Vitamin C attenuates the cytotoxic effects of Porphyromonas gingivalis on human gingival fibroblasts

https://doi.org/10.1016/j.archoralbio.2009.11.009Get rights and content

Abstract

Objective

Periodontitis is induced by an imbalance between bacterial virulence and host defense ability involving increased levels of oxidative stress. The aim of this study was to investigate the influence of vitamin C on the cytotoxic effects of Porphyromonas gingivalis on human gingival fibroblasts (HGF).

Methods

This in vitro study observed the interaction between HGF and P. gingivalis. HGF were cultured with medium containing vitamin C and exposed to P. gingivalis ATCC 33277 for a maximum of 180 min. The assessment of cell viability was followed by a 3-(4,5-dimethylthiazol-2-ly)-2,5-diphenyltetrazolium-bromide (MTT) assay. The apoptosis rate was detected by flow cytometry using Annexin-V-FITC and propidium iodide. Superoxide as an oxidative stress factor was measured photometrically by the reduction of ferricytochrome C.

Results

Vitamin C reduced the cytotoxic effects of P. gingivalis on HGF. Vitamin C-treated HGF showed significantly higher cell viability rates (89.0 ± 5.7%) in comparison to untreated HGF (77.0 ± 5.0%; p < 0.05). In vitamin C-treated HGF, lower apoptosis rates (40.0 ± 2.2%) were observed after P. gingivalis exposure than in untreated HGF (66.1 ± 1.6%; p < 0.05). The exposure of HGF to P. gingivalis led to a significant increase of superoxide concentration, but this effect was not influenced by vitamin C.

Conclusion

Vitamin C reduces the cytotoxic and apoptotic effects of P. gingivalis on HGF in vitro. These results suggest that the benefit of vitamin C should be further investigated clinically.

Introduction

Epidemiological studies indicate a low vitamin C concentration in plasma as a risk factor for periodontitis.1, 2, 3, 4 Our group recently reported that patients with periodontitis – in contrast to healthy subjects – had decreased vitamin C levels in their plasma.5 Although the specific role of vitamin C deficiency is widely unknown, this vitamin has long been a candidate for modifying periodontal diseases.6 This could be due to the various biological functions which are also connected with the periodontal tissues. For example, studies have shown that vitamin C increases the number of collagen bundles in the regenerating periodontal tissue,7 detoxifies histamine in gingival inflammation,8, 9 and reduces gingival oxidative stress.10 Particularly, the role of oxidative stress in periodontal disease is well established.11 The investigation of oxidative stress factors in the saliva by our group resulted in increased levels of reactive oxygen species in periodontitis patients corresponding with decreased anti-oxidative capacities.12 In this connection the antioxidant properties of vitamin C produced interest. As a water-soluble vitamin it protects especially aqueous environments against oxidative attacks. For example, leucocytes accumulate vitamin C to protect themselves during phagocytosis against oxidative species.13 Fibroblasts also require vitamin C for normal cell growth in the context of collagen synthesis14 and as an antioxidant.15 Gingival fibroblasts play a key role in the regulation of the physiological turnover of connective tissues by depleting collagen and remodelling new collagen fibrils in the periodontal tissue.16 Contact with periodontal-pathogenic bacteria disturbs this sensitive balance resulting in collagen degradation and consequently in pocket formation.17 One of the invasive bacteria in the plaque biofilm is Porphyromonas gingivalis, a black-pigmented, anaerobic gram-negative species.18 It produces a broad array of potential virulence factors, including lipopolysaccharides, fimbriae and proteases that affect the host response and damage oral cells.19, 20, 21 In periodontal fibroblasts it suppresses the proliferation and induces a reduction in collagen content.22 Because vitamin C stimulate the collagen synthesis in fibroblasts it might prevent the negative effects of P. gingivalis. A current study demonstrated that antibodies to P. gingivalis were negatively correlated with the plasma vitamin C concentration,3 which confirmed the hypothesis of a special interaction between P. gingivalis and vitamin C. The purpose of the present study was to investigate the influence of vitamin C on the cytotoxic effects of P. gingivalis on human gingival fibroblasts in vitro.

Section snippets

Culture of human gingival fibroblasts

Primary cultures of human gingival fibroblasts were obtained from a gingival biopsy of a donator with clinically healthy periodontium23 after the positive vote of the ethical committee of the Friedrich Schiller University Jena (B1881-10/06). Briefly, cells were cultured in Dulbecco's modified Eagle medium (DMEM; PAA Laboratories, Linz, Austria) supplemented with 10% fetal calf serum (FCS; PAA Laboratories, Linz, Austria) and antibiotics (10 U ml−1 penicillin, 10 mg/ml streptomycin, 25 μg/ml

Measurement of cell viability

Co-incubation with vitamin C significantly increased the viability rate of HGF (Fig. 1). The highest viability rate was observed with a vitamin C concentration of 12 μg/ml and exceeded the values of untreated HGF by 29.2 ± 6.4% (p < 0.05). The viability rates were lower with concentrations about 12 μg/ml, but the rates increased also the level of untreated controls.

Fig. 2 shows the viability rates varying with exposure to viable P. gingivalis ATCC 33277 and exposure time. In comparison to controls

Discussion

The present results demonstrate that vitamin C decreases apoptosis and prevents viability loss in HGF exposed to viable P. gingivalis.

Several studies have investigated the effect of antioxidants on bacteria-induced apoptosis in different cell lines. For example, researchers found that the treatment with tocopherol (vitamin E) increased the survival of Pseudomonas aeruginosa-infected cells.24 Before this investigation, there were no studies describing the effect of antioxidants in a context with

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