1. Introduction
Periodontitis, a common disease, is the inflammation of periodontium caused by oral microorganisms [
1]. Numerous cytokines have been detected in the gingival crevicular fluid (GCF). Interleukin-1β (IL-1β) and tumor necrosis-α (TNF-α) levels have been found in increased GCF from inflamed gingival tissues [
2,
3]. IL-1β, matrix metalloproteinase-8, and IL-8 levels in GCF were significantly higher in diseased sites than in healthy subjects, and those levels in GCF were significantly decreased after periodontal treatment [
4]. In a recent study, GCF volume and IL-1β levels in GCF reflected the disease severity, and these parameters were suggested to be better than probing pocket depth (PPD) and bleeding on probing (BOP) as markers of gingival inflammation [
5]. Therefore, it could be helpful to investigate the inflammatory cytokine levels in GCF for diagnosis of the active phase of periodontal disease.
Heat shock proteins (HSPs) act as molecular chaperones, which are enhanced proteins for protecting cells immediately after cells are exposed to heat shock stress [
6]. The 70-kDa HSPs (HSP70s) comprising various isoforms are involved in a number of human pathologies, ranging from cancer to neurodegenerative diseases [
7,
8]. HSP70 levels were higher in protein extract from inflamed gingiva collected during periodontal surgery, and HSP60 and HSP65 levels were higher in serum from the patients with periodontitis [
9]. In addition, expression levels of IL-1β, IL-8, and HSP70 were increased in GCF from patients with periodontitis compared to healthy subjects [
10]. Serum concentration of chitinase-3-like-1 (YKL-40), which is a novel marker of acute and chronic inflammation, was significantly higher in patients with periodontitis and diabetes compared to healthy groups. However, GCF concentration of YKL-40 was similar in patients with periodontitis, patients with diabetes, and healthy subjects. YKL-40 levels were significantly increased in diabetes patients with periodontitis [
11]. Therefore, HSP70 can be considered potentially as a marker for the severity of periodontal disease. However, there is no report on changes in HSP70 concentration in GCF before and after initial periodontal therapy, or its correlation with clinical parameters. The aim of this study was to elucidate the HSP70 levels in GCF from patients with periodontitis, and to compare their concentrations in GCF at first visit, after initial periodontal therapy, and at a three month follow-up.
3. Results
The patient characteristics such as age, sex, PPD, CAL, PlI, GI, and BOP distributions for the 10 patients in this study are listed in
Table 1. Average PPD and CAL at the HC sites (PPD ≤ 3 mm) were 2.7 ± 0.2 mm and 3.9 ± 0.4 mm, and at the diseased sites (PPD ≥ 5 mm) were 6.5 ± 0.5 mm and 7.7 ± 0.7 mm, respectively. GI and BOP scores at the diseased sites (1.7 ± 0.2 and 80%) were higher than those at the HC sites (0.3 ± 0.2 and 0%). PlI at the HC and diseased sites were the same score (1.1 ± 0.2).
The concentrations of HSP70 in GCF from the HC and diseased sites at each point of examination during initial periodontal therapy are shown in
Table 2. The average HSP70 level at the 1st visit in GCF from diseased sites was significantly higher than HC sites. Moreover, the concentration of HSP70 at diseased sites was significantly decreased at 3rd examination (a three month follow-up after initial periodontal therapy) as compared to the 1st examination. The concentration of HSP70 at HC sites did not change through the periodontal therapy (1st, 2nd, and 3rd examinations) (
Table 2).
Changes in five kinds of clinical parameters (PPD, CAL, PlI, GI, and BOP scores) at the HC and diseased sites during initial periodontal therapy are listed in
Table 3 and
Table 4. At HC sites, PlIs were significantly decreased at the 2nd and 3rd examinations as compared to the 1st examination (
Table 3). On the other hand, PPD and PlI at diseased sites were significantly decreased at the 2nd and 3rd examinations compared to the 1st examination. Furthermore, GI and BOP scores at diseased sites were significantly decreased at the 3rd examination compared to the 1st examination. GCF volumes from HC and diseased sites were measured by Periotron 4000 during the course of periodontal therapy (
Table 5). GCF volumes from HC and diseased sites did not change during the periodontal therapy, however, the volumes of GCF from the diseased sites were significantly higher than those in the HC sites at the 1st and 2nd visits (
Table 5).
4. Discussion
In this study, we have shown that there was a significant difference in HSP70 concentration in GCF between HC and diseased sites at the first visit. The concentration of HSP70 in GCF from diseased sites was significantly decreased at the three month follow-up after initial periodontal therapy (3rd examination;
Table 2). At HC sites, PlI was significantly decreased at the 2nd and 3rd examinations (
Table 3). At diseased sites, PPD and PlI were significantly decreased at the 2nd and 3rd examinations, whereas GI and BOP were significantly decreased only at the 3rd examination as compared to the 1st visit (
Table 4). These results suggest that initial periodontal therapy is effective in improving inflammation of periodontal tissues and there is an association between the level of HSP70 and periodontitis. In addition, improvements of GI, BOP, and HSP70 levels were found to take longer than improvements of PPD and PlI.
Intracellular HSP levels are elevated immediately after exposed to stresses such as high temperature. HSPs are involved in the maintenance of cellular homeostasis and protein repair in damaged cells [
18]. However, there are several unclear points in the relationship between HSPs and periodontitis. Inflammatory periodontal pockets have a higher temperature than healthy pockets [
19]. Inflammatory cytokines, such as IL-1, TNF-α, and INF-γ, are produced in inflamed periodontal tissues [
20], and they might act as stressors to induce the expression of HSPs. Lipopolysaccharide (LPS) and IL-1 increased hyperthermia-induced HSP70 in monocyte/macrophage-like RAW264.7 cells [
21]. However, one study described how HSP70 dramatically down-regulated in the inflamed periodontal tissues [
22]. Another study showed that GCF volume at the first visit decreased significantly after initial periodontal therapy [
23]. However, in this study, GCF volumes from HC and diseased sites did not change during periodontal therapy (
Table 5), although the GCF volumes from diseased sites at the 1st and 2nd visits were significantly higher than the GCF volumes from HC sites (
Table 5). Therefore, further study is necessary to elucidate the involvement of HSP70 in the onset and progression of periodontitis.
Stress and smoking are environmental factors for periodontitis [
24,
25]. Several studies have shown that smoking has an adverse effect on the incidence and progression of periodontitis [
25]. In the synovial tissues of smokers with rheumatoid arthritis (RA), HSP70 levels were significantly higher than in the synovial tissues of non-smokers with RA [
26]. Therefore, smoking could increase the expression of HSP70. There are several studies describing the association between HSP70 and cancer. Malignant cells, such as osteosarcoma derived cells, expressed higher levels of HSP70 during tumor progression compared to normal cells [
27]. Moreover, HSP70 has been assessed as a marker for oral epithelial dysplasia such as oral leukoplakia [
28]. Therefore, various studies have been conducted to develop the HSP70 inhibitors for cancer therapy [
29]. We have previously shown that the anti-HSP70 antibody levels were significantly higher in GCF from HC sites than diseased sites, and the anti-HSP70 antibody levels were increased after initial periodontal therapy in both HC and diseased sites [
30]. Therefore, these results suggest that anti-HSP70 antibody may reduce inflammation of periodontal tissues via decreasing the HSP70 levels.
In conclusion, GCF volumes from the diseased sites were significantly higher than those in the HC sites at the 1st and 2nd visits. HSP70 concentration in GCF from diseased sites was significantly higher than the concentration of HSP70 from HC sites at the 1st visit. Moreover, the HSP70 concentration at the 1st visit was significantly decreased at the three month follow-up after initial periodontal therapy together with clinical parameters, such as PPD, GI, PlI, and BOP. These results suggest that the HSP70 concentration could become an appropriate indicator for the healing process of periodontitis.