The Anti-Periodontitis Effects of Ethanol Extract Prepared Using Lactobacillus paracasei subsp. paracasei NTU 101

Abstract

Poor oral health and related diseases, including caries, periodontal disease, and oral cancer, are highly prevalent across the world, particularly in the elderly. This study aimed to investigate the anti-periodontitis activity of fermented skim milk produced using the promising probiotic Lactobacillus paracasei subsp. paracasei NTU 101 (NTU101FM). An initial analysis found that an ethanol extract of NTU101FM displayed anti-oxidative activities. Further investigation of pathogen growth inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs) revealed that the NTU101FM ethanol extract also had anti-periodontal pathogen activities. In addition, the NTU101FM ethanol extract significantly decreased the release of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) in RAW 264.7 macrophage cells. Finally, the NTU101FM ethanol extract was found to inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by reducing tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-positive multinucleated osteoclasts. In summary, our study demonstrated that ethanol extract prepared from NTU101FM has potential use as an anti-periodontitis agent.

Keywords: Lactobacillus paracasei subsp. paracasei NTU 101; anti-inflammation; anti-periodontitis; fermented skim milk; osteoclast differentiation.

Figure 1

Effects of NTU101FM ethanol extract on cell viability of RAW 264.7 cell. Cells were treated with various concentrations of NTU101FM ethanol extract (50 to 1000 µg/mL) for 24 h. Cell viability was measured by MTT assay and represented as percent of control cell viability. The data are presented as means ± SD (n = 3). Values with different uppercase letters were significant by Duncan’s multiple range tests (p < 0.05). NTU101FM, NTU 101-fermented skim milk; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

Effects of NTU101FM ethanol extract on: (A) NO; (B) TNF-α; (C) IL-6; (D) IL-1β; and (E) IL-17 of LPS-induced inflammation RAW 264.7 cell. Cells were treated with various concentrations of NTU101FM ethanol extract (25 to 500 µg/mL) for 24 h. The data are presented as means ± SD (n = 3). Values with different uppercase letters were significant by Duncan’s multiple range tests (p < 0.05). NTU101FM, NTU 101-fermented skim milk; NO, nitric oxide; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-17, interleukin-17; LPS, lipopolysaccharide.

Figure 3

Effect of NTU101FM ethanol extract on RANKL-induced osteoclast differentiation: (A) photograph of TRAP-stained osteoclasts; (B) number of TRAP-positive multinucleated osteoclasts; and (C) TRAP activity. The RAW 264.7 cells were cultured with the indicated dose of NTU101FM ethanol extract (25 to 500 μg/mL) in the presence of RANKL (100 ng/mL) for four days. The data are presented as means ± SD (n = 3). Values with different uppercase letters were significant by Duncan’s multiple range tests (p < 0.05). NTU101FM, NTU 101-fermented skim milk; RANKL, receptor activator of nuclear factor kappa-β ligand; TRAP, tartrate-resistant acid phosphatase.

Figure 4

Effect of NTU101FM ethanol extract on RANKL-induced bone resorption: (A) photograph; and (B) the area in osteoclast. The RAW 264.7 cells were cultured with the indicated dose of NTU101FM ethanol extract (25 to 500 μg/mL) in the presence of RANKL (100 ng/mL) for four days. The data are presented as means ± SD (n = 3). Values with different uppercase letters were significant by Duncan’s multiple range tests (p < 0.05). NTU101FM, NTU 101-fermented skim milk; RANKL, receptor activator of nuclear factor kappa-β ligand.

Figure 5

The proposed mechanism of NTU101FM ethanol extract to attenuate periodontal disease and its associated symptoms. LPS, lipopolysaccharide; IL-17, interleukin 17; T_H 17 cell, T helper type 17 cell; RANKL, receptor activator of nuclear factor kappa-B ligand; IL-6, interleukin 6; IL-8, interleukin 8; MMP, matrix metallopeptidase; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor-α.