口腔Lactobacillus zeae加劇小鼠中牙周病的病理表現

Abstract

牙周病於全球具有相當高的盛行率，患者又以老年人口佔有較高的比例。其病症包括牙齦發炎、齒槽骨喪失甚至是牙齒的脫落，近來更是有許多研究顯示牙周病與全身性系統疾病的高度相關，使其對人體健康的影響更加不容小覷。造成牙周病常見的主要原因是口內微生物的菌群失衡，致病菌持續激化宿主的免疫發炎反應，進而導致牙周組織的喪失。因此，在牙周病新興治療的研究中，口腔微生物學便成為現今牙周病的重要主題。在我們過去的研究裡，以第三代定序的系統(Oxford Nanopore Technologies)對牙周病及非牙周病患者的牙齦下牙菌斑進行總體基因體定序分析，其中，我們發現乳桿菌屬中的Lactobacillus zeae (L. zeae) 在牙周病患者的數量比非牙周病患者高出24倍；然而，其他研究則提出數種乳酸桿菌對口腔致病菌有抑制的效果，Lactobacillus rhamnosus GG更是顯著減少了牙周病已知致病菌Porphyromonas gingivalis (P. gingivalis)所造成的牙周損傷，被視為牙周益生菌。因此，關於L. zeae在牙周病中所扮演的角色有待研究證實。 我們在結紮線誘導牙周炎的小鼠模型中，於牙齦單獨或同時接種L. zeae 以及 P. gingivalis，並取下小鼠的上顎骨作為後續實驗分析之檢體，利用微電腦斷層掃描、免疫組織染色法與抗酒石酸酸性磷酸酶染色法分別對齒槽骨的喪失程度、嗜中性白血球浸潤程度以及活化蝕骨細胞的數量進行檢測。同時，採集小鼠口內患處的牙齦溝液進行蛋白質多重免疫分析，觀測其免疫發炎因子的分泌。結果顯示，接種L. zeae 的組別會引起更嚴重的齒槽骨喪失、嗜中性白血球浸潤以及活化蝕骨細胞的數量。在蛋白質多重免疫分析結果中，免疫發炎因子(腫瘤壞死因子-α、白血球介素-6、白細胞介素-1β、干擾素-γ、單核細胞趨化蛋白-1) 在接種L. zeae 的小鼠中也有較高的表現量。在本研究中，L. zeae 於誘導牙周炎的小鼠口內顯著地增加了牙周病的病理表現，因此，我們認為L. zeae在牙周病患者口腔中可能扮演著加劇牙周病進程的角色。

The prevalence of periodontal disease (PD) is considerably high worldwide. Besides, the elderly accounts for a greater proportion among the PD patients. The main symptoms of PD including inflamed gums, reduced alveolar bone, and even the tooth loss. PD is taken much more seriously by people not only because of these influences but also its close association with numerous systemic diseases indicated in recent studies. The main cause of PD development is the imbalanced of oral microbiota which is called dysbiosis. The pathogens interfere with the host immune system and cause the consequent tissue destruction. Therefore, oral microbiology undoubtedly becomes a major topic in the novel therapeutics for PD. In our previous study, we used the third-generation sequencing system (the Oxford Nanopore Technologies) to analyze metagenome in the subgingival dental plaque from PD and non-PD patients. Notably, the number of Lactobacillus zeae (L. zeae) in PD group is 24 times more than the non-PD group. However, multiple species of Lactobacillus are said to have inhibition effects against oral pathogens. Lactobacillus rhamnosus GG (LGG) even reduces the periodontal damage caused by the well-known PD pathogen Porphyromonas gingivalis (P. gingivalis) and LGG is further regarded as periodontal probiotics. As a result, researches for uncovering the roles of L. zeae in PD are unquestionably needed. We inoculated L. zeae and P. gingivalis independently and also simultaneously into the gingiva of the ligature-induced periodontitis mice. The maxillae tissues of mice were collected for further analysis. Afterwards, micro computed tomography (micro-CT) analysis, immunohistochemistry (IHC) staining, and tartrate-resistant acid phosphatase (TRAP) staining were respectively used for evaluating the loss extent of alveolar bone, the infiltration extent of neutrophils, and the number of activated osteoclasts. Also, gingival crevicular fluid (GCF) in the lesion site of mice was collected for Multiplex assay to assess the secretion of inflammatory cytokines. The result showed that L. zeae inoculated group exhibited more serious alveolar bone resorption, significant neutrophil infiltration, and elevated activated osteoclast number. Additionally, the high expression of inflammatory cytokines including tumor necrosis factor-alpha, interleukin-6, interleukin-1 beta, interferon-gamma, and monocyte chemoattractant protein-1 could also be seen in L. zeae inoculated mice. In this study, L. zeae exacerbates the pathological manifestation of periodontal disease in ligature-induced periodontitis mice. We deduced that L. zeae might play roles that accelerate the progression of PD.