以飽和脂肪酸與誘導型一氧化氮生合成的觀點探討代謝體的調控對於根尖牙周炎的治療潛能

To explore the treatment potential of modulating metabolisms of saturated fatty acid and inducible nitric oxide in apical periodontitis

王翰偉(Han-Wei Wang)

指導教授 ： 林立德

共同指導教授 ： 林思洸(Sze-Kwan Lin)

國立臺灣大學/醫學院/臨床牙醫學研究所/博士(2021年)

https://doi.org/10.6342/NTU202100970

摘要

目的：研究棕櫚酸（palmitic acid，PA）與一氧化氮（NO）對於單核球/巨噬細胞的趨化性、以及根尖牙周炎的信息傳遞所造成的影響，並提出可能的治療方針。
方法：實驗使用J774與Mono-Mac-6等單核球/巨噬細胞（monocyte/macrophage）族系之細胞株。以西方點墨法分析固醇調節元件結合蛋白-1c（sterol regulatory element binding protein-1c，SREBP-1c），脂肪酸合酶（fatty acid synthase，FASN）以及誘導型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的表現；以反相高效液相層析質譜儀測定PA含量；以small interfering RNA（siRNA）靜默SREBP-1c及FASN的表現；CC-趨化因子配體2（CC-chemokine ligand 2，CCL2）的分泌是以酶聯免疫吸附測定法（ELISA）量化；細胞趨化性則是以transwell細胞遷徙的方式量化；以測量培養液的亞硝酸鹽含量來量化產生的一氧化氮（NO）。以實驗誘發大鼠根尖牙周炎（induced apical periodontitis）模型來評估磺基-N-琥珀酰亞胺基油酸酯（sulfo-N-succinimidyl oleate，SSO）與二甲雙胍（N, N-dimethylbiguanide，藥品名metformin）抑制脂肪酸與iNOS，對於輔助根尖牙周炎治療所帶來的效益，再以根尖放射線造影（periapical x-ray）和微米電腦斷層攝影（micro-CT）評估病灶大小，以免疫組織化學染色評估monocyte/macrophage的數量與iNOS表現量。
結果：LPS刺激巨噬細胞的SREBP-1c成為活化態、增加FASN表現，並增強PA合成。靜默SREBP-1c可減弱脂多糖（lipopolysaccharide，LPS）強化的FASN表現。靜默FASN會抑制LPS增強的棕櫚酸合成。LPS和外源加入的棕櫚酸會提高CCL2和巨噬細胞的趨化性。抑制FASN表現可顯著減輕LPS增強的CCL2分泌。SSO可抑制LPS和PA增強的CCL2分泌和巨噬細胞趨化性。Metformin能抑制monocytes因LPS刺激產生的iNOS及NO，而且透過調節iNOS/NO路徑來抑制LPS增強的CCL2表現。在實驗誘發大鼠根尖牙周炎模型中，使用SSO治療以及根管內給予metformin，都能顯著抑制發炎擴展及monocyte/macrophage聚集。
結論：LPS/SREBP-1c/FASN/PA的訊息傳遞路徑會惡化細菌感染引起的根尖牙周組織破壞。控制脂肪酸的代謝與訊息傳遞有利於根尖牙周炎的治療。而在根管內使用metformin治療根尖牙周炎，其機制是抑制iNOS/NO的產生來減緩monocytes聚集與發炎反應。調控飽和脂肪酸與NO等代謝體的產生，對於根尖牙周炎的治療確實能發揮相當的助益。

關鍵字

根尖牙周炎 ； 脂多糖 ； 飽和脂肪酸 ； 誘導型一氧化氮合酶 ； 單核球／巨噬細胞 ； 磺基-N-琥珀酰亞胺基油酸酯 ； 二甲雙胍

並列摘要

Aim: To examine the role of palmitic acid (PA) and inducible nitric oxide (NO) in lipopolysaccharide-stimulated chemotaxis of monocytes/macrophages and their potential contribution to the pathogenesis of apical periodontitis.
Methodology: The mouse macrophage cell line, J774, and the human monocyte cell line, Mono-Mac-6, were used in the experiments. After treatment with lipopolysaccharide, sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FASN), and inducible nitric oxide synthase (iNOS) were examined by Western blot. The levels of palmitic acid were measured by reverse phase-high performance liquid chromatography-mass spectrometry. Small interfering RNAs targeting SREBP-1c and FASN were applied to knockdown respective gene expression. C-C motif chemokine ligand 2 (CCL2) excretion was measured by enzyme-linked immunosorbent assay (ELISA), and cell chemotaxis was quantified by transwell migration assay. Nitric oxide production was quantified by measuring the amount of nitrite in culture medium. Sulfo-N-succinimidyl oleate (SSO) and metformin were used in induced apical periodontitis in rats for evaluation of inhibiting fatty acid signaling and iNOS/NO pathway in vivo. Lesion progression was evaluated both by conventional periapical radiography and micro-computed tomography. The recruited monocytes/macrophages were examined by immunohistochemistry under a microscope.
Results: LPS stimulated the maturation of SREBP-1c, the expression of FASN and iNOS, and the synthesis of PA in monocyte/macrophage lineage. Knockdown of SREBP-1c attenuated LPS-enhanced FASN expression. Knockdown of FASN attenuated LPS-enhanced PA synthesis. LPS and exogenous PA significantly enhanced CCL2 excretion and macrophage chemotaxis. Inhibition of FASN expression attenuated LPS-induced CCL2 excretion. SSO suppressed CCL2 excretion and macrophage chemotaxis augmented by LPS and exogenous PA. Metformin suppressed LPS-induced iNOS and NO production by monocytes. Moreover, metformin inhibited LPS-enhanced CCL2 synthesis through iNOS/NO pathway. In a rat model of induced apical periodontitis, SSO treatment significantly attenuated the progression of apical periodontitis and macrophage recruitment. Intracanal metformin treatment in rat model also significantly reduced bone resorption associated with apical periodontitis.
Conclusions: The signal transduction of LPS/SREBP-1c/FASN/PA contributes to the tissue destruction resulted from bacterial infection. The iNOS/NO pathway also plays a significant role in monocyte/macrophage recruitment and inflammatory cascade. Modulation of fatty acid and NO through the use of SSO and intracanal metformin had been proved helpful in managing apical periodontitis. Therefore, the concept of modulating metabolites may serve as a beneficial adjunct in treating inflammatory diseases.

並列關鍵字

apical periodontitis ； lipopolysaccharide ； saturated fatty acid ； inducible nitric oxide synthase ； monocyte/macrophage ； sulfo-N-succinimidyl oleate ； metformin

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