J Periodontal Implant Sci.  2012 Dec;42(6):185-195.

Anti-inflammatory effect of (-)-epigallocatechin-3-gallate on Porphyromonas gingivalis lipopolysaccharide-stimulated fibroblasts and stem cells derived from human periodontal ligament

• Jung IH
• Lee DE
• Yun JH
• Cho AR
• Kim CS
• You YJ
• Kim SJ
• Choi SH

Abstract

PURPOSE
(-)-epigallocatechin-3-gallate (EGCG) has been reported to exert anti-inflammatory and antibacterial effects in periodontitis. However, its exact mechanism of action has yet to be determined. The present in vitro study evaluated the anti-inflammatory effects of EGCG on human periodontal ligament fibroblasts (hPDLFs) and human periodontal ligament stem cells (hPDLSCs) affected by bacterial lipopolysaccharide (LPS) extracted from Porphyromonas gingivalis.
METHODS
hPDLFs and hPDLSCs were extracted from healthy young adults and were treated with EGCG and/or P. gingivalis LPS. After 1, 3, 5, and 7 days from treatment, cytotoxic and proliferative effects were evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and bromodeoxyuridine assay, respectively. And then, the gene expressions of hPDLFs and hPDLSCs were observed for interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, osteoprotegerin (OPG), receptor activator of nuclear factor kappa-B ligand (RANKL), and RANKL/OPG using real-time polymerase chain reaction (PCR) at 0, 6, 24, and 48 hours after treatment. The experiments were performed with the following groups for hPDLFs and hPDLSCs; 1) No treat, 2) EGCG alone, 3) P. gingivalis LPS alone, 4) EGCG+P. gingivalis LPS.
RESULTS
The 20 microM of EGCG and 20 microg/mL of P. gingivalis LPS had the lowest cytotoxic effects, so those concentrations were used for further experiments. The proliferations of hPDLFs and hPDLSCs increased in all groups, though the 'EGCG alone' showed less increase. In real-time PCR, the hPDLFs and hPDLSCs of 'EGCG alone' showed similar gene expressions to those cells of 'no treat'. The gene expressions of 'P. gingivalis LPS alone' in both hPDLFs and hPDLSCs were highly increased at 6 hours for IL-1beta, IL-6, TNF-alpha, RANKL, and RANKL/OPG, except the RANKL/OPG in hPDLSCs. However, those increased gene expressions were down-regulated in 'EGCG+P. gingivalis LPS' by the additional treatment of EGCG.
CONCLUSIONS
Our results demonstrate that EGCG could exert an anti-inflammatory effect in hPDLFs and hPDLSCs against a major pathogen of periodontitis, P. gingivalis LPS.

Keyword

Anti-inflammatory agents; Lipopolysaccharide; Periodontal ligament; Periodontitis; Porphyromonas gingivalis

MeSH Terms

Anti-Inflammatory Agents
Bromodeoxyuridine
Fibroblasts
Gene Expression
Humans
Interleukin-6
Interleukins
Osteoprotegerin
Periodontal Ligament
Periodontitis
Porphyromonas
Porphyromonas gingivalis
Real-Time Polymerase Chain Reaction
Stem Cells
Tetrazolium Salts
Thiazoles
Tumor Necrosis Factor-alpha
Young Adult
Anti-Inflammatory Agents
Bromodeoxyuridine
Interleukin-6
Interleukins
Osteoprotegerin
Tetrazolium Salts
Thiazoles
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Effects of (-)-epigallocatechin-3-gallate (EGCG) and Porphyromonas gingivalis lipopolysaccharide (LPS) on the viability of human periodontal ligament fibroblasts (hPDLFs) and human periodontal ligament stem cells (hPDLSCs). Most of the hPDLFs (A) and hPDLSCs (B) exhibited comparable cell viability in the EGCG-containing media (0, 0.1, 1, 10, and 20 µM) at days 3 to 7, except in 50 and 100 µM (P<0.1). The proliferation of hPDLFs (C) and hPDLSCs (D) treated with 50 µg/mL P. gingivalis LPS exhibited the most significant down-regulation at day 7, while remained unaffected for hPDLFs and less affected for hPDLSCs in other concentrations (i.e., 0, 0.1, 0.5, 1, 5, 10, and 20 µg/mL) (P<0.05). MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, OD: optical density. a), b), or c)There is a statistically significant difference; a) vs. b), a) vs. c), and b) vs. c) (P<0.1). d), e), or f)There is a statistically significant difference; d) vs. e), d) vs. f), and e) vs. f) (P<0.1).

• 

  Figure 2 Proliferative changes of human periodontal ligament fibroblasts (hPDLFs) and human periodontal ligament stem cells (hPDLSCs) treated with Porphyromonas gingivalis lipopolysaccharide (LPS) and (-)-epigallocatechin-3-gallate (EGCG). In general, hPDLFs (A) and hPDLSCs (B) proliferated continuously during the experimental period, with an exponential increase in cell growth being observed from days 5 to 7. The cellular proliferation was generally higher in hPDLSCs than hPDLFs. However, for both hPDLFs and hPDLSCs, the degree of conjugation with bromodeoxyuridine (BrdU) was significantly lower in EGCG-treated groups than in EGCG-untreated groups. Conjugation with BrdU was not affected by treatment with P. gingivalis LPS (P<0.05). OD: optical density. a), b), or c)There is a statistically significant difference; a) vs. b), a) vs. c), and b) vs. c) (P<0.05).

• 

  Figure 3 Effects of Porphyromonas gingivalis lipopolysaccharide (LPS) and/or (-)-epigallocatechin-3-gallate (EGCG) on inflammatory mRNA expressions. EGCG-treated cells (human periodontal ligament fibroblasts [hPDLFs] and human periodontal ligament stem cells [hPDLSCs]) showed similar or lower gene expressions than those cells of 'No treat'. The gene expressions of 'P. gingivalis LPS' alone were highly increased at 6 hours, but those increased gene expressions were down-regulated in 'EGCG+P. ginvialis LPS' group by the additional treatment of EGCG. IL-1β: interleukin 1β, IL-6: interleukin 6, TNF-β: tumor necrosis factor-α.

• 

  Figure 4 Effects of Porphyromonas gingivalis lipopolysaccharide (LPS) and (-)-epigallocatechin-3-gallate (EGCG) on osteoclastogenesis-related mRNA expression. (A, B, and C) The gene expression of 'P. gingivalis LPS alone' in human periodontal ligament fibroblasts (hPDLFs) was increased at 6 hours in receptor activator of nuclear factor kappa-B ligand (RANKL) and RANKL/osteoprotegerin (OPG), but it was down-regulated in 'EGCG+P. gingivalis LPS' group. (D, E, and F) The 'P. gingivali LPS alone' of human periodontal ligament stem cells (hPDLSCs) showed much higher gene expressions at 6 hours in both of RANKL and OPG, but those up-regulated gene expressions of RANKL and OPG were not increased in 'EGCG+P. gingivalis LPS' group.

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