Bueno Manuela R, Ishikawa Karin H, Almeida-Santos Gislane, Ando-Suguimoto Ellen S, Shimabukuro Natali, Kawamoto Dione, Mayer Marcia P A
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Department of Stomatology, School of Dentistry, University of São Paulo, São Paulo, Brazil.
Front Microbiol. 2022 May 4;13:846192. doi: 10.3389/fmicb.2022.846192. eCollection 2022.
Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by . OBA-9 GECs were infected with strain JP2 at an MOI of 1:100 and/or co-infected with La5 (La5) or Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1β, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of to GECs was 1.8 × 10 (± 1.2 × 10) cells/well in the mono-infection but reduced to 1.2 × 10 (± 1.5 × 10) in the co-infection with Lr32 and to 6 × 10 (± 1 × 10) in the co-infection with La5 (p < 0.05). GECs mono-infected with produced CXCL-8, GM-CSF, and IL-1β, and the co-infection with both probiotic strains altered this profile. While the co-infection of with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1β. The probiotics upregulated the expression of and downregulated in cells co-infected with . induced the upregulation of was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene was upregulated, whereas the pro-apoptotic was downregulated in cells co-infected with and the probiotics. Infection with induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, La5 properties suggest that this strain has a higher potential to control associated periodontitis than Lr32.
益生菌可被视为在牙周炎中实现微生物群平衡的一种额外策略。然而,益生菌用于预防或控制牙周炎的潜在机制仍未完全阐明。本研究旨在评估两种市售乳酸杆菌菌株对受牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)OBA - 9菌株攻击的牙龈上皮细胞(GECs)的影响。将GECs以1:100的感染复数(MOI)用Pg JP2菌株感染,并以1:10的MOI与La5或Lr32共感染2小时和24小时。通过定量聚合酶链反应(qPCR)确定粘附/内化到GECs的细菌数量。通过酶联免疫吸附测定(ELISA)确定GECs产生的炎症介质(CXCL - 8、白细胞介素 - 1β(IL - 1β)、粒细胞 - 巨噬细胞集落刺激因子(GM - CSF)和白细胞介素 - 10(IL - 10)),并通过逆转录 - qPCR确定编码细胞受体和参与细胞凋亡的基因的表达。还通过膜联蛋白V染色分析细胞凋亡。感染Pg或测试的益生菌2小时后,OBA - 9细胞活力略有下降,24小时共感染后这种下降更为明显。在单感染中,Pg粘附到GECs上的数量为1.8×10(±1.2×10)个细胞/孔,但在与Lr32共感染时降至1.2×10(±1.5×10)个细胞/孔,在与La5共感染时降至6×10(±1×10)个细胞/孔(p < 0.05)。单感染Pg的GECs产生CXCL - 8、GM - CSF和IL - 1β,与两种益生菌菌株的共感染改变了这种情况。虽然Pg与La5共感染导致所有介质水平降低,但与Lr32共感染促进了CXCL - 8和GM - CSF水平降低,但增加了IL - 1β的产生。在与Pg共感染的细胞中,益生菌上调了某些基因(此处原文未明确写出基因名称)的表达并下调了另一些基因(此处原文未明确写出基因名称)的表达。Pg诱导的某些基因(此处原文未明确写出基因名称)的上调被La5减弱,但被Lr32增强。此外,在与Pg和益生菌共感染的细胞中,抗凋亡基因(此处原文未明确写出基因名称)的转录上调,而促凋亡基因(此处原文未明确写出基因名称)的转录下调。感染Pg诱导GECs凋亡,而与乳酸杆菌共感染减弱了凋亡表型。两种测试的乳酸杆菌都可能通过降低Pg与牙龈上皮细胞相互作用的能力并调节细胞反应来干扰其在口腔中的定植。然而,La5的特性表明该菌株比Lr32具有更高的控制相关牙周炎的潜力。