Profiles of subgingival microbiomes and gingival crevicular metabolic signatures in patients with amnestic mild cognitive impairment and Alzheimer's disease.

BACKGROUND: The relationship between periodontitis and Alzheimer's disease (AD) has attracted more attention recently, whereas profiles of subgingival microbiomes and gingival crevicular fluid (GCF) metabolic signatures in AD patients have rarely been characterized; thus, little evidence exists to support the oral-brain axis hypothesis. Therefore, our study aimed to characterize both the microbial community of subgingival plaque and the metabolomic profiles of GCF in patients with AD and amnestic mild cognitive impairment (aMCI) for the first time. METHODS: This was a cross-sectional study. Clinical examinations were performed on all participants. The microbial community of subgingival plaque and the metabolomic profiles of GCF were characterized using the 16S ribosomal RNA (rRNA) gene high-throughput sequencing and liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) analysis, respectively. RESULTS: Thirty-two patients with AD, 32 patients with aMCI, and 32 cognitively normal people were enrolled. The severity of periodontitis was significantly increased in AD patients compared with aMCI patients and cognitively normal people. The 16S rRNA gene sequencing results showed that the relative abundances of 16 species in subgingival plaque were significantly correlated with cognitive function, and LC-MS/MS analysis identified a total of 165 differentially abundant metabolites in GCF. Moreover, multiomics Data Integration Analysis for Biomarker discovery using Latent cOmponents (DIABLO) analysis revealed that 19 differentially abundant metabolites were significantly correlated with Veillonella parvula, Dialister pneumosintes, Leptotrichia buccalis, Pseudoleptotrichia goodfellowii, and Actinomyces massiliensis, in which galactinol, sn-glycerol 3-phosphoethanolamine, D-mannitol, 1 h-indole-1-pentanoic acid, 3-(1-naphthalenylcarbonyl)- and L-iditol yielded satisfactory accuracy for the predictive diagnosis of AD progression. CONCLUSIONS: This is the first combined subgingival microbiome and GCF metabolome study in patients with AD and aMCI, which revealed that periodontal microbial dysbiosis and metabolic disorders may be involved in the etiology and progression of AD, and the differential abundance of the microbiota and metabolites may be useful as potential markers for AD in the future.

Abnormal amyloid precursor protein processing in periodontal tissue in a murine model of periodontitis induced by Porphyromonas gingivalis.

OBJECTIVE: The study aimed to investigate the change of amyloid precursor protein (APP) processing and amyloid ß (Aß) metabolites in linking periodontitis to Alzheimer's disease (AD). BACKGROUND: Aß is one of the main pathological features of AD, and few studies have discussed changes in its expression in peripheral tissues or analyzed the relationship between the peripheral imbalance of Aß production and clearance. METHODS: A murine model of periodontitis was established by oral infection with Porphyromonas gingivalis (P. gingivalis). Micro-computed tomography (Micro-CT) was used to observe the destruction of the alveolar bone. Nested quantitative polymerase chain reaction (qPCR) was used to measure small quantities of P.gingivalis DNA in different tissues. Behavioral experiments were performed to measure cognitive function in the mice. The mRNA levels of TNF-α, IL-6, IL-8, RANKL, OPG, APP695, APP751, APP770, and BACE1 in the gingival tissues or cortex were detected by RT-PCR. The levels of Aß1-40 and Aß1-42 in gingival crevicular fluid (GCF) and plasma were tested by ELISA. RESULTS: P. gingivalis oral infection was found to cause alveolar bone resorption and impaired learning and memory. P.gingivalis DNA was detected in the gingiva, blood and cortex of the P.gingivalis group by nested qPCR (p < .05). The mRNA expression of TNF-α, IL-6, IL-8, RANKL/OPG, and BACE1 in the gingival tissue was significantly higher than that in the control group (p < .05). Similarly, upregulated mRNA levels of APP695 and APP770 were observed in the gingival tissuses and cortex of the P. gingivalis group (p < .05). The levels of Aß1-40 and Aß1-42 in the GCF and plasma of the P. gingivalis group were significantly higher than those in the control group (p < .05). CONCLUSION: P. gingivalis can directly invade the brain via hematogenous infection. The invasion of P. gingivalis could trigger an immune response and lead to an imbalance between Aß production and clearance in peripheral tissues, which may trigger an abnormal Aß metabolite in the brain, resulting in the occurrence and development of AD.

Periodontitis Salivary Microbiota Aggravates Ischemic Stroke Through IL-17A.

Although epidemiological studies suggest that periodontitis is tightly associated with ischemic stroke, its impact on ischemic stroke and the underlysing mechanisms are poorly understood. Recent studies have shown that alteration in gut microbiota composition influences the outcomes of ischemic stroke. In the state of periodontitis, many oral pathogenic bacteria in the saliva are swallowed and transmitted to the gut. However, the role of periodontitis microbiota in the pathogenesis and progression of ischemic stroke is unclear. Therefore, we hypothesized that the periodontitis salivary microbiota influences the gut immune system and aggravates ischemic stroke. Mice receiving gavage of periodontitis salivary microbiota showed significantly worse stroke outcomes. And these mice also manifested more severe neuroinflammation, with higher infiltration of inflammatory cells and expression of inflammatory cytokines in the ischemic brain. More accumulation of Th17 cells and IL-17+ γδ T cells were observed in the ileum. And in Kaede transgenic mice after photoconversion. Migration of CD4+ T cells and γδ T cells from the ileum to the brain was observed after ischemic stroke in photoconverted Kaede transgenic mice. Furthermore, the worse stroke outcome was abolished in the IL-17A knockout mice. These findings suggest that periodontitis salivary microbiota increased IL-17A-producing immune cells in the gut, likely promoted the migration of these cells from the gut to the brain, and subsequently provoked neuroinflammation after ischemic stroke. These findings have revealed the role of periodontitis in ischemic stroke through the gut and provided new insights into the worse outcome of ischemic stroke coexisting with periodontitis in clinical trials.

The imbalance of Th17/Treg via STAT3 activation modulates cognitive impairment in P. gingivalis LPS-induced periodontitis mice.

Periodontitis is one of the most common oral diseases worldwide, and it is associated with various systemic diseases, including cognitive diseases. STAT3 regulates the inflammatory cascade and influences adaptive immunity by modulating Th17/Treg cell differentiation. In this study, we aimed to explore the effect of adaptive immunity inside and outside the brain on the association between periodontitis and cognitive impairment and understand the role of the STAT3 signaling pathway. We established Porphyromonas gingivalis LPS-induced periodontitis mice models by injecting P. gingivalis LPS into the gingival sulcus of mice. Behavioral tests showed that learning and memory abilities were impaired. The flow cytometry data showed an imbalance in the Th17/Treg ratio in the blood and brain samples of the mice. The expression of Th17-related cytokines (IL-1ß, IL-17A, IL-21, and IL-22) increased, whereas that of Treg-related cytokines (IL-2 and IL-10) decreased in both the blood and the brain. The level of LPS increased and the STAT3 signaling pathway was activated during this process. These effects were reversed by C188-9, a STAT3 inhibitor. In conclusion, P. gingivalis LPS-induced periodontitis may promote the occurrence and progression of cognitive impairment by modulating the Th17/Treg balance inside and outside the brain. The STAT3 signaling pathway may have immunoregulatory effects on the mouth-to-brain axis.

Lipopolysaccharide Preparation Derived From Porphyromonas gingivalis Induces a Weaker Immuno-Inflammatory Response in BV-2 Microglial Cells Than Escherichia coli by Differentially Activating TLR2/4-Mediated NF-κB/STAT3 Signaling Pathways.

Alzheimer's disease (AD) is a degenerative disease of the central nervous system with unclear etiology and pathogenesis. In recent years, as the infectious theory and endotoxin hypothesis of AD has gained substantial attention, several studies have proposed that Porphyromonas gingivalis (P. gingivalis), one of the main pathogenic bacteria of chronic periodontitis, and the lipopolysaccharide (LPS) of P. gingivalis may lead to AD-like pathological changes and cognition impairment. However, research on the relationship between P. gingivalis-LPS and neuroinflammation is still lacking. Our study aimed to investigate the effects of P. gingivalis-LPS preparation on immuno-inflammation in microglial cells and further compared the differential inflammatory response induced by P. gingivalis-LPS and Escherichia coli (E. coli) LPS preparations. The results showed that P. gingivalis-LPS could upregulate the gene expression and release of pro-inflammatory factors in BV-2 microglial cells, including IL-1ß, IL-6, TNF-α, IL-17, and IL-23. We also observed an increase in the level of Toll-like receptor 2/4 (TLR2/4) and NF-κB/STAT3 signaling. Moreover, the changes mentioned above were more significant in the E. coli-LPS group and the effects of both kinds of LPS could be differentially reversed by the administration of the TLR2 inhibitor C29 and TLR4 inhibitor TAK-242. The molecular simulation showed that the binding affinity of P. gingivalis-lipid A to TLR4-MD-2 was weaker than E. coli-lipid A, which was probably due to the presence of fewer acyl chains and phosphate groups of P. gingivalis-lipid A than E. coli-lipid A. We conclude that P. gingivalis-LPS could activate TLR2/4-mediated NF-κB/STAT3 signaling pathways, which ultimately resulted in an immune-inflammatory response in BV-2 microglia. In contrast to E. coli-LPS, P. gingivalis-LPS is a weaker TLR2/4 agonist and NF-κB/STAT3 signaling activator. Furthermore, the different fatty acid chains and phosphate groups between P. gingivalis-lipid A and E. coli-lipid A may be the reason for the weaker activating properties of P. gingivalis-LPS.

Activated STAT3 signaling pathway by ligature-induced periodontitis could contribute to neuroinflammation and cognitive impairment in rats.

BACKGROUND: Increasing evidence suggests a causal link between periodontitis and cognitive disorders. Systemic inflammation initiated by periodontitis may mediate the development of cognitive impairment. Our study aims to investigate the effect of ligature-induced periodontitis on cognitive function and the role of signal transducers and activators of transcription 3 (STAT3) in this process. MATERIALS AND METHODS: Ligature-induced periodontitis was established, and the rats were treated intraperitoneally with/without the pSTAT3 inhibitor cryptotanshinone (CTS). Alveolar bone resorption and periodontal inflammation were detected by micro-computed tomography analysis and histopathological evaluation. Locomotor activity and cognitive function were evaluated by the open field test and the Morris water maze test, respectively. The activation of microglia and astrocytes in the hippocampus and cortex was assessed by immunohistochemistry (IHC). The expression of interleukins (IL-1ß, IL-6, IL-8, IL-21) in both the periphery and cortex was evaluated by RT-PCR and ELISA. The expression of TLR/NF-κB and ROS cascades was evaluated by RT-PCR. The expression of pSTAT3 and the activation of the STAT3 signaling pathway (JAK2, STAT3, and pSTAT3) in the periodontal tissue and cortex were assessed by IHC and Western blot. The expression of amyloid precursor protein (APP) and its key secretases was evaluated by RT-PCR. The level of amyloid ß-protein (Aß) and the ratio of Aß1-40/1-42 were measured via ELISA in the plasma and cortex while IHC was used to detect the level of Aß1-42 in the brain. RESULTS: In periodontal ligature rats, significant alveolar bone resorption and local inflammatory cell infiltration were present. Apparent increases in inflammatory cytokines (IL-1ß, IL-6, IL-8, and IL-21) were detected in peripherial blood and brain. Additionally, spatial learning and memory ability was impaired, while locomotor activity was not affected. Activated microglia and astrocytes were found in the cortex and hippocampus, presenting as enlarged cell bodies and irregular protrusions. Levels of TLR/NF-kB, PPAR and ROS were altered. The STAT3 signaling pathway was activated in both the periodontal tissue and cortex, and the processing of APP by ß- and γ-secretases was promoted. The changes mentioned above could be relieved by the pSTAT3 inhibitor CTS. CONCLUSIONS: Ligature-induced periodontitis in rats resulted in systemic inflammation and further abnormal APP processing, leading to cognitive impairments. In this progress, the activation of the STAT3 signaling pathway may play an important role by increasing inflammatory load and promoting neuroinflammation.

Periodontitis Induced by P. gingivalis-LPS Is Associated With Neuroinflammation and Learning and Memory Impairment in Sprague-Dawley Rats.

BACKGROUND: Periodontitis is one of the most common oral diseases and is a potential risk factor for systemic diseases. In this study, we aimed to investigate the association between periodontitis and learning and memory impairment. METHODS: We established a periodontitis model by topical application of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) into the palatal gingival sulcus of the maxillary first molars of 10-week-old male rats for a 10-week period. We assessed alveolar bone resorption using micro-computed tomography analysis and learning and memory ability using the Morris water maze test. We determined the levels of cytokines [interleukin (IL)-1ß, IL-6, IL-8, and IL-21] and LPS in the peripheral blood and cortex, as well as toll-like receptor 4 (TLR4)/NF-κB signaling pathway activation, using reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot. We determined activation of microglia and astrocytes, expression of Aß1-42, APP and Tau by immunohistochemistry. Finally, we measured the expression of amyloid precursor protein (APP) and its key secretases, as well as the Aß1-40/1-42 ratio, by RT-PCR, western blot, and ELISA. RESULTS: We found that periodontitis induced learning and memory impairment in the rats. Further, we observed that it induced significant alveolar bone resorption. There was an increase in the levels of inflammatory cytokines and LPS. Moreover, we confirmed TLR4/NF-κB signaling pathway activation. We also observed activated microglia and astrocytes with enlarged cell bodies and irregular protrusions. Finally, we observed the promotion of ß- and γ-secretases APP processing. CONCLUSION: Our findings indicated that periodontitis was associated with learning and memory impairment, probably induced by neuroinflammation via activating the TLR4/NF-κB signaling pathway. Furthermore, abnormal APP processing could be involved in this progress.

[Association between periodontitis and Alzheimer disease: a meta analysis].

PURPOSE: To evaluate the association between periodontitis and Alzheimer's disease. METHODS: Databases of PubMed, Embase, CNKI, VIP and WanFang databases were searched for the relevant observational studies focusing on the association between periodontitis and Alzheimer's disease. The deadline was January 2019. Data quality evaluation and extraction were independently conducted by two authors. Meta analysis was performed using RevMan 5.2 software. RESULTS: Four case-control, five cross-sectional and two cohort studies were included. One cohort study and four case-control studies treated periodontitis as the exposure factor, all five cross-sectional studies and the other cohort study treated Alzheimer's disease as the exposure factor. The results of meta analysis showed that patients with periodontitis had a higher risk of Alzheimer's disease (RR=1.22, 95%CI: 1.13-1.33, P<0.00001), and the risk was more higher in patients with severe periodontitis(RR=1.54, 95%CI:1.05-2.26, P=0.03<0.05); but there was no significant difference in the risk of Alzheimer's disease in patients with moderate periodontitis (RR=1.19, 95%CI: 0.98-1.44, P=0.07>0.05). The results of meta-analysis also showed that probing depth in patients with Alzheimer's disease was significantly higher than that of the control group (MD=2.58, 95%CI: 0.17-4.99, P=0.04<0.05), as well as clinical attachment loss (MD=1.27, 95%CI: 0.43-2.10, P=0.003<0.05), plaque index (MD=1.14, 95%CI: 0.85-1.44, P<0.00001) and the percentage of bleeding on probing (MD=21.11%, 95%CI: 18.23%-23.99%, P<0.00001). Furthermore, the number of present teeth in patients with Alzheimer's disease was significantly less than that of the control group (MD=-3.77, 95% CI: -6.89- -0.65, P=0.02<0.05). CONCLUSIONS: The current evidence indicates that periodontitis is associated with Alzheimer's disease and patients with periodontitis (especially severe periodontitis) probably have a higher risk of developing Alzheimer's disease, and patients with Alzheimer's disease tend to have poorer periodontal health. However, the number of existing studies is limited and more clinical evidences are needed to support the correlation between these two diseases.

Applicability and accuracy of Demirjian and Willems methods in a population of Eastern Chinese subadults.

BACKGROUND: To assess the validity of the Demirjian and Willems dental age estimation methods in a population of Eastern Chinese 11-18-year-old subadults. SAMPLES AND METHODS: A total of 1622 orthopantomograms (787 boys and 835 girls) aged from 11.00 to 18.99 years old from a Chinese Han population were evaluated in the study. Dental ages were calculated using both Demirjian and Willems method. Statistical significance was set at p<0.05 and for each method, discrepancies between chronological age (CA) and dental age (DA) were statistically analyzed by paired t-tests. Special age interval analysis was conducted by Chi-square tests to compare the accuracy of the Demirjian and the Willems methods. RESULTS: Demirjian method underestimated CA 0.66±1.45 y (p<0.001) in boys and 0.62±1.61 y (p<0.001) in girls while Willems method underestimated 0.90±1.32 y (p<0.001) and 0.97±1.56 y (p<0.001) in boys and girls, respectively. The mean absolute error (MAE) value between the CA and the DA (CA-DA) using the Demirjian method and the Willems method were 1.31 and 1.29 y for boys, respectively, and 1.35 and 1.43 y for girls, respectively. CONCLUSIONS: It is suggested that the Demirjian method was found to be more accurate compared with the Willems method. However, the Demirjian method may not be satisfactory for 11-18-year-old children of Eastern China. It is recommended that a modified Demirjian method for better dental age estimation for Eastern Chinese juvenile individuals.