益生菌透過抑制牙周病原菌Porphyromonas gingivalis預防牙周病的潛力

Abstract

牙周病 (Periodontal disease) 是由口腔病原菌引起的發炎反應，不僅影響人類，也是犬隻常見的口腔疾病，在三歲以上的犬隻中有約三分之二患有某些程度上的牙周病。牙周病的起因是由於口腔菌群的失衡，導致微生物堆積在牙齒邊緣產生生物膜及牙菌斑，造成牙齦發炎。若不將發炎反應加以治療，感染區域會持續擴大，並且可能由牙齦炎發展為牙周病，造成骨骼破壞及流失最終可能導致牙齒鬆動及脫落，而口腔問題不只影響口腔還可能引起全身性健康問題。

Porphyromonas gingivalis是牙周病的關鍵病原菌，被認為是牙周病發展的關鍵因素。這種細菌能夠貼附並侵入宿主細胞，降低免疫系統對細菌的殺傷能力，並引發持續的炎症。目前常見的治療方法包括洗牙 (scaling) 及根面平整術 (planing)，並使用抗生素和抗炎藥物進行治療，但這些方法可能導致抗藥性的產生，並破壞口腔細菌的平衡。此外，犬隻在進行洗牙時還需承受麻醉的風險，因此維護犬隻口腔健康是很重要的議題。近年來，研究人員正在嘗試使用益生菌作為一種替代治療方法，在過去研究中顯示出作為輔助療法的可能，但需要更多研究來探討預防牙周病的的功效。因此本次研究目的為使用體外試驗及動物試驗篩選可能可以預防或延緩牙周病的益生菌。

在本次研究中，首先從動物產品實驗室中選出可食用的乳酸菌 (lactic acid bacteria, LAB) 並且透過紙錠擴散試驗 （disc diffusion method）篩選可以抑制牙周病原菌P. gingivalis生長的菌株，其中選出了4株抑制效果最顯著者作為候選菌株，四株菌皆為Ligilactobacillus salivarius。接著進入抗生物膜實驗，發現四株菌皆有良好的生物膜抑制效果，所以所有菌株進入細胞實驗。而在細胞實驗中選擇牙齦上皮細胞及小鼠巨噬細胞進行實驗。在牙齦上皮細胞的實驗中，結果表明四株益生菌顯著減少P. gingivalis的貼附和侵入能力，其中APL 25表現出了良好的細胞貼附能力。此外，益生菌還能防止由病原菌引起的免疫抑制反應，並且在巨噬細胞上可以有效增加IL-10的產生並引起免疫反應。

在候選菌株中，APL25在所有細胞實驗中表現良好抑制病原菌貼附及侵入，同時改善其引起的免疫反應，並且對於細胞有較小的損傷，因此本研究選擇APL 25進行小鼠實驗。在小鼠試驗中，先在益生菌組小鼠牙齦邊緣給予一周的益生菌進行防禦，接著再使用牙周病原菌P. gingivalis進行直接塗抹的牙周病誘導。在最後結果中有先給予益生菌預防的組別有較少的骨質流失情況，並且維持良好的骨礦物質密度，同時具較低的牙齦發炎評分。此外，有給予益生菌的小鼠體內白血球數量較少，並減少脾臟及牙齦組織中的促發炎因子濃度，在血清中則可以改善病原菌引起的不良免疫抑制。不只如此，在最後一次塗抹益生菌後四周，有先給予L. salivarius的小鼠組別在口腔、牙齦組織及糞便中仍具較少的病原菌，並且與病原菌組相比具較多的乳酸菌，顯示出長時間的保護效果。

綜合本次研究結果，APL 25在體內外實驗中皆展現良好的預防或是延緩牙周病的潛力，期望在未來可以實際應用於人及犬隻，減少牙周病的發生。

Periodontal disease is an inflammatory response caused by oral pathogens, affecting not only humans but also being a common oral disease in dogs, affecting over two-thirds of dogs over 3 years old having some degree of periodontal disease. The cause of periodontal disease is an imbalance in the oral microbiota, leading to the accumulation of microbes at the tooth margin, forming biofilm and dental plaque, and causing gingival inflammation. If untreated, the infection area will continue to expand, and gingivitis may develop into periodontitis, leading to bone destruction and loss, ultimately causing tooth loosening and falling out. Oral problems can not only affect the oral cavity but may also lead to systemic health issues. Porphyromonas gingivalis is the key pathogen of periodontal disease and is considered a critical factor in the development of periodontal disease. This bacterium can adhere to and invade host cells, reducing the immune system's ability to kill the bacteria, and triggering sustained inflammation. The common treatment methods include scaling and root planing, along with the use of antibiotics and anti-inflammatory drugs and substances, but these methods may lead to the development of antibiotic resistance and disrupt the balance of oral bacteria. Additionally, dogs undergoing scaling require anesthesia, making the maintenance of canine oral health an important issue. Recent research has explored the potential of probiotics as an adjunctive therapy for periodontal disease. Although promising results have been observed, further investigation is needed to establish their efficacy. This study aimed to screen probiotic strains that might prevent or delay periodontal disease using both in vitro and in vivo experiments, with the ultimate goal of reducing its incidence in humans and dogs. In this study, edible lactic acid bacteria (LAB) were selected from the animal product laboratory. The disc diffusion method was used to screen for strains capable of inhibiting the growth of the periodontal pathogen Porphyromonas gingivalis. Four strains of Ligilactobacillus salivarius demonstrated the most significant inhibitory effects and were chosen as candidate strains. Next, anti-biofilm experiments were conducted, showing that all four strains effectively inhibited biofilm formation. These strains were then tested in cell experiments using gingival epithelial cells and mouse macrophages. The results indicated that the four probiotic strains significantly reduced the adhesion and invasion capabilities of P. gingivalis, with strain APL 25 exhibiting excellent cell adhesion. Furthermore, the probiotics prevented the immunosuppressive response in gingival cells induced by the pathogen and increased the production of IL-10, triggering a beneficial immune response in macrophages.

Among the candidate strains, APL 25 excelled in all cell experiments by inhibiting pathogen adhesion and invasion, enhancing the immune response, and causing minimal cell damage. Consequently, APL 25 was selected for further testing in mouse experiments. In the mouse experiment, the probiotic group received APL 25 for one week as a preventive measure before inducing periodontal disease with P. gingivalis. The final results showed that the group pre-treated with probiotics had less bone loss, maintained good bone mineral density, and had lower gum inflammation scores. In addition, mice given probiotics had fewer white blood cells and reduced the concentration of pro-inflammatory factors in the spleen and gingival tissues, and serum levels improved the adverse immune suppression caused by the pathogen. Not only that, but four weeks after the last application of probiotics, the group given L. salivarius APL 25 still had fewer pathogens in the oral cavity, gingival tissues, and feces, along with a higher presence of lactic acid bacteria compared to the pathogen group, indicating a long-lasting protective effect. In summary, the results of this study indicate that APL 25 has strong potential to prevent or delay the onset of periodontal disease in both in vitro and in vivo experiments. This strain shows promise for future application in humans and dogs to reduce the occurrence of periodontal disease.