Porphyromonas gingivalis induced periodontitis exacerbates progression of non-alcoholic steatohepatitis in rats

Abstract

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease that can develop into hepatocirrhosis and hepatic carcinoma. In recent years, epidemiological and animal studies have reported that Porphyromonas gingivalis (P. gingivalis), a known periodontopathic bacteria, is closely related to NASH. However, previous studies could not demonstrate a direct relationship between periodontitis, P. gingivalis infection, and NASH. The purpose of the present study was to examine the impact of P. gingivalis-associated periodontitis on the onset and progression of NASH. Forty-two male Wistar rats were used in this study. Rats were fed a high-fat diet (HFD) for 12 weeks in order to induce fatty liver. At 4 weeks from the start of feeding, the animals were performed ligature placement around the maxillary first molar tooth in order to induce experimental periodontitis, and then a P. gingivalis slurry was applied around the ligature twice in a week for 8 weeks (HFD/Pg(+) group). Controls were given the slurry without P. gingivalis after ligature placement using the same protocol (HFD/Pg(-) group). Significant increases in alveolar bone resorption and inflammation in periodontal tissue around the molar tooth in the HFD/Pg(+) group were observed when compared with the HFD/Pg(-) group. Moreover, histological images showing NASH characterized by perivenular lipid deposition including big fatty drops, ballooning degeneration, and focal necrosis with inflammatory cells were confirmed in the liver of rats in the HFD/Pg(+) group. Significant increases in alanine aminotransaminase, aspartate aminotransferase, and C-reactive protein levels were observed in the HFD/Pg(+) group. Furthermore, endotoxin levels in serum in the HFD/Pg(+) group were significantly higher than those in the HFD/Pg(-) group. The present study demonstrated that experimental periodontitis induced by P. gingivalis led to the progression of NASH in rats with fatty liver. Increased levels of endotoxin derived from P. gingivalis infection appear to play a considerable role in the progression of NASH.

Keywords: animal model; endotoxemia; high‐fat diet; non‐alcoholic fatty liver disease; periodontal bacteria.

Figure 1

Study design. BD, basal diet; HFD, high‐fat diet; P.g, Porphyromonas gingivalis; CMC, sodium carboxymethyl cellulose

Figure 2

Fatty liver after high‐fat diet (HFD) feeding. In order to evaluate the effects of HFD feeding on the liver, rats fed HFD (60% fat, 20% carbohydrate, and 20% protein) were classed as the (a) HFD group. Rats fed basal diet (BD) (10% fat, 70% carbohydrate, and 20% protein) were defined as the (b) BD group (control). Sudan III‐stained images of the hepatic lobulus were also observed

Figure 3

Experimental periodontitis induced by ligature placement and P Porphyromonas gingivalis infection. Rats fed high‐fat diet (HFD) for 4 weeks were treated around the ligature of the first molar with or without P. gingivalis. After 12 weeks, photographs of the right maxillary molars were taken of (a) Pg(−) group and (b) Pg(+) group), and microfocus X‐ray computed tomography (μCT) analysis was performed on (c) Pg(−) group and (d) Pg(+) group. Subsequently, the average distance from cement‐enamel junction (CEJ) to alveolar bone crest in (e) three palatal regions (mesial, central, and distal sites) of right maxillary first molar M1 was measured on (f) three‐dimensional images produced by μCT imaging. Data are shown as means ± SD (n = 6). The results of observation on hematoxylin and eosin (H‐E) staining images showed that progression of attachment loss, infiltration of inflammatory cells, and disorder of collagen fibers in the (h) Pg(+) group were more severe when compared with those in the (g) Pg(−) group (forked arrowhead, CEJ; triangle arrowhead, epithelial junction). Inflammatory cells mainly composed of (i) lymphocytes and (j) the appearance of osteoclasts on the alveolar bone crest (round‐tipped arrow) were confirmed when magnifying the black frame in the (h) Pg(+) group

Figure 4

Serum levels of liver function parameters. Serum (a) alanine aminotransaminase (ALT) and (b) aspartate aminotransferase (AST) were measured using the enzymatic method, after 12 weeks of feeding with basal diet (BD) or high‐fat diet (HFD). Data are shown as means ± SD (n = 6)

Figure 5

Hematoxylin and eosin (H‐E) stained images of liver. The caudate lobes of liver were extracted from rats in the high‐fat diet (HFD)/Pg(−) group, (a) ×200 and (c) ×400, and the HFD/Pg(+) group, (b) ×200 and (d) ×400 (arrow: focal necrosis), for diagnosis of NASH, and fields with a focus on the central vein were observed in H‐E stained images. Large fatty drops and focal hepatocyte necrosis (b, arrow) were confirmed in the HFD/Pg(+) group, and ballooning degeneration with (e) Mallory−Denk bodies (arrow) was observed on magnified images.

Figure 6

Lipid and fibrosis stained images in the liver after high‐fat diet (HFD) feeding and Porphyromonas gingivalis infection. The lipid staining images with Sudan III on (a) Pg(−) group and (b) Pg(+) group were observed to specifically evaluate lipid deposition and large fatty drops after 12 weeks of feeding with HFD. The orange‐stained area was quantitatively measured using ImageJ in (c) three randomly selected fields. Data are shown as means ± SD (n = 6). In addition, Azan–Mallory stained images of (d) Pg(−) group and (e) Pg(+) group were observed to evaluate hepatic fibrosis

Figure 7

Serum levels of endotoxin and C‐reactive protein (CRP) after high‐fat diet (HFD) feeding and Porphyromonas gingivalis infection. (a) Serum endotoxin and (b) CRP levels were respectively measured using ELISA and enzyme colorimetry after 12 weeks of feeding with HFD. Data are shown as means ± SD (n = 4).