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標題: | 副乾酪乳酸桿菌副乾酪亞種 NTU 101 發酵產物抗牙周病之研究 Study on anti-periodontitis effect of Lactobacillus paracasei subsp. paracasei NTU 101-fermented products |
作者: | Te-Hua Liu 劉德華 |
指導教授: | 潘子明 |
關鍵字: | 抗氧化,抗牙周病,抗發炎,副乾酪乳酸桿菌副乾酪亞種 NTU 101,酪胺酸與乳酸混合物, anti-oxidant,anti-periodontitis,anti-inflammation,Lactobacillus paracasei subsp. paracasei NTU 101,a mixture of tyrosine and lactic acid, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 牙周病為牙周致病菌引發牙周組織慢性發炎之疾病,牙周致病菌會於牙齒表面形成牙菌斑,且最終導致齒槽骨流失。此外,氧化壓力亦為導致牙周病之關鍵因子,雖然已有許多文獻指出益生菌具有改善牙周發炎之效果,但乳酸菌發酵產物與牙周病相關之研究並不多。故本研究旨在探討乳酸菌發酵產物於體內及體外模式中抗牙周病之效果。首先,檢測副乾酪乳酸桿菌副乾酪亞種 NTU 101 (Lactobacillus paracasei subsp. paracasei NTU 101, NTU 101) 與胚芽乳酸桿菌 TWK 10 (L. plantarum TWK 10, TWK 10) 發酵脫脂牛奶與豆奶萃取物之抗氧化及抗菌活性以進行初步篩選,結果顯示,NTU 101 發酵脫脂牛奶乙醇萃取物 (NTU101FMEE) 除了表現良好之抗氧化活性外,亦具有抗牙周致病菌 Actinomyces actinomycetemcomitans 與 Porphyromonas gingivalis 之能力。利用脂多醣 (lipopolysaccharide, LPS) 誘發牙周病之動物模式探討 NTU101FMEE 抑制牙周病之效果,實驗結果顯示,NTU101FMEE 可顯著改善因牙周發炎所導致的體重減輕情況 (p < 0.05),此外,LPS 誘發牙周病大鼠經不同劑量之 NTU101FMEE 處理後,可顯著減少 0.99-2.02 log CFU/g 組織之口腔菌數以及 26.41-37.92% 之齒槽骨流失程度 (p < 0.001),同時亦可發現,NTU101FMEE 可藉由降低因 LPS 所誘發之促發炎細胞激素生成及牙周組織中之氧化壓力,並減緩牙周病大鼠牙周組織中基質金屬蛋白酶-9 (matrix metalloproteinases-9, MMP-9) 活性,進而達到改善牙周發炎與損傷之情形。NTU101FMEE 於 LPS 所誘導發炎之小鼠巨噬細胞 RAW 264.7 模式中,可顯著降低促發炎細胞激素生成量 (p < 0.05);此外,NTU101FMEE 亦可藉由顯著減少 27.22-55.31% 之抗酒石酸酸性磷酸酶 (tartrate-resistant acid phosphatase, TRAP) 活性與 30.99-49.07% 之 TRAP-positive 細胞數目 (p < 0.05),達到抑制核因子 κB 配體受體致活劑 (receptor activator of nuclear factor κB ligand, RANKL) 誘導蝕骨細胞生成之效果,並可減緩骨吸收面積,上述結果證實 NTU101FMEE 於體外模式中具有抗牙周病之潛能。進一步探討 NTU101FMEE 中具有抗牙周病活性之有效成分,經管柱層析可得細分層 (fraction) 1-7 (F1-7),當 F4 濃度為 100 μg/mL 時與 LPS 組相比顯著下降 IL-6 生成量達 50.84% (p < 0.05),經核磁共振儀 (nuclear magnetic resonance, NMR) 及超高效液相層析串聯質譜儀 (ultra-performance liquid chromatography-mass spectrometry, UPLC-MS) 之結果鑑定其為酪胺酸與乳酸之混合物,且經 1H-NMR 圖譜積分值判定其混合比例約為 3:1 (3T1L)。此外,經 UPLC 定量後可發現 NTU101FMEE 中之酪胺酸與乳酸含量高於未發酵脫脂牛奶乙醇萃取物。進一步利用 RAW 264.7 與人類牙齦纖維細胞 (human gingival fibroblasts-1, HGF-1) 探討 3T1L 抗牙周病之可能機轉。於 RAW 264.7 細胞模式中,3T1L 可減緩因 LPS 所誘發之促發炎細胞激素含量,同時可有效調節 MMP-9 與基質金屬蛋白酶組織抑制劑-1 (tissue inhibitor of matrix metalloproteinase-1, TIMP-1) 含量,並抑制經 RANKL 誘導蝕骨細胞分化相關因子。而在 HGF-1 細胞模式中,3T1L 可減緩 LPS 引起胞內活性含氧物、介白素 (interleukin, IL)-6 與 IL-8 生成量。亦可藉由調控磷酸化之細胞外信號調節激酶 (extracellular signal-regulated kinases, ERK)、Jun 胺基末端激酶 (Jun N-terminal kinases, JNK) 與 p38 表現量與核因子-κB (nuclear factor-κB, NF-κB) 含量,降低 LPS 所誘發之發炎反應與氧化壓力。綜上所述,自 NTU101FMEE 分離純化出之 3T1L 具有良好抗氧化與抗發炎之效果,具開發成抗牙周病保健成分之潛能。 Periodontitis is a chronic inflammatory disease of the supporting tissues of the teeth associated with periodontal pathogens, and they initiated by the plaque biofilm, that leads to loss of periodontal attachment to the root surface and adjacent alveolar bone and which results in tooth loss. There are evidences that oxidative stress is also a key factor contributing to periodontitis. Although, numerous studies have confirmed that probiotics can improve periodontal inflammation, the specific effects of lactic acid bacteria (LAB) fermented products have not been studied. The current study aimed to assess the anti-periodontitis effect of LAB-fermented products on in vivo and in vitro. In preliminary screening, the extracts from Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101)- and L. plantarum TWK 10 (TWK 10)-fermented skim milk and soy milk were used to evaluate the ability of anti-oxidation and anti-microbial activities. NTU 101-fermented skim milk ethanol extract (NTU101FMEE) displayed a significant effect not only on anti-oxidation activity but also on ability to against Actinomyces actinomycetemcomitans and Porphyromonas gingivalis. And we further investigated the prevention effect of NTU101FMEE on lipopolysaccharide (LPS)-induced periodontal disease in rats. NTU101FMEE significantly ameliorated weight loss caused by periodontal inflammation (p < 0.05). Moreover, different dosage of NTU101FMEE treatment also reduced the oral microbial levels by 0.99 to 2.02 log CFU/g tissue and decreased the levels of alveolar bone loss by 26.41 to 37.92% (p < 0.001). We also found that NTU101FMEE improved periodontal inflammation by decreasing the levels of pro-inflammatiory cytokines and reducing oxidative stresses induced by LPS. The matrix metalloproteinases-9 (MMP-9) activity of gingival tissue which increased by LPS was also significantly reduced by NTU101FMEE (p < 0.05) to improve the periodontal inflammation and damage. In addition, the NTU101FMEE significantly decreased pro-inflammatory cytokines release induced by LPS in RAW 264.7 cells (p < 0.05). Furthermore, NTU101FMEE was found to inhibit receptor activator of nuclear factor κΒ ligand (RANKL)-induced osteoclast differentiation by reducing tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-positive multinucleated osteoclasts by 27.22 to 55.13% and 30.99 to 49.07%, respectively (p < 0.05). And NTU101FMEE also reduced the bone resorption area in pit formation assay. Our results demonstrate that ethanol extract prepared from NTU101FM has excellent anti-periodontitis potential in vitro. Therefore, we further investigated the anti-periodontitis effect ingredient(s) in NTU101FMEE. There were 1-7 fractions (F1-F7) which obtain from column chromatography. At a concentration of 100 μg/mL, F4 significantly reduced IL-6 level by 50.48%, compared with LPS group (p < 0.05). The F4 was identified as a mixture of tyrosine and lactic acid using nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). And the ratio of these two mixtures was about 3:1 (3T1L) which determined by peak area of 1H-NMR spectrum. Moreover, the contents of tyrosine and lactic acid in NTU101FMEE were higher than unfermented skim milk ethanol extract according to the result of UPLC quantitative analysis. And we used the RAW 264.7 and human gingival fibroblasts-1 (HGF-1) cells to assess the anti-periosontits effcts and probably mechanism of 3T1L. In the RAW 264.7 cell model, 3T1L ameliorated LPS-induced the contents of pro-inflammatory cytokines. And 3T1L not only regulated the contents of MMP-9 and tissue inhibitor of matrix metalloproteinase-1, but also inhibited the RANKL-induced osteoclastogenesis-related factors. In the HGF-1 cell model, 3T1L attenuated reactive oxygen species generation, IL (interleukin)-6 and IL-8 levels. Treatment with 3T1L decreased inflammatory response and oxidative stress which induced by LPS via down-regulating the phosphorylation of extracellular signal-regulated kinases, Jun N-terminal kinases and p38 and nuclear factor-κB level. In conclusion, our findings demonstrate that 3T1L derived from NTU101FMEE showed favorable anti-oxidation and anti-inflammation effects and could be developed as a functional ingredient with anti-periodontitis effect. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21799 |
DOI: | 10.6342/NTU201900228 |
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顯示於系所單位: | 生化科技學系 |
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