Detecting the salivary protein profile of patients with DM2 and periodontitis can help find possible biomarkers for the diseases and also provide a better understanding of the relationship between both conditions, which can serve as an important tool to guide diagnosis, prognosis, and treatment of the affected population. Under the author’s knowledge, this is the first study that used mass spectrometry to compare the salivary proteomic profile of individuals with DM2 and periodontitis in relation to their respective controls. The main results of this study showed that uncontrolled DM2 and periodontitis can alter the salivary protein profile.
Our results showed a large number of proteins present in the salivary samples, with more diversity found in the diseased groups with DM2 and periodontitis. When evaluating the groups DM2 + P and DM2, 163 common proteins were found. Bioinformatics analysis showed that the main processes involved were related to alpha-amylase activity (17.2%), mesenchyme migration (13.7%), and several immune-inflammatory processes such as humoral immune response (13.79%), downregulation of complement system activation (6.9%), immunoglobulin complexes (41.37%), and neutrophil aggregation (6.9%). Furthermore, it was possible to observe a high ratio of S100 family proteins within the processes involved. Protein S100-A8 was 6-fold upregulated in the group DM2 + P when compared to DM2. Other studies have related S100 family proteins as biomarkers of inflammation in patients with periodontal disease [26, 27]. In addition, S100 proteins are characterized by two calcium binding sites and exert diverse regulatory activities on monocytes/macrophages, neutrophils, lymphocytes, mast cells, endothelial cells and epithelial cells, participating in the regulation of proliferation, differentiation, apoptosis, Ca+ 2 homeostasis, specific and non-specific inflammatory responses and inflammation [26].
The S100A8 protein is related to regulating inflammation, and its primary sources are monocytes and macrophages. S100A8 is reported to induce TNF-α and IL-1β production in bone marrow cells via TLR-4 activation [28]. Further, in vitro studies have shown that S100A8 can directly induce osteoclast activity, suggesting a possible mechanism by which the immune system can regulate bone resorption [29]. Thus, our results indicate that the S100 A8 protein may play an important role in the relationship between periodontal disease and DM2, suggesting that it may serve as a possible biomarker for periodontal disease in this population. Concerning S100A9, it can induce the production of IL-6, IL-8, and RANKL in human periodontal ligament fibroblasts and osteocyte cells, respectively, acting in inflammation and destruction of periodontal tissue [30–32]. Also, in agreement with our results, studies have shown significantly higher levels of S100A8 and A9 in the saliva of systemically healthy patients with periodontitis [33–35]. Noteworthy, the S100 family proteins can bind to TLR-4 and RAGEs, activating a signaling cascade and inducing a pro-inflammatory response [36], which provides an additional biological link between periodontitis and DM2.
When comparing the groups DM2 + P and P, 87 common proteins were found. The main processes involved were alpha-amylase activity, TLR-4 receptor binding, IL-8 binding, B cell apoptosis process, macrophage chemotaxis regulation, neutrophil aggregation, and CXC-2 upregulation. The protein-protein network interaction showed a high involvement of Immunoglobins, S100 family proteins, and defensins. Immunoglobulin lambda constant 7 was more than 2 times fold upregulated in participants with DM2 and periodontitis compared to periodontitis patients without DM2. Immunoglobulin lambda constant 7 is an antibody released by B lymphocytes and is part of the specific humoral immune response [37]. NF-κB plays an essential role in B cell activation and development, and NF-κB activation is critical in inflammation and DM2 development [38]. In this context, the increased B lymphocyte response is related to a greater Th2 response over the Th1, which is associated with the development and progression of periodontitis [39, 40]
In the evaluation of the groups without DM2 (P and H), out of 115 common proteins, 22 proteins were upregulated. The processes involved were mesenchyme migration, neutrophil aggregation, antioxidant activity, secretion of prostaglandin involved in the immune response, B cell apoptosis, and S-nitrosylation of peptide cysteine. Network protein analysis showed the involvement of S100 family proteins (S100 A8 and A9) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was more than 2-fold upregulated in participants with periodontitis. GAPDH represents one of the main receptors for the fimbriae of Porphyromonas gingivalis, one of the main pathogens related to periodontitis [41]. Indeed, the literature has suggested that GAPDH likely plays a role in adhesion and colonization of the oral cavity by P. gingivalis, as well as triggering host cellular processes involved in the pathogenesis of periodontal diseases[42, 43]. Furthermore, our results showed that S100 family proteins were also upregulated in participants without DM2 but with periodontitis. In 2019, a cross-sectional study observed increased S100A8 and A9 proteins in the saliva of patients with periodontitis but without DM2 in proteomic analysis, thus corroborating our results [33].
Similarly to our results, a clinical study found a significant increase in the levels of S100A8, S100A9, and immunoglobulins proteins in the diseased groups after analyzing the gingival crevicular fluid proteome of 20 DM2 and periodontitis individuals and controls [44].
In participants without periodontitis (DM2 and H groups), of the 119 common proteins found, 23 proteins were upregulated. The processes involved were complement system activity, oxygen-carrying activity, glomerular filtration, regulation of senescence activity, neutrophil aggregation, and regulation of prostaglandin involved in immune activity. The protein network analysis showed a great interaction among upregulated proteins of the hemoglobin family. Hemoglobin subunit alpha is a hemoglobin protein that in humans is encoded by the HBA1 gene [45]. An increase in hemoglobin has been reported in patients with insulin resistance and DM2 in serum proteomic analysis studies [45]. Thus, our data suggest this salivary protein as a possible biomarker for DM2. In addition, several inflammatory markers were altered in the DM2 group, which corroborates this population's increased risk for systemic and periodontal diseases.
Some limitations can be highlighted in our study. Although gender and age-matched provided some bias control, other factors may have influenced our results. We did not assess obesity status, physical activity, diet, and other oral diseases, which can also change the salivary proteomic profile of patients. In addition, the study design does not allow for further conclusions. Thus, prospective studies should assess the influence of DM2 and periodontitis on the salivary proteomic profile in order to confirm our findings.