrent aphthous stomatitis, and the only available treatments are palliative. This is partly due to a poor understanding of its etiopathogenesis. In this case-control study, we characterized the salivary proteome of patients with recurrent aphthous stomatitis in the presence and absence of lesions. Through mass spectrometry-based proteomics and bioinformatics tools, we identified that the presence of oral ulcers is associated with several specific biological processes, including the metabolic pathways of vitamin B9, B12, nitrogen, selenium, and the bacterium Neisseria meningitidis. These changes occurred only in the presence of clinically visible lesions, and there were no relevant differences between patients in anatomical regions unaffected by ulcers. Additionally, using western blot and ELISA assays, we verified that carbonic anhydrase 1 (CA1) and hemoglobin subunit beta (HBB) proteins are highly expressed during the ulcerative and remission phases of recurrent aphthous stomatitis. Our results cumulatively support saliva as an indicator of the pathophysiological changes, which occur during the clinical course of lesions. From a clinical perspective, we suggest that recurrent aphthous stomatitis is a condition triggered by temporary biological changes in people with lesions.

Figure 1

Clinical manifestations of recurrent aphthous stomatitis. (A) Representative images of ulcers. Lesions were ovoid with an erythematous halo. (B) Anatomical sites where lesions occurred. The most affected regions were labial mucosa, vestibule, and cheeks. (C) Clinical history associated with ulcers. Lesions were smaller than 5 mm accompanied by moderate pain.

Figure 2

Salivary proteome. (A) We assessed samples integrity by SDS-PAGE gel electrophoresis (20 μg/lane protein concentration). (B) Venn diagram shows that most of the salivary proteins identified were proteins in common. (C) Volcano graphs show the windows considered in the analysis, constructed considering statistical significance and fold change. Proteins up- and down- regulated are enclosed by a green square. The fold change limits were − 0.5 and 0.5. The established p value was 0.05.

Figure 3

The presence or absence of recurrent aphthous ulcers reveals metabolic pathways for vitamins and nutrients. Each pathway on the right corresponds to a database on the left. The existence of proteins in these pathways is indicated by dark gray squares. APOB and SERPINA3, for example, are proteins involved in the vitamin B9 and B12 metabolism pathways. (A) Pathways enriched from the list of differentially expressed proteins between healthy controls and patients at the ulcerative stage. (B) Enrichment analysis corresponding to the contrast between the ulcerative and remission-stage patients. The absence of oral ulcers, i.e., the contrast between healthy controls and remission-stage patients, did not show enriched pathways. For enrichment analysis, g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) and WebGestalt (http://www.webgestalt.org/) were used.

Figure 4

Salivary proteins and vitamins during the clinical course of recurrent aphthous stomatitis. Verification of proteomic data points. (A) Western blotting showed higher CA1 protein levels during the ulcerative and remission stages. Ponceau red were used as loading control. (B) ELISA showed a higher amount of HBB protein in ulcerative and remission stages. (C) Samples from patients with recurrent aphthous stomatitis show lower levels of folate (vitamin B9) and vitamin B12, but without statistically significant differences. Data are represented by mean ± SD (Student’s t-test, different letters indicate statistically difference at p value ≤ 0.05). CA1, carbonic anhydrase 1; SLURP1, secreted Ly-6/uPAR-related protein 1; HBB, hemoglobin subunit beta; CST1, cystatin-SN; A2M, alpha-2-macroglobulin.

Figure 5

Presence of recurrent aphthous ulcers reveals Neisseria meningitidis proteins. (A) Volcano graphs show the windows considered in the analysis, constructed considering statistical significance and fold change. Proteins up- and down-regulated are enclosed by a green square. Fold change limits were − 0.5 and 0.5. The established p value was 0.05. (B) Table details the proteins expressed differentially in the comparison between groups. The presence of proteins and strains of Neisseria meningitidis (serogroup B, strain MC58 and serogroup C, strain 053442) during ulcerative activity stands out. Information of oral bacteria was obtained from The Human Oral Microbiome Database (http://www.homd.org/).

Figure 6

Recurrent aphthous stomatitis did not leave a mark on affected sites. Selected peaks with CFS (see table S4 in supplementary material) from proteomic profiles were used to perform PCA graphics. (A) During the ulcerative stage, healthy and affected tissues are completely separated. (B) As the oral mucosa regenerates, healthy and recovered tissues fail to separate. (C) When an injury occurs again, the sites separate again. (D) All intact oral mucosa tissues fail to separate, regardless of health and disease status.