흰쥐에서 LPS로 유도된 치주염에 동반된 이틀뼈 흡수에 대한 SLPI의 억제 효과

Abstract

Periodontitis is an infectious disease induced by gram-negative bacteria in dental plaque. Lipopolysaccharide (LPS), one of the cell wall components of gram-negative bacteria, induces the inflammatory response around tissue due to stimulation with inflammatory cells such as macrophage and thereby, it causes formation of periodontal pocket and alveolar bone resorption. Secretory leukocyte protease inhibitor in (SLPI) is known as anti-bacterial and anti-inflammatory cytokine inflammatory response. In addition, the expression of SLPI is increased by LPS as well as proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukine-1β (IL-1β) secreted from LPS-activated inflammatory cells. Increased SLPI by inflammatory response decreased arthritis and destruction of cartilage and bone through inhibition of TNF-α and nitric oxide secretion. Therefore, this study investigates whether SLPI inhibits inflammatory response and alveolar bone resorption in LPS-induced periodontitis. In the SLPI-injected group after LPS injection (LPS/SLPI), the bone volume and trabecular bone number were higher, but the ratio of bone surface area to bone volume, trabecular bone space, and trabecular bone structure model index were lower than those of the LPS-injected (LPS) group in analysis of the ROI parameter using micro-CT. The alveolar bone resorption in the LPS/SLPI group was decreased compared to that of the LPS group through histological analysis. In the LPS/SLPI group, protein expression of TNF-α, IL-1β and RANKL was decreased compared to that of the LPS group but SLPI, OPG, and Runx2 were increased in the tissues by immunohistochemical reaction. In LPS-treated MC3T3-E1 cells (MC3T3-E1/LPS), the mRNA and protein expression of TNF-α, IL-1β, and RANKL were increased compared to those of the control, but OPG and Runx2 were decreased. In LPS/SLPI-treated MC3T3-E1 cells (MC3T3-E1/LPS/SLPI), the mRNA and protein expression of TNF-α, IL-1β, and RANKL were decreased compared to those of MC3T3-E1/LPS but SLPI, OPG, and Runx2 were increased. In summary, Micro-CT and histological analysis showed that SLPI inhibits inflammation and decreases alveolar bone resorption in LPS-induced periodontitis rat. The expression of TNF-α, IL-1β, and RANKL is decreased, and that of SLPI, OPG, and Runx2 is increased in periodontal tissue of LPS/SLPI group and in MC3T3-E1/LPS/SLPI. Therefore, SLPI might be the signal molecule that can inhibit osteoclast activity and induce bone formation through the inhibition of osteoclast activation and increase of osteoblast activation.| 치주염은 주로 치면세균막에 존재하는 그람음성 세균들에 의해 유도되는 감염성 질환이다. 그람음성세균의 세포벽 구성성분중 하나인 LPS는 대식세포와 같은 염증세포들을 자극해 주변 조직에 염증반응을 유도함으로써 치주낭 형성과 이틀뼈 흡수를 일으킨다. 분비백혈구단백분해효소억제제(SLPI)는 염증반응부위에서 항세균 및 항염증작용을 하는 물질로 알려져 있다. SLPI 발현은 LPS 뿐만 아니라 TNF-α와 IL-1β 같은 전염증사이토카인에 의해서도 증가된다. 염증반응에 의해 증가된 SLPI는 TNF-α와 NO분비를 억제함으로서 관절염증, 연골 및 뼈의 파괴를 감소시킨다. 따라서, 본 연구에서는 SLPI가 LPS 처리에 의해 유도된 치주염에 동반되는 치조골 흡수 억제에 대한 효과를 규명하고자 하였다. Micro-CT를 이용한 ROI 분석에서 LPS 주사 후 SLPI를 주사한 군(LPS/SLPI군)은 LPS를 주사한 군(LPS군)에 비해 뼈 부피(bone volume)와 잔기둥뼈 개수(trabecular bone number)가 높았고, 뼈 복잡성(ratio of bone surface area to bone volume), 잔기둥뼈 사이 공간(trabecular bone space) 그리고 잔기둥뼈 형태(trabecular bone structure model index)는 낮았다. 조직학적 분석을 통해 LPS/SLPI군은 LPS군에 비해 이틀뼈 흡수가 감소하였다. 면역조직화학적 분석에서 LPS/SLPI군은 LPS군에 비해 TNF-α, IL-1β 그리고 RANKL 단백질이 낮게 발현되었으며, SLPI, OPG 그리고 Runx2 단백질은 높게 발현됐다. LPS를 처리한 MC3T3-E1 세포(MC3T3-E1/LPS)에서는 대조군에 비해 TNF-α, IL-1β 그리고 RANKL의 mRNA와 단백질 발현이 증가됐고, OPG와 Runx2는 감소됐다. LPS/SLPI를 처리한 세포(MC3T3-E1/LPS/SLPI)에서는 MC3T3-E1/LPS에 비해 TNF-α, IL-1β 그리고 RANKL의 mRNA와 단백질 발현이 감소됐고, OPG와 Runx2는 증가됐다. 본 결과를 요약하면, Micro-CT 및 조직학적 분석 결과로 SLPI는 흰쥐의 LPS로 유도된 치주염 억제 및 이틀뼈 흡수를 감소시켰다. LPS/SLPI군의 치주조직 면역화학분석 및 MC3T3-E1/LPS/SLPI에서는 TNF-α, IL-1β, 그리고 RANKL이 감소됐고 SLPI, OPG 그리고 Runx2가 증가됐다. 따라서 SLPI는 염증성 사이토카인 및 뼈파괴세포 활성 인자를 억제하고, 뼈모세포 활성인자들을 증가시킴으로써 뼈파괴세포 활성을 억제하고, 뼈형성 유도를 조절하는 신호분자 일 것으로 생각된다.