Gingipains may be one of the key virulence factors of Porphyromonas gingivalis to impair cognition and enhance blood-brain barrier permeability: An animal study.

AIM: Blood-brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process. MATERIALS AND METHODS: C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav-1 levels were measured. The mechanisms of Ddx3x-induced regulation on Mfsd2a by arginine-specific gingipain A (RgpA) in BMECs were explored. RESULTS: P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up-regulated Cav-1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav-1 regulatory axis was activated. CONCLUSIONS: Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav-1 axis.

The role of periodontitis in the development of atherosclerotic cardiovascular disease in participants with the components of metabolic syndrome: a systematic review and meta-analysis.

OBJECTIVES: Prevention of atherosclerotic cardiovascular disease (ASCVD) is important in individuals with metabolic syndrome components (MetS), and periodontitis may play an important role in this process. This study aims to evaluate the association between periodontitis and ASCVD in participants with the components of MetS, including obesity, dysglycemia, hypertension, and dyslipidemia. MATERIALS AND METHODS: This study conducted followed the MOOSE reporting guidelines and the PRISMA 2020 guidelines. EMBASE, MEDLINE, Web of Science, Cochrane Library, PubMed and OpenGrey were searched for observational studies about the linkage of periodontitis to ASCVD in people with MetS components up to April 9, 2023. Cohort, case-control and cross-sectional studies were included after study selection. Quality evaluation was carried out using the original and modified Newcastle-Ottawa Scale as appropriate. Random-effects model was employed for meta-analysis. RESULTS: Nineteen studies were finally included in the quality analysis, and all of them were assessed as moderate to high quality. Meta-analyses among fifteen studies revealed that the participants with periodontitis were more likely to develop ASCVD in those who have dysglycemia (RR = 1.25, 95% CI = 1.13-1.37; p < 0.05), obesity (RR = 1.13, 95% CI = 1.02-1.24; p < 0.05), dyslipidemia (RR = 1.36, 95% CI = 1.13-1.65; p < 0.05), or hypertension (1.20, 95% CI = 1.05-1.36; p < 0.05). CONCLUSIONS: Periodontitis promotes the development of ASCVD in participants with one MetS component (obesity, dysglycemia, hypertension or dyslipidemia). CLINICAL RELEVANCE: In people with MetS components, periodontitis may contribute to the ASCVD incidence.

Vitamin D decreases Porphyromonas gingivalis internalized into macrophages by promoting autophagy.

OBJECTIVES: This paper aims to study the effect of the active form of vitamin D (calcitriol) on the internalized Porphyromonas gingivalis in macrophages and to assess the role of autophagy during this process. MATERIALS AND METHODS: Quantitative RT-PCR and bacteria culture were used to quantify live P. gingivalis internalized into U937-derived macrophages. Western blot assays were performed to detect the effect of P. gingivalis and calcitriol on autophagy in macrophages. Transmission electron microscope was used to observe the effect of calcitriol on the status of internalized P. gingivalis. Colocalization of P. gingivalis with the autophagosome and lysosome markers was observed by confocal laser scanning microscopy. RESULTS: Calcitriol caused a dose-dependent decrease in live P. gingivalis numbers and promoted both the endogenous and P. gingivalis-induced autophagy in macrophages. Calcitriol significantly promoted the destruction of P. gingivalis and the colocalization of P. gingivalis with autophagosome and lysosome markers. Conversely, with 3-MA, live P. gingivalis numbers in macrophages increased significantly and inhibition effect of calcitriol on the number of live P. gingivalis was attenuated. CONCLUSION: In U937-derived macrophages, calcitriol may promote colocalization of P. gingivalis with autophagosomes and lysosomes, namely autophagy process, to degrade live P. gingivalis.

The prevalence rate of periodontal pathogens and its association with oral squamous cell carcinoma.

Mounting evidence suggests a causal relationship between specific bacterial infections or microbial compositions and the development of certain malignant neoplasms. In this study, we performed research through 16S rRNA amplicon sequencing, qPCR and fluorescence in situ hybridization to certify the relationship between periodontal pathogens and oral squamous cell carcinoma (OSCC). Subgingival plaque, cancer and paracancerous tissues from 6 patients with OSCC were selected for mapping bacterial profiles by 16S rRNA amplicon sequencing. The research showed that periodontal pathogens were enriched in cancer and paracancerous tissues, while the bacterial profiles were similar between the cancer tissues and subgingival plaque. Furthermore, the relative abundance of Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus sanguinis was detected in 61 cancer tissues, paracancerous tissues and subgingival plaque samples and in 30 normal tissues by qPCR. The results revealed that P. gingivalis and F. nucleatum existed at higher levels in cancer tissue than in normal tissues and were correlated with subgingival plaques. P. gingivalis was detected using a special oligonucleotide probe in 60.7% of OSCC tissues, 32.8% of paracancerous tissues and 13.3% of normal tissues. Relevance analysis showed that P. gingivalis infection was positively associated with late clinical staging, low differentiation and lymph node metastasis in patients with OSCC, which was accompanied by deeper periodontal pockets, severe clinical attachment loss and loss of teeth. This study revealed that there might be a close relationship between oral microorganisms, particularly periodontal pathogens, and OSCC, which might enrich the pathogenesis of oral squamous carcinoma.

Persistent infection with Porphyromonas gingivalis increases the tumorigenic potential of human immortalised oral epithelial cells through ZFP36 inhibition.

The association between Porphyromonas gingivalis infection and oral squamous cell carcinoma (OSCC) has been established by numerous epidemiological studies. However, the underlying mechanism specific to this connection remains unclear. By bioinformatical analysis, we identified ZFP36 as a potentially significant co-expressed gene in both the OSCC gene database and the persistent infection model of P. gingivalis. To further investigate the role of ZFP36, we established a cell model that human immortalized oral epithelial cells (HIOECs) that were sustainedly infected by P. gingivalis (MOI = 1) for a duration of 30 weeks. Our findings indicated that sustained infection with P. gingivalis inhibited the expression of ZFP36 protein and induced changes in the biological behaviour of HIOECs. The mechanism investigation demonstrated the potential role of ZFP36 in regulating the cancer-related biological behaviour of HIOECs. Subsequent studies revealed that highly expressed CCAT1 could serve as a molecular scaffold in the formation of the ZFP36/CCAT1/MK2 complex. This complex formation enhanced the binding abundance of MK2 and ZFP36, thereby promoting the inhibition of ZFP36 protein phosphorylation. To summarize, low expression of ZFP36 protein under persistent P. gingivalis infection enhances the cancer-related biological behaviour of HIOECs.

Role of ferroptosis in Porphyromonas gingivalis-induced impairment of epithelial junction.

Objectives: This study aims to clarify the effect of ferroptosis by P. gingivalis on periodontal epithelium impairment and potential mechanisms. Materials and methods: The expression of epithelial junction proteins (CDH1, OCLN, ZO-1), FTL and GPX4 in healthy and periodontitis tissues was analyzed using bioinformatics analysis and validated in vivo. An in vitro model was constructed to evaluate ferroptosis by mitochondria morphology, content of iron and GSH, and level of lipid peroxidation, FTL, GPX4 and SLC7A11. The iron concentration was changed with iron chelator DFO and iron supplementation FAC. The epithelial impairment was assessed by protein expression. To investigate the mechanism, si-MYB (a negative transcription factor of SLC7A11) and GPX4 inhibitor RSL3 were employed. Results: CDH1, OCLN, ZO-1 and GPX4 expression was decreased, while FTL expression was elevated in periodontitis tissues. Infected cells showed ferroptosis change of the mitochondria with higher level of lipid peroxidation, iron, FTL and lower level of GPX4, GSH, SLC7A11. FAC augmented ferroptosis and weakened epithelial junction, while DFO exhibited a counteractive effect. Silencing MYB rescued SLC7A11, GPX4 and epithelial junction proteins, which was hindered by RSL3. Conclusions: Our study demonstrated that P. gingivalis weakened the oral epithelial barrier by causing ferroptosis via inhibiting SLC7A11/GSH/GPX4 axis.

Association between periodontitis and inflammatory comorbidities: The common role of innate immune cells, underlying mechanisms and therapeutic targets.

Periodontitis, which is related to various systemic diseases, is a chronic inflammatory disease caused by periodontal dysbiosis of the microbiota. Multiple factors can influence the interaction of periodontitis and associated inflammatory disorders, among which host immunity is an important contributor to this interaction. Innate immunity can be activated aberrantly because of the systemic inflammation induced by periodontitis. This aberrant activation not only exacerbates periodontal tissue damage but also impairs systemic health, triggering or aggravating inflammatory comorbidities. Therefore, innate immunity is a potential therapeutic target for periodontitis and associated inflammatory comorbidities. This review delineates analogous aberrations of innate immune cells in periodontitis and comorbid conditions such as atherosclerosis, diabetes, obesity, and rheumatoid arthritis. The mechanisms behind these changes in innate immune cells are discussed, including trained immunity and clonal hematopoiesis of indeterminate potential (CHIP), which can mediate the abnormal activation and myeloid-biased differentiation of hematopoietic stem and progenitor cells. Besides, the expansion of myeloid-derived suppressor cells (MDSCs), which have immunosuppressive and osteolytic effects on peripheral tissues, also contributes to the interaction between periodontitis and its inflammatory comorbidities. The potential treatment targets for relieving the risk of both periodontitis and systemic conditions are also elucidated, such as the modulation of innate immunity cells and mediators, the regulation of trained immunity and CHIP, as well as the inhibition of MDSCs' expansion.

P. gingivalis alters lung microbiota and aggravates disease severity of COPD rats by up-regulating Hsp90α/MLKL.

Background: Epidemiological evidence has confirmed that periodontitis is an essential and independent risk factor of chronic obstructive pulmonary disease (COPD). Porphyromonas gingivalis, a major pathogen implicated in periodontitis, may make a vital contribution to COPD progression. However, the specific effects and molecular mechanism of the link between P. gingivalis and COPD are not clear. Methods and Results: A COPD rat model was constructed by smoke exposure combined intratracheal instillation of E. coli-LPS, then P. gingivalis was introduced into the oral cavity of COPD rats. This research observed that lower lung function, more severe alveolar damage and inflammation occurred in COPD rats with P. gingivalis group. Meanwhile, P. gingivalis/gingipains could colonize the lung tissues and be enriched in bronchoalveolar lavage fluid (BALF) of COPD rats with P. gingivalis group, along with alterations in lung microbiota. Proteomic analysis suggested that Hsp90α/MLKL-meditated necroptosis pathway was up-regulated in P. gingivalis-induced COPD aggravation, the detection of Hsp90α and MLKL in serum and lung tissue verified that Hsp90α/MLKL was up-regulated. Conclusion: These results indicate that P. gingivalis could emigrate into the lungs, alter lung microbiota and lead to aggravation of COPD, which Hsp90α/MLKL might participate in.

Porphyromonas gingivalis lipopolysaccharide induced RIPK3/MLKL-mediated necroptosis of oral epithelial cells and the further regulation in macrophage activation.

Necroptosis, a new type of regulated cell death with massive release of damage-associated molecular patterns (DAMPs), is involved in the pathogenesis of periodontitis. However, the role of necroptosis in oral epithelial cells and the following effect on macrophages activation remain unknown. Human immortalized oral epithelial cells were stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Cell death was assessed while expressions of RIPK3/MLKL and toll-like receptors (TLRs) were evaluated. Necrosulfonamide (NSA), an inhibitor of MLKL was applied to block necroptosis. The expression of DAMPs and the epithelial connection protein were evaluated by qPCR and immunofluorescence, respectively. Immortalized human monocytes U937 were induced into the M0 or M2 subset, and influences of HIOECs-derived DAMPs on macrophage polarization as well as activation of the Mincle/SYK axis were assessed. P. gingivalis LPS could be recognized by TLR2 and regulates necroptosis of HIOECs by activating RIPK3/MLKL. NSA inhibited cell death of HIOECs, alleviated impaired epithelial connection, and inhibited expressions of DAMPs. Low dose of DAMPs derived from HIOECs promoted M2-like polarization by activating the Mincle/SYK axis, which was significantly suppressed with increased doses of DAMPs. P. gingivalis LPS destructed oral epithelial cells via RIPK3/MLKL-mediated necroptosis, which further regulated macrophage activation via DAMPs from oral epithelial cells.

LL-37-Induced Autophagy Contributed to the Elimination of Live Porphyromonas gingivalis Internalized in Keratinocytes.

Porphyromonas gingivalis (P. gingivalis), one of the most important pathogens of periodontitis, is closely associated with the aggravation and recurrence of periodontitis and systemic diseases. Antibacterial peptide LL-37, transcribed from the cathelicidin antimicrobial peptide (CAMP) gene, exhibits a broad spectrum of antibacterial activity and regulates the immune system. In this study, we demonstrated that LL-37 reduced the number of live P. gingivalis (ATCC 33277) in HaCaT cells in a dose-dependent manner via an antibiotic-protection assay. LL-37 promoted autophagy of HaCaT cells internalized with P. gingivalis. Inhibition of autophagy with 3-methyladenine (3-MA) weakened the inhibitory effect of LL-37 on the number of intracellular P. gingivalis. A cluster of orthologous groups (COGs) and a gene ontology (GO) functional analysis were used to individually assign 65 (10%) differentially expressed genes (DEGs) to an "Intracellular trafficking, secretion, and vesicular transport" cluster and 306 (47.08%) DEGs to metabolic processes including autophagy. Autophagy-related genes, a tripartite motif-containing 22 (TRIM22), and lysosomal-associated membrane protein 3 (LAMP3) were identified as potentially involved in LL-37-induced autophagy. Finally, bioinformatics software was utilized to construct and predict the protein-protein interaction (PPI) network of CAMP-TRIM22/LAMP3-Autophagy. The findings indicated that LL-37 can reduce the quantity of live P. gingivalis internalized in HaCaT cells by promoting autophagy in these cells. The transcriptome sequencing and analysis also revealed the potential molecular pathway of LL-37-induced autophagy.

Calcitriol decreases live Porphyromonas gingivalis internalized into epithelial cells and monocytes by promoting autophagy.

BACKGROUND: The present study aimed to explore the effects of the active form of vitamin D (calcitriol, 1α,25-dihydroxyvitamin D3 , 1α,25 (OH)2 D3 , 1,25D) on live Porphyromonas gingivalis internalized into KB cells and U937 cells. METHODS: Quantitative real-time polymerase chain reaction method was used to evaluate the number of surviving P. gingivalis internalized into KB cells and U937 cells. Transmission electron microscopy was used to detect P. gingivalis in cells. A western blot analysis was performed to observe LC3 expressions. RESULTS: 1) Treatment with 1,25D decreased the number of live P. gingivalis in KB cells and U937 cells in a dose-dependent manner. 2) Dividing P. gingivalis were found only in KB cells but not in U937 cells. The cell walls of most P. gingivalis in KB cells were intact, while those in U937 cells were disrupted. Treatment with 1,25D promoted the encapsulation of P. gingivalis in autophagosomes in both KB and U937 cells. 3) Both 1,25D treatment and P. gingivalis infection increased the LC3 II/I ratio. Furthermore, 1,25D treatment increased the P. gingivalis-upregulated LC3 II/I ratio. 4) Treatment with 3-methyladenine (3-MA) decreased the number of P. gingivalis by 11.41% in KB cells, while increased that by 121.51% in U937 cells. Under 1,25D treatment conditions, 3-MA treatment increased the number of P. gingivalis by 88.71% in KB cells and by 284.70% in U937 cells. CONCLUSIONS: Autophagy may facilitate P. gingivalis survival in KB cells and eliminate P. gingivalis in U937 cells. Treatment with 1,25D may help decrease the number of live P. gingivalis in KB cells and U937 cells by promoting functional autophagy.

Identification of Potential Candidate Genes of Oral Cancer in Response to Chronic Infection With Porphyromonas gingivalis Using Bioinformatical Analyses.

Recent investigations revealed the relationship between chronic periodontitis, Porphyromonas gingivalis and cancer. However, host genes that change in response to chronic infection with P. gingivalis and may contribute to oral cancer have remained largely unknown. In the present study, we aimed to comprehensively analyze microarray data obtained from the chronic infection model of immortalized oral epithelial cells that were persistently exposed to P. gingivalis for 15 weeks. Using protein-protein interaction (PPI) networks and Ingenuity Pathway Analysis (IPA), we identified hub genes, major biological processes, upstream regulators and genes potentially involved in tumor initiation and progression. We also validated gene expression and demonstrated genetic alteration of hub genes from clinical samples of head and neck cancer. Overall, we utilized bioinformatical methods to identify IL6, STAT1, LYN, BDNF, C3, CD274, PDCD1LG2, and CXCL10 as potential candidate genes that might facilitate the prevention and treatment of oral squamous cell carcinoma (OSCC), the most common type of head and neck squamous cell carcinoma (HNSCC).

Persistent Exposure to Porphyromonas gingivalis Promotes Proliferative and Invasion Capabilities, and Tumorigenic Properties of Human Immortalized Oral Epithelial Cells.

Recent epidemiological studies revealed a significant association between oral squamous cell carcinoma (OSCC) and Porphyromonas gingivalis, a major pathogen of periodontal disease. As a keystone pathogen of periodontitis, P. gingivalis is known not only to damage local periodontal tissues, but also to evade the host immune system and eventually affect systemic health. However, its role in OSCC has yet to be defined. To explore the underlying effect of chronic P. gingivalis infection on OSCC and to identify relevant biomarkers as promising targets for therapy and prevention, we established a novel model by exposing human immortalized oral epithelial cells (HIOECs) to P. gingivalis at a low multiplicity of infection (MOI) for 5-23 weeks. The P. gingivalis infected HIOECs were monitored for tumor biological alteration by proliferation, wound healing, transwell invasion, and gelatin zymography assays. Microarray and proteomic analyses were performed on HIOECs infected with P. gingivalis for 15 weeks, and some selected data were validated by quantitative real-time PCR and (or) western blot on cells infected for 15 and 23 weeks. Persistent exposure to P. gingivalis caused cell morphological changes, increased proliferation ability with higher S phase fraction in the cell cycle, and promoted cell migratory and invasive properties. In combining results of bioinformatics analyses and validation assays, tumor-related genes such as NNMT, FLI1, GAS6, lncRNA CCAT1, PDCD1LG2, and CD274 may be considered as the key regulators in tumor-like transformation in response to long-time exposure of P. gingivalis. In addition, some useful clinical biomarkers and novel proteins were also presented. In conclusion, P. gingivalis could promote tumorigenic properties of HIOECs, indicating that chronic P. gingivalis infection may be considered as a potential risk factor for oral cancer. The key regulators detected from the present model might be used in monitoring the development of OSCC with chronic periodontal infection.

Porphyromonas gingivalis promotes the cell cycle and inflammatory cytokine production in periodontal ligament fibroblasts.

OBJECTIVE: The infection of Porphyromonas gingivalis (P. gingivalis) modulates host immune-inflammatory responses and destructs homeostasis of normal cell cycle, thereby leading to periodontal tissue destruction. Human periodontal ligament fibroblasts (PDLFs) are key players in the host immune responses and periodontal tissue regeneration. The aim of the present study was to discover the effects of P. gingivalis infection on the cell cycle and inflammatory cytokine production in PDLFs. DESIGN: P. gingivalis infection model into PDLFs was established. The effect of P. gingivalis on the cell proliferation and cell cycle were detected by MTT and flow cytometry. The p21, cyclin D1 and cyclin E mRNA expression, p21 protein expression, as well as IL-6 and IL-8 protein levels were analyzed by RT-qPCR, Western blot and ELISA, respectively. RESULTS: P. gingivalis promoted proliferation and G1 phase of PDLFs. G1 phase promotion was associated with the decreased level of p21 and the up-regulation of cyclin D1 at 6h, and with the increased level of cyclin E at 12h. Simultaneously, the immune-inflammatory response of PDLFs was initiated by P. gingivalis during the initial stage of infection, including the increased expressions of IL-6 and IL-8. CONCLUSION: We confirmed that the infection of P. gingivalis could modulate the expression of PDLF genes, which control cell cycle and inflammatory cytokine production. Thus, P. gingivalis may contribute to the proliferation and inflammation of periodontal tissue.

The sociodemographic characteristics, periodontal health status, and subgingival microbiota of patients with chronic periodontitis and type 2 diabetes mellitus: a case-control study in a Chinese population.

BACKGROUND: In China, chronic periodontitis (CP) is common in patients with type 2 diabetes mellitus (T2DM). The purpose of this study is to identify the sociodemographic characteristics associated with such patients and to assess the periodontal health status and subgingival microbiota of patients with CP and T2DM (T2DMCP) in the Chinese population. METHODS: A total of 150 patients with T2DMCP and 306 patients with CP without any systemic disease completed questionnaires, underwent clinical periodontal examinations and participated in diabetes-related parameter examinations. Subgingival plaques were obtained to determine the prevalence and amounts of selected oral bacterial species using polymerase chain reaction (PCR) and real-time PCR, respectively. RESULTS: The income level and mean body mass index (BMI) of the patients with T2DMCP were significantly higher than those of the patients with CP. Additionally, the patients with T2DMCP were more likely to be urban residents, and they had significantly more severe periodontitis than did the patients with CP. In the patients with T2DMCP, the prevalence and amounts of Treponema denticola and Tannerella forsythia were significantly higher than those in the patients with CP. Finally, compared with the patients with CP, the patients with T2DMCP had a significantly lower prevalence and amount of Prevotella intermedia. CONCLUSIONS: Compared with the patients with CP, the patients with T2DMCP were more likely to be urban residents and generally had higher incomes, higher mean BMI, and poorer periodontal health status. Higher levels of T. denticola and T. forsythia and lower levels of P. intermedia were identified in the subgingival plaque of the patients with T2DMCP.

[Evaluation on the application of mechanical toothguide training box to chromatics teaching of prosthodontics].

OBJECTIVE: To evaluate the effectiveness on the application of mechanical toothguide training box (TTB) to chromatics teaching of prosthodontics. METHODS: 12 preclinical undergraduate students were chosen to participate in the VITA 3D-Master shade-matching system simulant toothguide training process using Linearguide Training Box program of TTB. Toothguide Trainer program considered as a test was performed on the trained undergraduate students group, the postgraduate students group as well as the clinical prosthodontists group with under 5-year clinical experience. The test scores, elapsed time and the coincidence of chromatics single factor matching were recorded each time, the shade-matching efficiency was calculated. The data was analyzed with SPSS 13.0 software. RESULTS: The shade-matching efficiency of undergraduate students trained by TTB (64.03 +/- 18.82) was significantly higher than before (P < 0.05), higher than the postgraduate students group and the clinical prosthodontists group (P < 0.05). The coincidence of chromatics single factor matching of trained undergraduate students were 64.64% (lightness), 88.57% (chroma), 87.70% (hue). CONCLUSION: TTB is a effective tool for shade-mathing practice in chromatics teaching of prosthodontics. The trained undergraduate students are less sensitive in lightness-matching.