Gingival fibroblasts resist apoptosis in response to oxidative stress in a model of periodontal diseases

Abstract

Periodontal diseases are classified as inflammation affecting the supporting tissue of teeth, which eventually leads to tooth loss. Mild reversible gingivitis and severe irreversible periodontitis are the most common periodontal diseases. Periodontal pathogens initiate the diseases. The bacterial toxin, lipopolysaccharide (LPS), triggers the inflammatory response and leads to oxidative stress. However, the progress of oxidative stress in periodontal diseases is unknown. The purpose of this study is to examine oxidative stress and cell damage in gingivitis and periodontitis. Our results showed that LPS increases reactive oxygen species (ROS) accumulation in gingival fibroblast (GF). However, oxidative stress resulting from excessive ROS did not influence DNA damage and cell apoptosis within 24 h. The mechanism may be related to the increased expression of DNA repair genes, Ogg1, Neil1 and Rad50. Detection of apoptosis-related proteins also showed anti-apoptotic effects and pro-apoptotic effects were balanced. The earliest damage appeared in DNA when increased γH2AX, an early biomarker for DNA damage, was detected in the LPS group after 48 h. Later, when recurrent inflammation persisted, 8-OHdG, a biomarker for oxidative stress was much higher in periodontitis model compared to the control in vivo. Staining of 8-OHdG in human periodontitis specimens confirmed the results. Furthermore, TUNEL staining of apoptotic cells indicated that the periodontitis model induced more cell apoptosis in gingival tissue. This suggested GF could resist early and acute inflammation (gingivitis), which was regarded as reversible, but recurrent and chronic inflammation (periodontitis) led to permanent cell damage and death.

Figure 1

Gingival fibroblast was treated with LPS (1 μg/ml) for 24 h. Reactive oxygen species (ROS) were determined by DCFH-DA staining. (a) LPS increases ROS accumulation in gingival fibroblast (magnification ×100). (b) The ROS-positive cells were counted by flow cytometry. The experiment was repeated three times with similar results (Ctrl, control; **P<0.01).

Figure 2

DNA damage appeared after stimulation of LPS for 48 h. Immunofluorescent localization of γH2AX in gingival fibroblasts was performed following 1 μg/ml LPS treatment for 6, 24 and 48 h. (a and b) γ-H2AX was not detected in 6 and 24 h, respectively. (c) But γ-H2AX was increased in cell nuclei by LPS in 48 h (red arrow; Ctrl, control; magnification ×400). (d) LPS (1 μg/ml) increased p53 and p21 in 24 h.

Figure 3

Even though ROS was increased and DNA damage appeared, cell apoptosis rate were not changed by LPS. Annexin V/7-AAD staining followed by flow cytometry was conducted to study the apoptotic rates. (a) LPS (1 μg/ml) had little influence on apoptosis rate and cell cycle in 24 h. The experiment was repeated three times and statistical analysis was performed. (b) Even in 48 h, LPS (1 μg/ml) had little influence on apoptosis rate (Ctrl, control). The experiment was repeated three times. (c) Cleaved caspase-3 is not obvious after stimulation of LPS (1 μg/ml) for 24 h.

Figure 4

Chronic periodontitis-induced apoptosis and DNA damage in gingiva. Approximately, 10 μg LPS in 10 μl PBS or 10 μl PBS vehicle were injected into mandibular buccal gingiva two times per week for 3 weeks to establish experimental periodontitis models or PBS vehicle models. (a) There were higher expressions of 8-OHdG in the nuclei in BalB/c mice periodontitis models compared to the PBS vehicle models (scale bar, 100 μm). (b) Paraffin-embedded human gingival samples were stained for 8-OHdG by IHC (other two healthy and four periodontitis specimens were shown in Supplementary Figure 1; scale bar, 50 μm). The percentage of positive cells was counted from four microscopic fields per sample. (c) TUNEL staining showed more apoptotic cells in BalB/c mice periodontitis model, especially in submucosal tissue (scale bar, 100 μm). (d) TUNEL staining in Rag2^−/− mice periodontitis model (scale bar, 100 μm). The percentage of positive cells (a, c and d) was counted from three to four mice samples and four microscopic fields per sample (**P<0.01).

Figure 5

The mechanisms of gingival fibroblast in cell apoptosis resistance. (a) Effects of LPS on the expressions of pro-apoptotic proteins and (b) anti-apoptotic proteins in vitro. Gingival fibroblasts were treated with different doses of LPS for 24 h (0, 0.001, 0.01, 0.1 and 1 μg/ml LPS). The results showed that LPS increased phosphor-ERK1/2 (p-ERK), phosphor-p38 (p-p38), phosphor-JNK (p-JNK), p53, BCL-2 and survivin in a dose-dependent manner. Simultaneously, phosphor-AKT (p-AKT) decreases in a dose-dependent manner. Error bars represent the S.D. of three different experiments (*P<0.05; **P<0.01).

Figure 6

Other possible mechanisms in cell survival process. (a) Gingival fibroblasts were treated with different doses of LPS for 24 h (0, 0.001, 0.01, 0.1 and 1 μg/ml LPS). LPS increased the ratio of LC3 ІI/LC3 I, indicating autophagy was involved. However, the protein level of p16 had not changed. Error bars represent the S.D. of three different experiments (**P<0.01). (b) Stimulation of LPS for 24 h increased the mRNA expressions of DNA repair genes (Ctrl, control).