The Anti-Inflammatory Effect of Human Telomerase-derived Peptide on Porphyromonas gingivalis Lipopolysaccharide-Induced Cytokine Production

Abstract

1. 목적 치수 및 치근단 질환의 주된 원인인 세균감염을 억제하고 조절하기 위해 항균, 항염증 펩타이드가 우식 및 근관치료 영역에 소개되고 있다. Human telomerase reverse transcriptase (hTERT) 유래의 항암 백신으로 개발된 GV1001 펩타이드는 세포내로 들어가 약리학적 효과를 나타내는 것과 함께, 항염 효과도 보고되고 있어, 치수 염증 질환 치료에의 적용 가능성을 제시하였다. 본 연구의 목적은 사람 치수 세포에서 P. gingivalis의 LPS로 유도된 염증에 대한 GV1001 펩타이드의 항염 효과를 알아보기 위함이다.

2. 재료 및 방법 성인의 매복 제3대구치에서 치수세포를 분리 배양하였다. P. gingivialis LPS (1 μg/ml)가 어떠한 Toll-like receptor (TLR)를 자극하는지 확인하기 위하여 CHO/CD14/TLR2와 CHO/CD14/TLR4를 사용하여 검증하였고 S. aureus LTA (1 μg/ml)와 E. coli LPS (100 ng/ml)는 대조군으로 사용하였다. Confocal microscopy로 GV1001 펩타이드의 세포질내 투과를 확인하였다. 사람 치수세포에 대한 GV1001 펩타이드 (1-50 µM)의 세포독성은 MTT assay로 분석하였고, P. gingivalis의 LPS로 유발된 염증 사이토카인인 TNF-α와 IL-6의 발현양을 real-time RT-PCR로 분석하였다. GV1001 펩타이드의 항염 효과에 MAP kinase (ERK, p38) 신호전달 과정이 관여하는지를 western blot 분석으로 확인하였다. 만-휘트니 U검증을 통하여 통계학적 유의성을 검증하였다 (P=0.05).

3. 결과 P. gingivalis LPS는 TLR2를 통해 CD25를 발현하였다. GV1001 펩타이드가 사람 치수 세포내로 들어가 세포질 내에 분포하였고, 사람 치수 세포에서 세포독성을 나타내지 않고 P. gingivalis LPS로 유도된 TNF-α와 IL-6의 생성을 유의하게 감소시켰다 (P < 0.05). 또한 GV1001 펩타이드는 LPS로 자극된 사람 치수 세포에서 MAP kinases (ERK, p38)의 인산화를 현저하게 억제하였다.

4. 결론 GV1001 펩타이드는 사람 치수 세포에서 P. gingivalis LPS로 유도된 ERK와 p38 MAPK의 인산화 억제를 통해 TNF-α와 IL-6의 발현을 감소시켜 항염 효과를 나타내었다.

Objectives. Therapeutic use of the antimicrobial and anti-inflammatory peptides to control microbial infection which is a major cause of pulpal and periapical diseases has attracted considerable interest in endodontic fields. GV1001 peptide is derived from human telomerase reverse transcriptase (hTERT) and was developed as a cancer vaccine. Due to its novel pharmaceutical potential with cell-penetrating ability, as well as anti-inflammatory activity, GV1001 peptide was suggested as a therapeutic agent to control pulpal inflammation. The purpose of this study was to evaluate the anti-inflammatory effect of GV1001 peptide and its related mechanism in P. gingivalis LPS-induced inflammation in human dental pulp cells (hDPCs).

Methods. Dental pulp cells from impacted third molars of human adults were isolated and cultivated. CHO/CD14/TLR2 and CHO/CD14/TLR4 were used to analyze which toll-like receptor (TLR) was stimulated by extracted LPS from P. gingivalis (1 μg/ml). S. aureus LTA (1 μg/ml) and E. coli LPS (100 ng/ml) were used as controls. The ability of intracellular penetration of GV1001 peptide was analyzed by confocal microscopy. The biocompatibility of the peptide (1-50 µM) was measured by MTT assay. Real-time RT-PCR was performed to investigate the expression levels of TNF-α and IL-6 from LPS-stimulated hDPCs. The role of MAP kinase (ERK, p38) signaling pathway on the anti-inflammatory effect of GV1001 peptide was analyzed by western blot analysis. Comparisons between 2 groups were analyzed using Mann-whitney U test (P=0.05).

Results. P. gingivalis LPS induced CD25 expression via TLR2. GV1001 peptide was predominantly located in the cytoplasm of hDPCs. The peptide down regulated P. gingivalis LPS-induced TNF-α and IL-6 production in hDPCs (P < 0.05) without showing significant cytotoxicity. Furthermore, the treatment of GV1001 peptide markedly inhibited the phosphorylation of MAP kinases (ERK and p38) in LPS-stimulated hDPCs.

Conclusions. GV1001 peptide inhibited TNF-α and IL-6 production through reducing P. gingivalis LPS-induced phosphorylation of ERK and p38 MAPK in hDPCs, which indicates that the peptide may have anti-inflammatory activity.