High Fat Diet Dysbiotic Mechanism of Decreased Gingival Blood Flow

Abstract

The gut microbiome has a very important role in human health and its influence on the development of numerous diseases is well known. In this study, we investigated the effect of high fat diet (HFD) on the onset of dysbiosis, gingival blood flow decreases, and the periodontal matrix remodeling. We established a dysbiosis model (HFD group) and probiotic model by Lactobacillus rhamnosus GG (LGG) treatment for 12weeks. Fecal samples were collected 24h before mice sacrificing, while short chain fatty acids (SCFA) analysis, DNA extraction, and sequencing for metagenomic analysis were performed afterwards. After sacrificing the animals, we collected periodontal tissues and conducted comprehensive morphological and genetic analyses. While HFD reduced Bacteroidetes, SCFA, and gingival blood flow, this type of diet increased Firmicutes, lipopolysaccharide (LPS) binding protein, TLR4, pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), matrix metalloproteinases (MMP-2 and MMP-9) expression, and also altered markers of bone resorption (OPG and RANKL). However, LGG treatment mitigated these effects. Thus, it was observed that HFD increased molecular remodeling via inflammation, matrix degradation, and functional remodeling and consequently cause reduced gingival blood flow. All of these changes may lead to the alveolar bone loss and the development of periodontal disease.

Keywords: Lactobacillus rhamnosus; Laser Doppler flowmetry; blood flow; gut microbiota; lipopolysaccharide; matrix metalloproteinases; periodontal disease.

Figure 1

Schematic representation of the study aim. High fat diet (HFD) causes gut dysbiosis (via decreased short chain fatty acids (SCFA) and Bacteroides, and increased Firmicutes and Proteobacteria) resulting in increased lipopolysaccharide (LPS) production and LPS/TLR4 pathway activation in the oral cavity and gastrointestinal tract. Consequently, molecular remodeling occurs by increasing proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and matrix metalloproteinases (MMP-2 and MMP-9). Due to the mentioned consequences, functional remodeling occurs, by an increase of proinflammatory cytokines, RANKL, and the decrease of OPG. Osteoclastogenesis and alveolar bone loss are promoted by disturbance in expression and activity of MMPs, matrix degradation, and gingival blood flow. This together cause periodontal disease and possibility of tooth loss. Probiotics maids mitigate all these consequences.

Figure 2

Effect of HFD and Lactobacillus rhamnosus GG (LGG) treatment on the glucose level (ng/ml). WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD, and LGG-supplemented wild-type mice. Values remain under defined diabetic values. All data represent means ± SD, WT vs. HFD group *p < 0.05; HFD vs. HFD + LGG ^nsp > 0.05, n = 5.

Figure 3

Microbiota analysis of stool in all examined groups. WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD and LGG-supplemented wild-type mice. (A) Relative abundance of total bacteria at the phylum level in each treatment group (%); (B) Relative abundance of Bacteroides and Firmicutes at the phylum level in each treatment group (%); (C) Analysis of acetic acid; (D) Analysis of propionic acid; (E) Analysis of butyrate acid; (F) Analysis of valeric acid; (G) Analysis of isovaleric acid. All data represent means ± SD, WT vs. HFD group *p < 0.05; HFD vs. HFD + LGG ^nsp > 0.05, HFD vs. HFD + LGG ^#p < 0.05, n = 5.

Figure 4

Effect of HFD and LGG treatment on the lipopolysaccharide binding protein concentration in mice serum (ng/ml). WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD, and LGG-supplemented wild-type mice. All data represent means ± SD, WT vs. HFD group *p < 0.05; HFD vs. HFD + LGG ^#p < 0.05, n = 5.

Figure 5

Effect of HFD and LGG treatment on the molecular remodeling in periodontal tissues. WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD, and LGG-supplemented wild-type mice. Quantitative RT-PCR analysis for (A) TLR₄, (B) TNFα, (C) IL-1β, (D) IL-6, (E) MMP-2, (F) MMP-9, (G) TIMP-2, (H) RANKL, and (I) OPG mRNA expression. The results are presented as the expression of the individual mRNAs with normalization GAPDH, using the 2^−ΔΔCq method. All data represent means ± SD, Fold change vs. WT group. All data represent means ± SD, WT vs. HFD group *p < 0.05; HFD vs. HFD + LGG; ^nsp > 0.05, HFD vs. HFD + LGG ^#p < 0.05, n = 5.

Figure 6

Effect of HFD and LGG treatment on the MMP activity in periodontal tissues. WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD and LGG-supplemented wild-type mice. (A) Original zymography membrane. (B) Gelatin zymography analysis in periodontal tissue. All data represent means ± SD, WT vs. HFD group *p < 0.05; HFD vs. HFD + LGG ^#p < 0.05, n = 5.

Figure 7

Effect of HFD and LGG treatment on the gingival blood flow. WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD, and LGG-supplemented wild-type mice. (A) Buccal view of the exposed right mandibular gingiva in first molar area. (B) Blood flow imaging at the surface of the buccal right mandibular gingiva in first molar area via Doppler laser. The black arrows indicate the gingival blood flow measurement site.

Figure 8

Representative images of hematoxylin-eosin (H&E) staining of periodontal tissue and histomorphometric analysis of alveolar bone loss, ED, and number of fibroblasts per 45 × 50μm. WT, Wild-type C57BJ/L6 mice; HFD, HFD-supplemented wild-type mice; LGG, LGG-supplemented wild-type mice; HFD + LGG, HFD, and LGG-supplemented wild-type mice. (A) Representative depiction of histological sections. Images were captured at 20× (200μm) magnification after H&E staining. Blue lines represent alveolar bone crest (ABC), and cement enamel junction (CEJ). Red lines represent the alveolar bone loss. Black arrows show the apical extent of the epithelial downgrowth (ED). (B) Alveolar bone-loss was measured as the distance between the CEJ and the ABC. (C) ED was defined by measuring the distance from the cementoenamel junction (CEJ) to the apical extent of the junctional epithelium. Results are expressed in μm. (D) Bars shows mean number of fibroblasts per 45 × 50μm. Scale bars = 200μm. All data represent means ± SD, WT vs. HFD group *p < 0.05; HFD vs. HFD + LGG ^#p < 0.05, n = 5.