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Oxidative damage of periodontal tissue in the rat periodontitis model: Effects of a high-cholesterol diet
Abstract
Studies suggest an association between consumption of a high-cholesterol diet and periodontitis. We addressed the mechanism by which high dietary cholesterol could be detrimental to periodontal health in a rat model. Feeding a high-cholesterol diet augmented the effects of bacterial pathogens and their products (e.g., lipopolysaccharide and proteases) on production of pro-inflammatory cytokines in fibroblasts. High dietary cholesterol also increased mitochondrial 8-hydroxydeoxyguanosine in the periodontal tissues. These results suggest that excessive tissue oxidative damage induced by high dietary cholesterol could potentiate pro-inflammatory cytokine production by fibroblasts stimulated with bacterial pathogens.
1 Introduction
Feeding rats a high-cholesterol diet impairs lipid metabolism, resulting in an increase in total serum cholesterol and triglycerides [1]. Higher serum levels of total cholesterol [2] and triglycerides [3] have been found in individuals with periodontal disease compared with healthy individuals, and periodontal patients with impaired cholesterol metabolism have deeper periodontal pocket depths than periodontal patients with normal metabolic status [4]. These results strongly suggest that high dietary cholesterol can influence the progression of periodontitis.
Oxidative stress is involved in the pathogenesis of a number of diseases, including periodontitis [5], and feeding a high cholesterol diet increases tissue oxidative stress in various organs [6, 7]. It is feasible then that tissue oxidative damage caused by high dietary cholesterol, as well as periodontopathic bacteria [8], may play an important role in periodontitis progression.
Oxidative stress induces tissue damage by injuring the cell both directly and indirectly and causing cell death [9]. Where fibroblasts in the periodontal tissues are injured by oxidative stress, the detrimental effects may result in periodontitis [10]. 8-Hydroxydeoxyguanosine (8-OHdG), a modified DNA base is generally accepted as a reliable indicator of oxidative stress [11]. The purpose of the present study was to assess the effects of high dietary cholesterol on 8-OHdG expression in fibroblasts in the rat periodontitis model. In addition, because pro-inflammatory cytokines such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) directly induce connective tissue breakdown and alveolar bone loss during periodontitis [12], the changes in IL-1β and TNF-α production were also evaluated.
2 Materials and methods
2.1 Animals
Eight-week-old male Wistar rats (n = 32) were housed two per cage in rooms maintained at 23–25 °C on a 12-h light/12-h dark cycle, with the light phase beginning at 6:00 AM. Rats had free access to food and water. All animal procedures were carried out in compliance with guidelines approved by the Animal Research Control Committee of Okayama University Dental School.
2.2 Experimental design
Rats were randomly divided into four groups of eight rats. During the experimental period, the first two groups were fed with a regular diet for eight weeks, and pyrogen free water (control group) or bacterial pathogens (periodontitis group) were applied into the gingival sulcus for four weeks prior to the end of the experimental period. The remaining two groups were fed a diet containing 1% cholesterol (w/w) and 0.5% cholic acid (w/w) (Oriental Yeast Co., Tokyo, Japan) for eight weeks, and were treated with pyrogen free water (cholesterol group) or bacterial pathogens (combination group) for four weeks prior to the end of the experimental period. Periodontitis was induced by bacterial pathogens [combination of a 25 μg/μL Escherichia coli lipopolysaccharide (LPS) and 2.25 U/μL proteases from Streptomyces griseus (Sigma Chemical Co., St. Louis, MO, USA) suspension in pyrogen-free water] [10]. LPS (0.5 μL × 3 times) and proteases (0.5 μL × 3 times), or pyrogen-free water (0.5 μL × 6 times) were introduced daily by micropipette into the palatal gingival sulcus of both maxillary first molars within 1 h of intraperitoneal anesthesia with sodium pentobarbital (0.5 mL/kg bodyweight).
2.3 Lipid assays
At eight weeks, blood samples were collected directly from the heart of 24-h-fasted animals. Blood was allowed to clot at room temperature for 1 h, and serum was separated by centrifugation at 1500 × g for 15 min. Levels of total serum cholesterol and triglycerides were determined using an enzymatic commercial kit (Cholesterol E-test Wako kit; Wako Pure Chemical Industries, Ltd., Osaka, Japan) [13].
2.4 Measurement of mitochondrial 8-OHdG
Mitochondrial DNA was isolated from rat gingiva using a DNA extractor kit (Wako Pure Chemical Industries). Isolated mitochondrial DNA was analyzed by a competitive enzyme-linked immunosorbent assay method using an “8-OHdG Check” kit (Japan Institute for the Control of Aging, Shizuoka, Japan) [14].
2.5 Histological and immunohistochemical analysis
The rats were sacrificed by intracardiac perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under intraperitoneal anesthesia. Teeth and gingivae were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C overnight, followed by decalcification with a 10% tetrasodium-EDTA aqueous solution (pH 7.4) for 2 weeks at 4 °C. Paraffin-embedded bucco-lingual sections (4 μm) of each tooth were then prepared for immunohistochemical staining for 8-OHdG, IL-1β and TNF-α.
8-OHdG, IL-1β and TNF-α were stained using a commercial kit (Nichirei Co., Tokyo, Japan). Polyclonal antibody against 8-OHdG (Chemicon International, CA, USA), IL-1β (Santa Cruz, Santa Cruz, CA, USA) and TNF-α (R & D Systems, Minneapolis, CA, USA) was diluted at 1/200, 1/50 and 1/200, respectively, in phosphate buffered saline. The color was developed with 3–3′-diamino benzidine tetrahydrochloride, and sections were counterstained with Mayer's hematoxylin. In addition, specificity of staining was established by showing isotype antibody controls and by confirming a prevention of positive staining when the antibody was previously treated with standard 8-OHdG (Japan Institute for the Control of Aging), IL-1β (Pierce Biotechnology, Inc., IL, USA) or TNF-α (Biosource International, Camarillo, CA, USA) in the immunohistochemical procedure.
Histometrical measurements were performed using a microscope at a magnification of 400×, and the examiner was blinded to which group each specimen belonged to. IL-1β positive fibroblasts, TNF-α positive fibroblasts and total fibroblasts were counted in standard areas (0.1 mm × 0.1 mm each) within the gingiva (the connective tissues subjacent to the junctional epithelium) and periodontal ligament [10].
2.6 Statistical analysis
Tukey's method was used to determine whether there were any statistically-significant differences between groups. All calculations were performed using a statistical software package (SPSS 10.0J for Windows, SPSS Japan, Tokyo, Japan).
3 Results
No significant differences were found among the four groups with regards to food consumption and body weight during the experimental period.
The mean values for total serum cholesterol for the cholesterol and combination groups were significantly higher than those for the control and periodontitis groups (Table 1 ). There were no statistical differences in mean serum triglycerides values among the four groups.
| Control | Cholesterol | Periodontitis | Combination | |
|---|---|---|---|---|
| Total cholesterol | 1.61 ± 0.30 a | 3.25 ± 0.94 b c | 1.70 ± 0.34 | 3.95 ± 1.52b, c |
| Triglyceride | 0.06 ± 0.03 | 0.06 ± 0.02 | 0.05 ± 0.02 | 0.07 ± 0.03 |
- a Values present means ± SD (n = 8).
- b p < 0.05, compared to the control group, using Tukey’s method.
- c p < 0.05, compared to the periodontitis group, using Tukey’s method.
8-OHdG staining was detected in the cytoplasm of gingival cells (Fig. 1 ). Compared with the control group, an increased number of 8-OHdG positive fibroblasts was observed in the cholesterol, periodontitis and combination groups (Table 2 ). The magnitude of this increase was greater in the combination group than in the cholesterol and periodontitis groups. The levels of mitochondrial 8-OHdG in the cholesterol, periodontitis and combination groups were higher than in the control group (Fig. 2 ). Furthermore, the levels of mitochondrial 8-OHdG for the combination group were higher than those for the cholesterol and periodontitis groups.
| Control | Cholesterol | Periodontitis | Combination | |
|---|---|---|---|---|
| Gingival fibroblasts | ||||
| IL-1β positive cells | 1.9 ± 1.5 a | 2.0 ± 1.7 | 3.0 ± 0.5 | 6.8 ± 2.2 b c d |
| TNF-α positive cells | 0.7 ± 0.5 | 0.7 ± 0.5 | 1.8 ± 0.9 | 3.6 ± 1.6d, b, c |
| 8-OHdG positive cells | 1.2 ± 0.7 | 3.2 ± 1.0 b | 3.5 ± 1.1 b | 7.0 ± 2.2d, b, c |
| Total cells | 19.6 ± 3.5 | 19.5 ± 3.2 | 21.8 ± 1.5 | 23.1 ± 4.0 |
| Periodontal ligament fibroblasts | ||||
| IL-1β positive cells | 2.7 ± 0.9 | 3.6 ± 1.6 | 4.9 ± 0.6 b | 7.4 ± 1.9d, b, c |
| TNF-α positive cells | 2.3 ± 0.9 | 3.0 ± 1.7 | 4.5 ± 0.6 b | 7.4 ± 1.0d, b, c |
| 8-OHdG positive cells | 3.0 ± 1.1 | 8.8 ± 1.7 b | 8.9 ± 1.0 b | 11.7 ± 1.0d, b, c |
| Total cells | 18.1 ± 2.2 | 19.7 ± 2.9 | 21.5 ± 2.6 | 21.5 ± 2.5 |
- a Values present means ± S.D. (number of cells/0.1 mm × 0.1 mm, n = 8).
- b p < 0.05, compared to the control group, using Tukey’s method.
- c p < 0.05, compared to the cholesterol group, using Tukey’s method.
- d p < 0.05, compared to the periodontitis group, using Tukey’s method.
Cell cytoplasm was stained positive for IL-1β and TNF-α (Fig. 1). The densities of IL-1β positive fibroblasts and TNF-α positive fibroblasts for the periodontitis group were higher than those for the control group in the periodontal ligament (Table 2). The densities of IL-1β positive fibroblasts and TNF-α positive fibroblasts for the combination group were higher than those for the periodontitis group in both the gingiva and periodontal ligament. There were no significant differences in the densities of total fibroblasts among the four groups.
4 Discussion
In the present study, feeding a high-cholesterol diet did not significantly affect production of IL-1β and TNF-α. However, high dietary cholesterol increased oxidative DNA damage and the effects of bacterial pathogens on IL-1β and TNF-α production in fibroblasts. Oxidative stress contributes to the impaired cellular function, including induction of nuclear factor κB activation, which stimulates pro-inflammatory cytokines production [15]. Excessive tissue oxidative damage by high dietary cholesterol may potentiate IL-1β and TNF-α production by fibroblasts stimulated with bacterial pathogens.
Bacterial pathogens can also enhance oxidative stress by potentiating inflammatory responses [16]. Thus, not only a hypercholestelomic condition but also bacterial pathogens can induce oxidative damage in tissues. In the current study, bacterial pathogens and high dietary cholesterol had an additive effect on oxidative DNA damage.
IL-1β and TNF-α can directly stimulate connective tissue breakdown and bone loss in the periodontal tissues [12]. In this study, bacterial pathogens increased IL-1β and TNF-α production in the periodontal ligament. Furthermore, the combination of high dietary cholesterol and bacterial pathogens elevated IL-1β and TNF-α production in the gingiva and periodontal ligament to a greater extent than bacterial pathogens alone. This enhanced production of IL-1β and TNF-α by high dietary cholesterol (tissue oxidative damage) may further augment periodontitis.
Expression of IL-1β and TNF-α in gingival fibroblasts did not increase following topical application of bacterial pathogens. Gingival fibroblasts may be relatively resistant to the induction of inflammatory cytokines by bacterial pathogens, compared to the periodontal ligament fibroblasts. This is consistent with the results of our previous study in which rat gingival fibroblasts exhibited less of an apoptotic response to lipopolysaccharide and proteases than rat periodontal ligament fibroblasts [10]. The difference in the composition of the fibroblast subpopulations may be responsible for this difference between gingival and periodontal ligament cell responses.
Excessive tissue oxidative damage induces impaired cellular function, contributing to periodontitis progression. Tissue destruction in periodontitis in turn produces oxidative stress [16], and such responses will further heighten oxidative damage in the periodontal tissues. Tissue oxidative damage, impaired cellular function and periodontitis progression thus seem to be a self-perpetuating process. It is feasible that high dietary cholesterol affects this process by increasing tissue oxidative damage.
Oxidative stress plays an important etiological role in a number of diseases, including arthritis, adult respiratory distress syndrome, heart disease, stroke, acquired immunodeficiency syndrome, Alzheimer's disease, Parkinson's disease and alcoholism [17]. Since periodontitis may influence general health [18], it is feasible that some of these diseases may be augmented by the oxidative stress induced in the periodontium. However, further studies are needed to clarify how elevation of oxidative stress in the periodontium may affect one's general health.
In the present study, we found no significant differences in serum triglyceride among the four groups. It has been reported that, in the rat model, there is less of an increase in serum triglyceride levels compared with total serum cholesterol following a high-cholesterol diet [19]. This may be because the high-cholesterol diet was used for too short a period of time to elevate serum triglycerides.
The present study used 0.5% cholic acid to induce hypercholesterolemia in rats [20]. While cholate may result in hepatic steatosis and cirrhosis, Lichtman et al. [21] reported that 0.5% cholic acid did not cause a significant elevation in serum levels of aspirate aminotransferase or alanine aminotransferase, both indicators of liver injury. The increased oxidative stress and levels of pro-inflammatory cytokine seen in the current study is most likely due to high cholesterol levels rather than the hepatotoxic effects of the cholic acid.
Using apolipoprotein E or low density lipoprotein receptor gene knock-out mice may be a more appropriate model for high cholesterol studies than the rat model, as these mice exhibit lipoprotein profiles more similar to humans [22]. However, mice have smaller mouths, making it difficult to apply bacterial pathogens to the gingival sulcus.
In conclusion, feeding a high-cholesterol diet could augment periodontitis via elevated IL-1β and TNF-α production by fibroblasts in response to bacterial pathogens, and this may be induced by excessive tissue oxidative damage.
Acknowledgement
This work was supported by a Grant in Aid from the Ministry of Education, Science, Sports and Culture of Japan (17209066 and 17791576).
