Periodontitis is a chronic inflammatory disease that involves hypoxia-induced apoptosis mechanisms [18]. Caspase activation has been reported to be associated with periodontitis through this mechanism, and few studies have investigated CASP8 in periodontitis [21, 22]. Also, it has been reported that SIRT6 modulates hypoxia-induced apoptosis in many studies, and this has been shown in endodontic periapical lesions [23, 24]. However, there is no study assessed the possible role SIRT6 in pathogenesis of periodontitis. In addition, LXA4, which has been associated with periodontitis through inflammatory pathways in various studies, has recently been shown to play a role in the mechanism of hypoxia-induced apoptosis. Therefore, we evaluated serum and saliva levels of SIRT6, CASP8 and LXA4 in patients with stage III/grade B periodontitis.
In this study, it was demonstrated that serum SIRT6 and saliva LXA4 levels were significantly lower in stage III/grade B periodontitis patients than healthy controls. We have shown decreased serum levels of SIRT6 were associated with advanced periodontitis for the first time. SIRT6 is known to be involved in the regulation of inflammation, stress, energy metabolism, and cell survival [29]. SIRT6 inhibits stress-mediated apoptotic cell death by cleaving BAX from mitochondria [7]. It has been shown that it is a critical regulator of glucose homeostasis and apoptosis, and protects the heart from hypoxic damage by reducing ROS production [8]. Balestrieri et al. (2015), reported that SIRT6 expression in atherosclerotic lesions of Type 2 diabetes (T2DM) patients was downregulated, and the decreased SIRT6 expression was related with increased oxidative stress and inflammation [30].
SIRT6 has anti-inflammatory effects by inhibiting the expression of NF-κB target genes and other pro-inflammatory cytokines [9]. It has been reported that overexpression of SIRT6 inhibits inflammatory responses and bone destruction in mice with collagen-induced arthritis [31]. Also, Woo et al. (2018), demonstrated that myeloid SIRT6 deficiency augments rheumatoid arthritis by enhancing macrophage activation and infiltration [32]. Other studies have associated SIRT6 deficiency with increased osteoclastic bone resorption and osteopenia [33, 34]. Hou et al. (2016), demonstrated that SIRT6 supresses hypoxia-induced inflammatory response in human osteoblasts by inhibition of ROS generation and glycolysis [35]. Periodontitis is characterized by increased inflammation, oxidative stress and bone destruction, and findings of these studies reported in SIRT6 deficiency are also involved in the pathogenesis of periodontitis and are consistent with our study.
Also, a study by Kok et al. (2015) revealed that SIRT6 modulates hypoxia-induced apoptosis in osteoblasts by inhibition of glycolysis and, it might alleviate periapical lesions [23]. Another study reported that SIRT6 supresses periapical lesion progression by modulating hypoxia-induced chemokine ligand 2 production in osteoblasts [24]. These findings may explain the low serum SIRT6 level in periodontitis, which is probably associated by hypoxia-induced apoptosis according to the previous studies.
Additionally, SIRT6 deficiency was associated with activation of NF-κB pathway in LPS-induced human dental pulp cell [36]. A recent study by Li et al. (2022), demonstrated that SIRT6 supresses the inflammatory response of LPS-induced PDLCs via inhibiting NF-κB pathway and, it promotes osteogenic differentiation and viability [25]. PDLCs represent local immune cells of the periodontal tissues and SIRT6 may have possible role of development periodontitis via NF-κB pathway. However, in the present study, we did not find a significant difference in salivary levels of SIRT6 between patients with periodontitis and healthy individuals. While SIRT6 in many diseases are mostly investigated in the serum [37–39], saliva SIRT6 level has been analyzed in only one study [40]. SIRT6 level fluctuation in saliva may not be detected easily because of the complex structure and content of saliva [41]. Thus, we think that serum level of SIRT6 is more important in monitoring the possible role of SIRT6 in periodontitis.
In the current study, we have also shown that LXA4 level in saliva decreased with periodontitis. Several studies have reported that LXA4 is related with periodontitis and its replacement therapy resolves periodontal inflammation in animal models [19, 20]. Consistent with our findings, a recent study by Tobon-Arroyave et al. (2019), reported that lower salivary LXA4 level was detected in periodontitis patients compared to healthy subjects [42]. However, we found no difference in serum levels of LXA4 in periodontitis than healthy individuals. Contrary to this study, few studies have reported increased serum LXA4 levels in chronic and aggressive periodontitis [43, 44].
In previous studies using GCF samples LXA4 level was reported to be lower in periodontitis, confirming that the findings of decrease in salivary LXA4 might be an indicator of susceptibility to periodontitis [45–47]. Therefore, saliva level of LXA4 may be more reliable than serum in evaluating the its role in periodontitis. LXA4 is a pro-resolving and anti-inflammatory mediator that acts inhibiting leukocyte-dependent inflammation. Similar to SIRT6, LXA4 inhibits the activation of NF-κB and this effect decreased the production of the inflammatory cytokines [48]. Liu et al. (2022), demonstrated that combination of Resolvin E1 and LXA4 treatment in pulpitis downregulated NF-κB activation and increased the expression of SIRT6 [49]. Our results might indicate that SIRT6 is involved in LXA4-mediated resolution and might synergistically promote resolution of inflammation in periodontitis. Also, it has been reported that LXA4 inhibits hypoxia-induced apoptosis and oxidative stress in human first trimester trophoblast cells [13]. Additionally, it has been shown that LXA4 reduced caspase 3, -8 and − 9 activation [14]. The role of LXA4 in periodontitis might also be related to apoptotic mechanisms.
It is thought that caspase activation might have an important role in periodontitis-associated tissue damage. Recently, several studies revealed that increased levels of caspase-3 is associated with periodontal disease progression [50–52]. We have evaluated saliva and serum CASP8 levels and found no significant differences between periodontitis patients and healthy individuals. Aral et al. (2017), reported that GCF levels of CASP8 similar between individuals with chronic periodontitis and healthy controls [21]. Also, Shi et al. (2019), evaluated CASP8 in gingival tissues and they found no differences between chronic periodontitis patients and healthy subjects [53]. These results are consistent with our study. Aral et al, (2017), also found that decreased saliva levels of CASP8 in aggressive periodontitis [21]. Manosudprasit et al. (2017), shown that in peripheral blood neutrophils, decreased CASP8 levels in chronic periodontitis with and without T2DM compared to healthy controls [22]. Several studies emphasize that caspase-3 and − 8 play an essential role in the butyric acid-induced apoptosis of inflamed gingival fibroblasts [54, 55]. Also, Zhou et al. (2018), reported that Porphyromonas gingivalis LPS induced apoptosis via increases expressions of caspase-3 and − 8 in osteoblasts [56]. ROS activate CASP8 to induce apoptosis and increases the BAX/BCL-2 ratio. Caspase-8, an initiator in extrinsic signaling pathways, cleaves caspase-3, an effector caspase, resulting in downstream events involved in apoptosis. Caspase-3 is rapidly activated by CASP8 [57]. The reason that we could not detect differences in CASP8 levels between groups might be due to the short half-life of this molecule and having a short detection window. SIRT6 has been shown to have an important role on caspase-8 in cancer cells by differentially regulating the expression and activity of pro-apoptotic and anti-apoptotic factors, depending on the type and stage of cancer [58]. The relationship of SIRT6 with CASP8 in its potential role in periodontitis can be better explained by using GCF samples as it may reflect the response in specific sites, and also by evaluating it together with effector caspase, caspase-3.
In the current study, negative correlations were found between all clinical parameters and saliva LXA4 level, and between PPD, CAL, and serum SIRT6 level. These findings regarding the correlation between salivary LXA4 and periodontal status is consistent with a previous study [42]. Our study findings demonstrated that decreased saliva LXA4 and serum SIRT6 levels are associated with periodontitis and indicating that decreased levels of these molecules would help to predict the clinical signs of the periodontal disease. Also we have found negative correlations between the SIRT6 serum and saliva levels, and CASP8 and LXA4 levels in saliva. The lack of previous studies on these correlations and also the fact that we have not seen significant differences between groups for these two molecules in our study, make it difficult to compare and interpret these correlations.
The main limitation of the current study is the relatively limited number of participants. Another limitation is the analysing only CASP8 as an indicator of apoptotic pathways. Also, the lack of a gingivitis group can be considered as a limitation.
In the current study, the results have underlined the importance of SIRT6, LXA4 and their correlation with periodontal status in periodontitis. Lower serum SIRT6 and saliva LXA4 levels in severe periodontitis may suggest SIRT6 and LXA4 are two of the many key molecules that are regulating periodontal health. Nonetheless, further analysis and larger studies are needed to clarify the role of SIRT6 in periodontal pathogenesis and resolution of inflammation processes.