Comparative Analyses of Subgingival Microbiome in Chronic Periodontitis Patients with and Without IgA Nephropathy by High Throughput 16S rRNA Sequencing

Background/Aims: Periodontitis is a prevalent chronic inflammatory disease caused by enhanced inflammation induced by dysbiotic microbes forming on subgingival tooth sites, which may disturb the balance of the microbial composition in the biofilm and finally result in the progressive destruction of the periodontal ligament and alveolar bone with periodontal pocket formation and/or gingival recession. Methods: To elucidate the correlation between subgingival microbiome and IgAN incidence in CP (chronic periodontitis at severe levels) patients, subgingival plaque samples were collected from CP patients without IgAN (Control) and CP patients with IgAN (Disease). 16S rRNA sequencing and comparative analyses of plaque bacterial microbiome between Control and Disease were performed. Results: Subgingival microbial diversity in Disease was a little higher than that in Control. Besides, significant differences were found in subgingival microbiome between Disease and Control. Compared with that in Control, at phylum level, the abundances of Proteobacteria and Actinobacteria were significantly higher while the abundances of Bacteroidetes, Fusobacteria, Spirochaetae, Synergistetes, and Saccharibacteria were significantly lower in Disease; at class level, the abundances of Betaproteobacteria, Bacilli, Actinobacteria, Flavobacteriia, and Gammaproteobacteria were significantly higher while the abundances of Bacteroidia, Fusobacteriia, Negativicutes, Clostridia, and Spirochaetes were significantly lower in Disease; at genus level, the abundances of Bergeyella, Capnocytophaga, Actinomyces, Corynebacterium, Comamonas, Lautropia, and Streptococcus were significantly higher while the abundances of Treponema and Prevotella were significantly lower in Disease. Conclusions: Our data indicated a correlation between the changes in subgingival microbial structure and IgAN incidence in CP patients, which might be used to predict IgAN incidence in CP patients

The cell cycle distribution in>4N phase and endoreduplication were interrupted by treatment with a DNA poly β inhibitor or by DNA poly β knockdown.

<p>(A) The expression of DNA poly β was examined by western blot as cells cultured with DDC or were transfected with siRNA DNA poly β. The results are presented as the ratio of the DNA poly β expression to β-actin expression. * <i>P</i><0.05 compared to the control group; ** <i>P</i><0.05 compared to the rotenone group; Rot: rotenone. (B) and (C) Cell cycle distribution and DNA replication were analyzed by flow cytometry using BrdU/PI staining after the addition of DDC or DNA poly β depletion. * <i>P</i><0.05 compared to the rotenone group; # <i>P</i>>0.05 compared to the rotenone group; R3 represents endoreduplication; R2-R3 represents S phase; The data represent three independent experiments and are expressed as the mean ± SD.</p

Expression of TH in the lesioned SN.

<p>Brain tissue sections were cut to a thickness of 5 µm and were immunostained using an anti-TH antibody. The TH-positive neurons were visualized using a Cy3-conjugated donkey-anti-sheep IgG antibody. The TH-positive SN neurons were counted in the same coordinates of two coronal sections from the right brain of two rats. The bottom graph is the selective enlargement. * <i>P</i><0.05 compared to the vehicle group; SNc: substantia nigra pars compacta; SNr: substantia nigra pars reticulate. Scale bar = 20 µm.</p

After stereotactic (ST) infusion rotenone, the increased expression of cyclin D was induced in the SN neurons.

<p>The immunoreactivity of cyclin D and TH was observed under the Laser confocal microscopy using DyLight 488-conjugated donkey-anti-rabbit IgG and Cy3-conjugated donkey-anti-sheep IgG, respectively, in the SN dopaminergic neurons of rats; The right graph is the selective enlargement; Scale bar = 20 µm.</p

Alterations in cell morphology and nucleus size caused by treatment with rotenone (0.25 or 2 µM) (x200 and selective enlargement).

<p>Cellular morphological changes and axons alterations were visualized using a light microscope after exposure to rotenone (0.25 or 2 µM). The toxic effect of chronic exposure to rotenone at low-dose levels was maintained for 1.5, 3 and 7 days. With exposure to rotenone (2 µM), cells were cultured for 24 and 36 h. Under the same conditions, cells were stained with Hoechst dye and the nucleus size was observed using a fluorescence microscope. The selective enlargement graph was at the bottom.</p

The expression of DNA poly β was enhanced in the lesioned brains of rats following rotenone infusion.

<p>Double immunohistostaining of DNA poly β and TH was performed. The right part represents the selective enlargement. Scale bar = 20 µm.</p

The selective increase of DNA poly β in the SN neurons in the ST-infused rats.

<p>After rotenone infusion, the immunoreactivity of DNA poly β was detected. B-D show selective enlargements of A. Scale bar = 20 µm.</p