藉由Metformin調節誘發性一氧化氮合成酶與一氧化氮合成減緩根尖病變進展

Abstract

根尖病變（periapical lesion）是當牙髓腔暴露、細菌入侵，導致牙髓感染發炎、壞死，而後蔓延至根尖部位的齒槽骨，造成骨頭破壞形成的骨吸收性病灶。在病灶區域內，細菌釋放內毒素(Lipopolysaccharide, LPS)引發一連串的發炎反應，除了引發成骨細胞凋亡，也吸引免疫細胞聚集、巨噬細胞活化與蝕骨細胞分化，造成骨吸收情形更加嚴重。Metformin是臨床上廣為被使用作為降血糖的藥物，但也具備抗發炎以及影響骨代謝的功能。先前的研究已經證明，發炎反應時產生的一氧化氮(Nitric oxide, NO)會誘發成骨細胞凋亡、刺激成骨細胞分泌膠原酶，加速根尖病變的進展；在動物實驗模型上也觀察到抑制一氧化氮的合成，可以減緩骨性吸收、縮小病灶範圍。另外，受到發炎因子刺激的成骨細胞也會分泌細胞趨化激素(C-C motif ligand 2，CCL2)，吸引巨噬細胞聚集浸潤，使骨吸收加劇。然而，對於發炎環境下，巨噬細胞產生一氧化氮後對於成骨細胞的刺激、以及Metformin在此機轉中扮演的抑制效果尚待闡明。 在本次研究中，利用人類類單核球細胞(MonoMac-6, MMC-6)在細菌內毒素刺激(LPS)後一氧化氮合成酶的活化以及一氧化氮的生成，透過西方點墨法與格里斯試劑(Greiss reagent)檢測一氧化氮合成酶的表現量與Nitrite濃度，並觀察Metformin在此路徑上是否具有抑制效果。另一方面，收集受到細菌內毒素刺激的MMC-6的conditioning medium後，觀察釋放至細胞培養液中的產物，是否會影響成骨細胞分泌CCL2的表現。最後，設計誘導根尖病變的大鼠模型，在經過標準的根管清創後給予Metformin治療，利用影像學分析以及組織免疫染色去探討對於根尖病灶的癒合狀況。 研究結果發現，在LPS刺激下，MMC-6的一氧化氮合成酶會被活化，並產生高濃度的Nitrite，而Metformin可以抑制一氧化氮合成酶的活性、和降低Nitrite的累積濃度。而受到LPS刺激後的MMC-6之conditioning medium會誘發成骨細胞生成CCL2，並且Metformin同樣可以對此有抑制效果。動物實驗方面，從組織切片染色的結果，我們觀察到在根尖發炎病變的區域中，有明顯的一氧化氮合成酶訊號和巨噬細胞浸潤。而影像學分析的結果顯示，Metformin以根管局部的投藥方式可以減緩巨噬細胞浸潤、降低一氧化氮合成酶的活化程度，有效減小根尖病變的範圍。總結以上所述，在in vitro的研究中我們驗證”因細菌感染所誘發的iNOS上升使巨噬細胞產生大量NO”可被Metformin抑制; 而在in vivo的研究中，我們也發現局部投予Metformin能減緩大鼠根尖病變的發炎性骨吸收。透過本次實驗，我們發現Metformin可以抑制巨噬細胞iNOS的表現量、抑制成骨細胞分泌CCL2、降低根尖病變區域巨噬細胞的浸潤，達到減緩根尖病變進展的效果。

Periapical lesion is an osteolytic lesion which is developed while the dental pulp is exposed to the oral environment, invaded by bacteria and lead to a non-reversible inflammation and destruction of periapical bone. Bacteria in the periapical lesion will cause a series of reaction including immune cell recruitment and infiltration, macrophage activation and osteoblast apoptosis. Previous studies show that nitric oxide (NO) produced by immune cells while inflammation progressed will cause the apoptosis of osteoblast and accelerate the progression of periapical lesion; it was also shown that inhibition of nitric oxide will attenuate periapical lesion progression. Furthermore, osteoblasts will produce C-C motif ligand 2 (CCL2) by the stimulation of lipopolysaccharide (LPS), lead to macrophage recruitment and exaggerated bone destruction. Metformin, a well-used anti-diabetic medication, has been shown to its anti-inflammatory effect and its ability to regulate bone metabolism. However, the effect of NO produced by macrophage on the osteoblast and the mechanism of Metformin attenuate the periapical lesion remain unclear. In our study, we use human monocytic cell and osteoblastic cell to investigate the activation of inducible nitric oxide synthase (iNOS) and the production of NO by macrophage and the synthesis of CCL2 by osteoblast respectively. The rat model with induced apical lesion were also designed by exposing the pulp chamber to oral cavity, and Metformin treatment was performed after standard root canal debridement, the healing of the apical lesion was investigated by imaging analysis and tissue immunostaining. We found NO production and iNOS expression was increased in LPS-treated macrophage, the activated macrophage stimulate the secretion of CCL2 in osteoblast. Metformin inhibited NO and iNOS expression in macrophage and reduced CCL2 in osteoblast by conditioning medium from LPS-treated macrophage. In the in vivo part of our study, bone destruction was significantly less in the Metformin- treated group. The infiltration of macrophage around the lesion was inhibited and the expression of iNOS was also reduced in the Metformin-treated group. Our study suggest that LPS-induced iNOS expression and NO production were inhibited by Metformin both in vitro and in vivo. Metformin inhibited the cell-cell communication between macrophage and osteoblast in periapical lesion. Therefore, Metformin may be promising for clinical application in patients with periapical lesion.