Gut microbiota dysbiosis links chronic apical periodontitis to liver fibrosis in nonalcoholic fatty liver disease: Insights from a mouse model.

AIM: In this study, we investigated the systemic implications of chronic apical periodontitis (CAP). CAP may contribute to the nonalcoholic fatty liver disease (NAFLD) progression through the gut microbiota and its metabolites, which are related to the degree of fibrosis. METHODOLOGY: Sixteen 7-week-old male apolipoprotein E knockout (apoE-/-) mice were randomly divided into two groups: the CAP and Con groups. A CAP model was established by sealing the first- and second-maxillary molars with bacterium-containing cotton balls. Apical lesions were evaluated by micro-CT. Histological evaluations of NAFLD were performed using second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) assays. Additionally, we comprehensively analyzed the gut microbiota using 16S rRNA gene sequencing and explored metabolic profiles by liquid chromatography-mass spectrometry (LC-MS). Immunofluorescence analysis was used to examine the impact of CAP on tight junction proteins and mucin expression. Transcriptome assays have elucidated gene expression alterations in liver tissues. RESULTS: Micro-CT scans revealed an evident periapical bone loss in the CAP group, and the total collagen percentage was increased (Con, 0.0361 ± 0.00510%, CAP, 0.0589 ± 0.00731%, p < .05). 16S rRNA sequencing revealed reduced diversity and distinct taxonomic enrichment in the CAP group. Metabolomic assessments revealed that differentially enriched metabolites, including D-galactosamine, were enriched and that 16-hydroxyhexadecanoic acid and 3-methylindole were depleted in the CAP group. Immunofluorescence analyses revealed disruptions in tight junction proteins and mucin production, indicating intestinal barrier integrity disruption. Liver transcriptome analysis revealed upregulation of Lpin-1 expression in the CAP group. CONCLUSION: This study provides comprehensive evidence of the systemic effects of CAP on liver fibrosis in NAFLD patients by elucidating alterations in the gut microbiota composition and metabolism.

Unveiling the oral-gut connection: chronic apical periodontitis accelerates atherosclerosis via gut microbiota dysbiosis and altered metabolites in apoE-/- Mice on a high-fat diet.

The aim of this study was to explore the impact of chronic apical periodontitis (CAP) on atherosclerosis in apoE-/- mice fed high-fat diet (HFD). This investigation focused on the gut microbiota, metabolites, and intestinal barrier function to uncover potential links between oral health and cardiovascular disease (CVD). In this study, CAP was shown to exacerbate atherosclerosis in HFD-fed apoE-/- mice, as evidenced by the increase in plaque size and volume in the aortic walls observed via Oil Red O staining. 16S rRNA sequencing revealed significant alterations in the gut microbiota, with harmful bacterial species thriving while beneficial species declining. Metabolomic profiling indicated disruptions in lipid metabolism and primary bile acid synthesis, leading to elevated levels of taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and tauroursodeoxycholic acid (TDCA). These metabolic shifts may contribute to atherosclerosis development. Furthermore, impaired intestinal barrier function, characterized by reduced mucin expression and disrupted tight junction proteins, was observed. The increased intestinal permeability observed was positively correlated with the severity of atherosclerotic lesions, highlighting the importance of the intestinal barrier in cardiovascular health. In conclusion, this research underscores the intricate interplay among oral health, gut microbiota composition, metabolite profiles, and CVD incidence. These findings emphasize the importance of maintaining good oral hygiene as a potential preventive measure against cardiovascular issues, as well as the need for further investigations into the intricate mechanisms linking oral health, gut microbiota, and metabolic pathways in CVD development.

Gut microbiota may mediate the impact of chronic apical periodontitis on atherosclerosis in apolipoprotein E-deficient mice.

AIM: There are growing evidences linking chronic apical periodontitis (CAP) to atherosclerosis. Gut microbiota is found to be involved in the development of atherosclerosis. Recent studies have shown that CAP could change the diversity and composition of the gut microbiota. It was therefore, we hypothesized that gut microbiota and its metabolites could mediate the impact of CAP on atherosclerosis. METHODOLOGY: Twenty-four 5-week-old lipoprotein E knockout (apoE-/- ) mice were randomly divided into four groups: the CAP group, Con group, Co-CAP (cohoused with CAP) and Co-Con (cohoused with Con) group. In the CAP group, sterile cotton wool containing P. gingivalis was placed into the exposed pulp chamber, followed by coronal resin-based composite restoration of the bilateral maxillary first and second molars. In the Con group, a sham operation was performed. Biweekly, mice in the CAP group were anaesthetised to check the sealing of coronal access. Meanwhile, the animals in the Con group were anaesthetised. The cohousing approach was used to introduce gut microbiota from the CAP and Con groups into the Co-CAP and Co-Con groups, respectively. Alterations in the abundance and diversity of the gut microbiota were detected using 16S rRNA sequencing, Oil-red O staining was used to demonstrate the extent of lesions, and serum levels of trimethylamine N-oxide (TMAO), and immunohistochemistry of flavin-containing monooxygenase 3 (FMO3) in liver were used to assess TMAO-related metabolic alterations. RESULTS: Alterations of alpha and beta diversity were shown both in the CAP and the Co-CAP groups. Moreover, the percentage of atherosclerotic lesion area increased in the CAP and Co-CAP groups (p < .05). Linear discriminant analysis effect size (LEfSe) at the family level found the increases of Lachnospiraceae and Ruminococcaceae (p < .05), which were positively correlated with serum TMAO levels (p < .05). In the redundancy analysis technique (RDA), serum levels of TMAO were positively associated with the atherosclerotic lesions. Co-occurrence analysis revealed that the relative abundances of Lachnospiraceae and Porphyromonadacae were positively correlated with both the percentage of lesion area and TMAO level (p < .05). CONCLUSION: Thus, within the limitations of this study, the data suggest that the gut microbiota can mediate the effects of CAP on atherosclerosis.

Chronic apical periodontitis exacerbates atherosclerosis in apolipoprotein E-deficient mice and leads to changes in the diversity of gut microbiota.

AIM: To investigate the impact of chronic apical periodontitis (CAP) on atherosclerosis and gut microbiota by establishing a Porphyromonas gingivalis (P. gingivalis)-induced CAP in an apolipoprotein E-deficient (apoE-/- ) mice model. METHODOLOGY: Twenty-eight male apoE-/- mice were divided into two groups with 14 in each: CAP group and control group. In the CAP group, sterile cotton wool containing 108 colony-forming units of P. gingivalis was placed into the pulp chamber after pulp exposure followed by coronal resin filling in bilateral maxillary first and second molars. The mice were fed with a chow diet to induce atherosclerosis. Animals were euthanized 16 weeks after the operation, and the periapical lesions of bilateral maxillary first and second molars were assessed by micro-CT. After collection of aortic arches, atherosclerotic lesions were measured by Oil Red O staining. Serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG) were measured. Stools were collected to detect alterations in gut microbiota by 16S rRNA gene sequencing. Independent samples t-test was used to calculate the difference between the two groups. RESULTS: CAP was observed in 98.2% of molars. A significant increase in atherosclerotic plaque formation in the aortic arches was found in the CAP groups (CAP: 2.001% ± 0.27%, control: 0.927% ± 0.22%, p = .005). No significant difference was observed between sevum level of HDL-C (CAP: 2.295 ± 0.31 mmol/L, Control: 3.037 ± 0.55 mmol/L, p = .264) or LDL-C (CAP: 17.066 ± 3.95 mmol/L, Control: 10.948 ± 1.69 mmol/L, p = .177) in CAP group and Control group. There were no significant differences in TG (CAP: 1.076 ± 0.08 mmol/L, control: 1.034 ± 0.13 mmol/L, p = .794) or TC (CAP: 6.372 ± 0.98 mmol/L, control: 6.679 ± 0.75 mmol/L, p = .72) levels between the two groups (p > .05). The alpha diversity was elevated in the CAP group. In terms of beta diversity, the CAP and control groups were clearly distinguished by the microbial community. CONCLUSION: In a mouse experimental model, pulp infection with P. gingivalis -induced CAP, thus aggravating the development of atherosclerosis. Meanwhile, CAP increased alpha diversity and altered the beta diversity of the gut microbiota.

Disruption of Hedgehog Signaling by Vismodegib Leads to Cleft Palate and Delayed Osteogenesis in Experimental Design.

The function of hedgehog signaling has previously been shown to be crucial for craniofacial development. In this study, we treated C57/BL6J mice with the hedgehog pathway inhibitor vismodegib by oral gavage to establish a stable vismodegib-induced cleft palate model. At E10.5 and E12.5, mice in the experimental group were treated with 100 mg/kg of vismodegib, whereas mice in the control group were treated with solvent. The treated pregnant mice were sacrificed on E13.5, E14.5, E15.5, and E16.5. Palatal shelf growth was evaluated via histological and immunohistochemical analyses as well as palatal organ culture. Immunohistochemical staining was performed to examine the expression of osteogenic proteins in the palatal tissue. A high proportion of the mice administered 2 doses of 100 mg/kg of vismodegib displayed a cleft palate. Histologic examination revealed severely retarded palatal shelf growth and thickened epithelium in the experimental group. Vismodegib exposure induced complete cleft palate, which was attributed to a reduced cell proliferation rate in the palatal mesenchyme along the anterior-posterior axis. Moreover, this model also showed delayed ossification in the region of palatine bone with downregulation of Indian hedgehog (Ihh) protein. Our results suggest that vismodegib can be used to inhibit hedgehog signaling to affect palatal morphogenesis. Under treatment with this exogenous inhibitor, the cell proliferation rate of the palatal shelves and the osteogenic potential of the hard palate were decreased, which likely contributed to the complete cleft palate.

Bone morphogenetic protein type I receptor inhibition induces cleft palate associated with micrognathia and cleft lower lip in mice.

BACKGROUND: Gain-of- and loss-of-function studies have demonstrated that changes in bone morphogenetic protein (BMP) signaling during embryo development cause craniofacial malformations, including cleft palate. It remains uncertain whether BMP signaling could be targeted pharmacologically to affect craniofacial morphogenesis. METHODS: Pregnant C57Bl/6J mice were treated with the BMP type I receptor inhibitor LDN-193189 at the dose of 3, 6, or 9 mg/kg twice a day by intraperitoneal injection from embryonic day 10.5 (E10.5) to E15.5. At E16.5, embryos were investigated by facial measurement analysis and histology to determine the optimal concentration for malformation. Subsequent embryonic phenotypes were analyzed in detail by histology, whole-mount skeletal staining, micro-computed tomography, and palatal organic culture. We further used immunohistochemistry to analyze protein expression of the BMP-mediated canonical and noncanonical signaling components. RESULTS: The optimal concentration of LDN-193189 was determined to be 6 mg/kg. In utero, LDN-193189 exposures induced partial clefting of the anterior palate or complete cleft palate, which was attributed to a reduced cell proliferation rate in the secondary palate, and delayed palatal elevation caused by micrognathia. Analysis of signal transduction in palatal shelves at E12.5 and E13.5 identified a significant reduction of BMP/Smad signaling (p-Smad1/5/8) and unchanged BMP noncanonical signaling (p-p38, p-Erk1/2) after treatment with LDN-193189. CONCLUSION: The results of this study indicate that LDN-193189 can be used to manipulate BMP signaling by selectively targeting the BMP/Smad signaling pathway to affect palatal morphogenesis and produce phenotypes mimicking those caused by genetic mutations. This work established a novel mouse model for teratogen-induced cleft palate. Birth Defects Research (Part A) 106:612-623, 2016. © 2016 Wiley Periodicals, Inc.