探討茶鹼衍生物KMUP-1抑制由RANKL/Pg LPS所誘導的發炎反應及蝕骨細胞分化之機轉

The Effect of Xanthine Derivativeon Inflammation and Osteoclast Differentiation Induced by RANKL/Pg LPS

張本樺(Pen-Hua Chang)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2020年)

摘要

在已發展國家和發展中的國家，牙周疾病是一種常見疾病，根據世界衛生組織2014年的統計數據，牙周疾病已經影響了全球20％-50％的人。牙周疾病是牙齒周圍的牙齦和骨支持物（牙周組織）的病理性炎症，骨頭的恆定是通過成骨細胞和蝕骨細胞維持平衡，而牙周病的病理性骨質流失情況是由於蝕骨細胞過度活化引起的。核因子κ-B配體受體激活劑（RANKL）活化NF-κB和MAPK途徑對於蝕骨細胞形成很重要。黃嘌呤衍生物在近幾年的大量論文中顯示出抗炎和抗氧化的功能。因此，本研究的目的是探討黃嘌呤衍生物(KMUP-1)在抑制RANKL /牙齦卟啉單胞菌脂多醣（Pg LPS）誘導炎症和蝕骨細胞分化的機轉。在體外試驗，將RAW264.7與KMUP-1和RANKL共同培養24小時，移除培養基後，使用KMUP-1預處理1小時，再加入Pg LPS。體外實驗顯示，KMUP-1可抑制RANKL / Pg LPS誘導的ROS生成和發炎性細胞因子含量。在西方墨點法中，KMUP-1抑制炎症反應是經由下調MAPK /NF-κB路徑表現，並且減少蝕骨細胞c-Fos、NFATc1和NF-κB的表現。除此之外，TRAP實驗也證明KMUP-1可抑制蝕骨細胞分化。在活體內實驗，實驗性牙周炎動物模式是使用來自Porphyromonas gingivalis (Pg) 的LPS注射到大鼠第1 (M1)和第2 (M2)臼齒之間的牙齦，藉由電腦斷層掃描以及TRAP分析可以發現KMUP-1顯著抑制牙周炎所引起牙槽骨質的流失。研究結果顯示，於體外實驗KMUP-1可以減少RANKL/Pg LPS誘導RAW264.7細胞的炎症和蝕骨細胞分化，在活體內實驗KMUP-1可預防牙周炎所導致的病理性骨質流失，因此KMUP-1有可能發展為預防及治療牙周炎的治療藥物。

關鍵字

牙齦卟啉單胞菌 ； 脂多醣 ； 發炎 ； 蝕骨細胞新生作用 ； 黃嘌呤衍生物

並列摘要

In the developed countries and developing countries the periodontal diseases (PD) is a common disease, according to the statistics of WHO in 2014, PD have an effect on the 20%-50% people in the world. PD are a pathological inflammatory condition of the gum and bone support (periodontal tissues) surrounding the teeth. Bone homeostasis is maintaining balance by osteoblasts and osteoclasts. Pathological alveolar bone loss situations result from the overactivated osteoclastogenesis. Receptor activator of nuclear factor κB ligand (RANKL)-activated NF-κB and MAPK pathways is important for osteoclastogenesis. In previous studies demonstrated that xanthine derivative (KMUP-1) have the biological properties such as anti-inflammation and anti-oxidation. Therefore, the aim of this study is investigative the effect of KMUP-1 in inhibiting inflammation and osteoclast differentiation induced by RANKL/Porphyromonas gingivalis lipopolysaccharide (Pg LPS). In vitro, RAW264.7 cells was co-tread with KMUP-1 and RANKL for 24 h. After removing the medium, KMUP-1was pretreated for 1 h, and the Pg LPS was added with KMUP-1. In vitro study, showed that KMUP-1 was suppressed the RANKL/Pg LPS induced the production of reactive oxygen species and inflammatory cytokines levels. In the Western blot, KMUP-1 inhibited inflammatory response through downregulating MAPK/NF-κB pathway expreesion and decrease the expression of c-Fos, NFATc1 and NF-κB in osteoclast precursor. Moreover, the TRAP assay also demonstrated that KMUP-1 inhibited osteoclast differentiation. In vivo study, the experiential periodontitis was repeated injections of Pg LPS into the gingiva between molar 1 (M1) and molar 2 (M2) of Wistar rats. KMUP-1 significantly inhibited alveolar bone loss in Pg LPS treatment rat through Micro-CT and TRAP assay. Collectively, this study demonstrates the KMUP-1 can decrease inflammation and osteoclast differentiation in vitro, and suppressed the bone loss by periodontitis in vivo. KMUP-1 may save as therapeutic drug in the prevention drug of periodontitis.

並列關鍵字

Porphyromonas gingivalis ； lipopolysaccharide ； inflammation ； osteoclastogenesis ； xanthine derivative

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