Gingival Epithelial Cells Research Articles

Surface pre-reacted glass-ionomer eluate protects gingival epithelium from penetration by lipopolysaccharides and peptidoglycans via transcription factor EB pathway.

Surface pre-reacted glass-ionomer (S-PRG) filler, produced by PRG technology for use with various dental materials, is bioactive and known to release ions from a glass-ionomer phase. We previously reported that coxsackievirus and adenovirus receptor (CXADR), a tight junction associated protein, was located in the epithelial barrier of gingival epithelium. In the present study, the tissue protective effects of an S-PRG eluate prepared with S-PRG filler were investigated using a three-dimensional human gingival epithelial tissue model. The results showed that the S-PRG eluate specifically induced CXADR expression at the transcriptional level of messenger RNA as well as the protein level, and also nuclear translocation of transcription factor EB (TFEB) in gingival epithelial cells. Furthermore, shigyakusan, a TFEB inhibitor, canceled induction of the CXADR protein by the S-PRG eluate. Additionally, gingival epithelial permeation by 40-kDa dextran, lipopolysaccharide, and peptidoglycan in the 3D-tissue models was prevented by the eluate, with those effects abrogated by knockdown of CXADR. These findings suggest that S-PRG eluate increases CXADR expression via the TFEB pathway, thus inhibiting penetration of bacterial virulence factors into subepithelial tissues.

Effects of potential probiotics on the expression of cytokines and human β-defensins in human gingival epithelial cells and in vivo efficacy in a dog model

ObjectivesThe aims of this study were to examine the effects of probiotic strains on the expression of cytokines and human β-defensins-2–4 in human gingival epithelial cells and to investigate the in vivo efficacy of probiotics in a dog model. DesignSelected probiotics, Lacticaseibacillus paracasei SD1, L. rhamnosus SD4, L. rhamnosus SD11 and Limosilactobacillus fermentum SD7, were examined for the expression of human β-defensins-2–4, and cytokine responses after stimulated by various periodontopathogens. Subsequently, an in vivo study was set as a randomized, double-blind, placebo-controlled intervention in a dog model. A total of 20 dogs with mild gingivitis were randomly assigned to either the probiotic or control group. The effects of probiotics on periodontopathogenic- and cytokine levels were analyzed after 4 weeks of intervention. ResultsIt showed that all probiotics could induce human β-defensins-2–4, interleukin-1β, interleukin-6, interleukin-8, and tumor necrosis factor-α expressions in human gingival epithelial cells; however, a significant difference had been found among the strains. When individual probiotic strain was combined to periodontopathogens, a significant reduction of IL-8 expression was found. Results of the in vivo study demonstrated that the bacterial levels, especially Porphyromonas gingivalis, and the interleukin-8 levels were significantly decreased after receiving the probiotic products compared to the controls. ConclusionThe results of the in vitro and in vivo studies in dogs are encouraging to support the effectiveness of a mixture of probiotic treatments for improvement of periodontal health by reducing periodontopathogens and interleukin-8 levels. Therefore, such probiotics preparation could possibly have therapeutic potential in human periodontitis.

Cold Atmospheric Pressure Plasma Is Effective against P. gingivalis (HW24D-1) Mature Biofilms and Non-Genotoxic to Oral Cells

The effects of helium cold atmospheric pressure plasma (He-CAPP) jet on Porphyromonas gingivalis (HW24D-1) biofilm, on human gingival fibroblasts (HGF) and human gingival keratinocytes (OBA-9) were assessed. Standardized suspension of P. gingivalis was obtained, and biofilms were grown anaerobically for 48 h. After exposition to He-CAPP, the biofilm viability was evaluated by XTT assay. HGF were grown at 37 °C, in an CO2 chamber in DMEM, while OBA-9 cells were cultured in keratinocyte serum-free medium. After 24 h, plates were exposed to He-CAPP for 1 to 7 min. Plasma was generated using a commercial AC power supply with amplitude modulated signal (voltage amplitude of 20 kVp-p, frequency of 31.0 kHz and duty cycle of 22%). The corresponding discharge power was 0.6W at He flow rate of 1 L/min. DNA damage was accessed by static cytometry. Data were analyzed by GraphPad Prism (p < 0.05). Significant reductions in P. gingivalis viability in relation to non-treated groups were detected (p < 0.0001), directly proportional to exposure time. Treated groups were slightly aneuploid after 5- and 7-min treatment in HGF, and for 3 min in OBA-9 cells, with 1.2 DNA index mean. Helium cold atmospheric pressure plasma jet showed inhibitory effect on P. gingivalis mature biofilm and was not genotoxic for epithelial gingival cells and human oral fibroblasts.

Cross Talk between Synthetic Food Colors (Azo Dyes), Oral Flora, and Cardiovascular Disorders

Synthetic food colors are important ingredients in the food industry. These synthetic food colorants are azo dyes, majorly acidic in nature such as Allura red and Tartrazine. They are present in sweets, carbonated drinks, meat products, and candies to attract the consumers. This review article is an attempt to explain the adverse effects of azo dyes and their association with oral cavities and cardiovascular disorders. These synthetic dyes (azo dyes) have staining effects on dentin. Poor dental care accelerates the bacterial accumulation on the dental crown (Gram-negative bacteria P. gingivalis, T. denticola, and T. forsythia and Gram-positive bacteria Strep. Gordonii), causing the washing of enamel, forming dental plaque. Bacterial pathogens (P. ginigivalis and F. nacleatum) release different chemicals (FadA and Fap2) that bind to protein on the cell by producing an inflammatory response through different line-host defenses, such as Gingival epithelial cells (ECs), Hemi-desmosomes, and desmosomes, which helps the bacterium migration from the cell–cell junction. This makes the junctions slightly open up and makes the whole vessel permeable, through which the bacterium enters into the blood stream line. This leads to different major arteries, such as the carotid artery, and causes the accumulation of plaque in major cardiac arteries, which causes different cardiovascular disorders. These bacterial species present in gums cause cardiovascular diseases, such as ischemic heart disease, coronary artery disease, heart attacks and strokes, and arrhythmias, which can lead to death.

Cannabis smoke condensate induces human gingival epithelial cell damage through apoptosis, autophagy, and oxidative stress

ObjectivesThis study aims to investigate the effects of cannabis smoke condensate (CSC) on the adhesion, growth, and signaling pathways of human gingival epithelial cells. DesignThe effects of CSC on cell shape and adhesion, and viability were evaluated after 30 min, 60 min, 2 h, and 24 h of exposure using microscopic observation, cell metabolic activity, and lactate dehydrogenase activity assays. The effects of CSC on cell apoptosis, necrosis, autophagy, and oxidative stress were determined through flow cytometry, while apoptotic and autophagic gene expression were identified via an RT2-PCR array. Phosphorylated signaling pathway proteins were measured using flow cytometry. ResultsCSC deregulated gingival epithelial cell shape and adhesion, decreased cell viability, and increased lactate dehydrogenase release. Its toxic effects included apoptosis, autophagy, and oxidative stress. Moreover, it modulated seven specific apoptotic and six autophagic genes. Furthermore, it decreased phosphorylation in signaling proteins, such as STAT5, ERK12, P38, and nuclear factor κB. ConclusionsCSC has notable adverse effects on gingival epithelial cells. This finding indicates that cannabis smoke could impair gingival epithelial cell innate immune function, leading to gingivitis and periodontitis. Oral health professionals may need to document observed modifications in the oral cavity of patients who smoke cannabis and consider these potential changes during clinical care.

MicroRNAs and Periodontal Disease: Helpful Therapeutic Targets?

Periodontal disease is the most common oral disease. This disease can be considered as an inflammatory disease. The immune response to bacteria accumulated in the gum line plays a key role in the pathogenesis of periodontal disease. In addition to immune cells, periodontal ligament cells and gingival epithelial cells are also involved in the pathogenesis of this disease. miRNAs which are small RNA molecules with around 22 nucleotides have a considerable relationship with the immune system affecting a wide range of immunological events. These small molecules are also in relation with periodontium tissues especially periodontal ligament cells. Extensive studies have been performed in recent years on the role of miRNAs in the pathogenesis of periodontal disease. In this review paper, we have reviewed the results of these studies and discussed the role of miRNAs in the immunopathogenesis of periodontal disease comprehensively. miRNAs play an important role in the pathogenesis of periodontal disease and maybe helpful therapeutic targets for the treatment of periodontal disease.

Establishment of Biomimetic Soft Tissue Integration with the Surface of Zirconia Fused with Platelet-Activating Peptide.

Soft tissue sealing around zirconia (ZrO2) abutment is critical for the long-term stability of dental implants. The goal of the study is to develop a strong basal lamina (BL)-mediated epithelial attachment to ZrO2 via a novel physicochemical immobilization method. An electrophoretic fusion (EPF) method was applied to fuse a phosphonic acid (PA) linker to ZrO2 discs. Bindings of the PA linker and the following protease activated receptor 4 (PAR4) were verified by Fourier-transform infrared spectroscopy (FITR). Then, ZrO2 discs were doped in platelet-rich plasma (PRP). Platelet-derived growth factor (PDGF) was measured to assess platelet activation. PRP-doped discs were subsequently co-cultured with human gingival epithelial cells (OBA9) to evaluate establishment of basal lamina-mediated epithelial attachment. The EPF method achieved robust immobilization of the PA linker and PAR4 onto the ZrO2 surface. The resultant PAR4-coupled ZrO2 successfully induced platelet aggregation and activation. Furthermore, a BL-mediated epithelial attachment was established. The results are significant for clinical application to minimize the risk of developing peri-implant diseases.

Characterization of a Treponema denticola ATCC 35405 mutant strain with mutation accumulation, including a lack of phage-derived genes.

Trepoenema denticola, a spirochetal bacterium, is associated with periodontal diseases. The type strain of the bacterium, ATCC 35405, is commonly used in a basic research. Here, we report that our stock strain derived from ATCC 35405 had a mutation on the chromosome and expressed differential characteristics from the original strain. Genome sequencing analysis revealed the lack of a phage-derived region, and over 200 mutations in the mutant strain. The mutant grew to a higher density in broth culture as compared with the origin. In addition, the mutant formed a colony on the surface of the agar medium, whereas the origin could not. On contrary, the mutant showed decreased motility and adhesion to gingival epithelial cells. There were no differences in the bacterial cell length and a chymotrypsin-like protease activity between the two strains. RNA and genome sequencing analysis could not identify the genes that introduced the phenotypic differences between the strains. This mutant is potentially useful for examining the genetic background responsible for the physiological and pathogenic characteristics of T. denticola.

Leuconostoc mesenteroides LVBH107 Antibacterial Activity against Porphyromonas gingivalis and Anti-Inflammatory Activity against P. gingivalis Lipopolysaccharide-Stimulated RAW 264.7 Cells.

Probiotics, active microorganisms benefiting human health, currently serve as nutritional supplements and clinical treatments. Periodontitis, a chronic infectious oral disease caused by Porphyromonas gingivalis (P. gingivalis), activates the host immune response to release numerous proinflammatory cytokines. Here, we aimed to clarify Leuconostoc mesenterica (L. mesenteroides) LVBH107 probiotic effects based on the inhibition of P. gingivalis activities while also evaluating the effectiveness of an in vitro P. gingivalis lipopolysaccharide-stimulated RAW 264.7 cell-based inflammation mode. L. mesenteroides LVBH107 survived at acid, bile salts, lysozyme, and hydrogen peroxide conditions, auto-aggregated and co-aggregated with P. gingivalis, exhibited strong hydrophobicity and electrostatic action, and strongly adhered to gingival epithelial and HT-29 cells (thus exhibiting oral tissue adherence and colonization abilities). Moreover, L. mesenteroides LVBH107 exhibited sensitivity to antibiotics erythromycin, doxycycline, minocycline, ampicillin, and others (thus indicating it lacked antibiotic resistance plasmids), effectively inhibited P. gingivalis biofilm formation and inflammation (in vitro inflammation model), reduced the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and inflammatory mediators (NO and PGE2), and decreased the expression levels of inflammation related genes. Thus, L. mesenterica LVBH107 holds promise as a probiotic that can inhibit P. gingivalis biofilm formation and exert anti-inflammatory activity to maintain oral health.

Green tea catechins inhibit Porphyromonas gulae LPS-induced inflammatory responses in human gingival epithelial cells

ObjectivesTo determine the anti-inflammatory effects of green tea catechins in immortalized human gingival epithelial cells (Ca9-22) stimulated with Porphyromonas gulae lipopolysaccharide (LPS). MethodsCa9-22 cells were incubated with P. gulae LPS (10 μg/ml) with or without green tea catechins, epigallocatechin-3-gallate (EGCg), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC) (each at 50 μM), for 6 or 24 h. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay were used to determine the induction of cyclooxygenase 2 (COX2), tumor necrosis factor alpha (TNF-ɑ), interleukin 6 (IL-6), and IL-8. Furthermore, the expression of toll-like receptors (TLRs) 2 and 4 was examined using real-time PCR and western blotting analysis, and phosphorylation of the p38 and ERK1/2 was examined using western blotting analysis. ResultsAt the mRNA and protein levels, EGCg, EGC, ECG, and EC were found to significantly inhibit COX2, TNF-ɑ, IL-6, and IL-8. Furthermore, the levels of ERK1/2 and p38 phosphorylation induced by P. gulae LPS were decreased following the addition of each of the catechins, as well as TLR2 and 4 mRNA and protein. ConclusionsThese findings indicate that green tea catechins are potent inhibitors of inflammatory responses induced by P. gulae LPS, and may also be useful for prevention and/or attenuation of periodontitis.

An SCPPPQ1/LAM332 protein complex enhances the adhesion and migration of oral epithelial cells: Implications for dentogingival regeneration

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e−8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm2 to 63 dyn/cm2). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. Statement of significanceThis work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration.

Promoting the adhesion of human gingival epithelial cells on titanium surface by non-thermal atmospheric plasma irradiation.

This work aimed to study the biological behavior of human gingival epithelial cells (HGECs) irradiated by non-thermal atmospheric plasma (NTAP) on a titanium surface. Cultured HGECs (3⁃5 generations) with the best activity were digested and treated for varying times (0, 10, 20, 30, and 60 s) by NTAP and then seeded on the surface of a titanium disc. The HGECs were cultured in oral keratinocyte medium and 1% penicillin/streptomycin solution. The cells were kept in an atmosphere of 5% CO2 at 37 ℃ and incubated for different times (4, 12, 24, and 48 h; n=5). Cell counting kit-8 (CCK-8) was used to detect cell adhesion capacity. Scanning electron microscopy (SEM) was conducted to observe the morphology of cells on titanium plates. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the gene expression of adhesion-related molecules, such as Laminin α3, Integrin β4, and Plectin. The number of adhered cells increased at 0‑20 s, whereas that gradually decreased at 20⁃60 s. Therefore, cell culture at the two time points showed that HGECs adhesion reached the maximum when NATP was irradiated for 20 s. Compared with the control group, more cells in the treatment group adhered to the titanium surface at each time point (P<0.05). Cells in the treatment group showed more irregular polygons, more protrusions and pseudopods, and a larger cell diffusion area on the titanium surface than those in the control group. qRT-PCR showed that the expression levels of Laminin α3, Integrin β4, and Plectin adhesion-related genes on the titanium surface in the treatment group were higher than those in the control group at each culture time point (P<0.05). Western blot showed that the expression levels of Laminin α3, Integrin β4, and Plectin adhesion-related proteins on the titanium surface were higher in the treatment group than in the control group at 4 and 12 h. After NTAP treatment, the results showed that 20 s of treatment time could maximize the number of adhered cells on the titanium surface; change the cell adhesion morphology; and significantly upregulate the expression of adhesion-related genes and proteins of Laminin α3, Integrin β4, and Plectin. Furthermore, it could promote the biological sealing effect of HGECs on the titanium surface.

Expression of human caspase-4 in the gingival epithelium affected with periodontitis: Its involvement in Porphyromonas gingivalis-challenged gingival epithelial cells

ObjectiveImplication of human caspase-4 in periodontitis and in sensing periodontal pathogens by gingival epithelial cells (GECs) is unclear. This study aimed to determine caspase-4 and interleukin (IL)-18 expressions in gingival tissues affected with periodontitis and to investigate caspase-4 involvement in mediating innate immune responses in GECs. DesignEx vivo, caspase-4 and IL-18 expressions in gingival biopsies, obtained from healthy participants with periodontitis or clinically healthy gingiva (N = 20 each), were determined by immunohistochemistry. In vitro, caspase-4 activation in cultured GECs stimulated with Porphyromonas gingivalis or Fusobacterium nucleatum was analyzed by immunoblotting. mRNA expressions of human β-defensin-2 (hBD-2), IL-8, and IL-18 in stimulated GECs in the presence or absence of a caspase-4 inhibitor were assayed by RT-qPCR. ResultsEx vivo, compared with healthy gingival epithelium, the epithelium affected with periodontitis displayed a significant decrease in caspase-4 expression (P = 0.015), whereas IL-18 expression was significantly increased (P = 0.012). Moreover, the expression of caspase-4, but not IL-18, was found to be a predictor of periodontitis (P = 0.007). In vitro, caspase-4 was activated in cultured GECs challenged with P. gingivalis, but not F. nucleatum. mRNA upregulations of hBD-2, IL-8, and IL-18 upon P. gingivalis stimulation were significantly reduced when caspase-4 was inhibited (P < 0.05), whereas the inhibitor failed to suppress those inductions by F. nucleatum. ConclusionsCaspase-4 expression is diminished in the epithelium affected with periodontitis while that of IL-18 is enhanced. Caspase-4 activation in P. gingivalis-infected GECs upregulates the three innate immune effector molecules, suggesting a possible sensing mechanism of caspase-4 in GECs in periodontal disease pathogenesis.

RNA Sequencing Reveals the Upregulation of FOXO Signaling Pathway in Porphyromonas gingivalis Persister-Treated Human Gingival Epithelial Cells.

Porphyromonas gingivalis as the keystone periodontopathogen plays a critical role in the pathogenesis of periodontitis, and crucially accounts for inflammatory comorbidities such as cardiovascular disease and Alzheimer′s disease. We recently identified the existence of P. gingivalis persisters and revealed the unforeseen perturbation of innate response in human gingival epithelial cells (HGECs) due to these noxious persisters. Herein, RNA sequencing revealed how P. gingivalis persisters affected the expression profile of cytokine genes and related signaling pathways in HGECs. Results showed that metronidazole-treated P. gingivalis persisters (M-PgPs) impaired the innate host defense of HGECs, in a similar fashion to P. gingivalis. Notably, over one thousand differentially expressed genes were identified in HGECs treated with M-PgPs or P. gingivalis with reference to the controls. Gene Ontology and KEGG pathway analysis demonstrated significantly enriched signaling pathways, such as FOXO. Importantly, the FOXO1 inhibitor rescued the M-PgP-induced disruption of cytokine expression. This study suggests that P. gingivalis persisters may perturb innate host defense, through the upregulation of the FOXO signaling pathway. Thus, the current findings could contribute to developing new approaches to tackling P. gingivalis persisters for the effective control of periodontitis and P. gingivalis-related inflammatory comorbidities.

Analyzing Human Periodontal Soft Tissue Inflammation and Drug Responses In Vitro Using Epithelium-Capillary Interface On-a-Chip.

The gingival epithelium–capillary interface is a unique feature of periodontal soft tissue, preserving periodontal tissue homeostasis and preventing microorganism and toxic substances from entering the subepithelial tissue. However, the function of the interface is disturbed in periodontitis, and mechanisms of the breakdown of the interface are incompletely understood. To address these limitations, we developed a microfluidic epithelium–capillary barrier with a thin culture membrane (10 μm) that closely mimics the in vivo gingival epithelial barrier with an immune micro-environment. To test the validity of the fabricated gingival epithelial barrier model, epithelium–capillary interface-on-a-chip was cultured with human gingival epithelial cells (HGECs) and human vascular endothelial cells (HUVEC). Their key properties were tested using optical microscope, transepithelial/transendothelial electrical resistance (TEER), and permeability assays. The clear expression of VE-cadherin revealed the tight junctions in endothelial cells. Live/dead assays indicated a high cell viability, and the astrocytic morphology of HGE cells was confirmed by F-actin immunostaining. By the third day of cell culture, TEER levels typically exceeded in co-cultures. The resultant permeability coefficients showed a significant difference between 70 kDa and 40 kDa FITC-dextran. The expression of protein intercellular cell adhesion molecule (ICAM-1) and human beta defensin-2 (HBD2) decreased when exposed to TNF-α and LPS, but recovered with the NF-κB inhibitor treatment- Pyrrolidinedithiocarbamic acid (PDTC), indicating the stability of the fabricated chip. These results demonstrate that the developed epithelium-capillary interface system is a valid model for studying periodontal soft tissue function and drug delivery.

Dental follicle cells-derived small extracellular vesicles inhibit pathogenicity of Porphyromonas gingivalis.

It aims to explore the effect of dental follicle cells-derived small extracellular vesicles (D-sEVs) with or without lipopolysaccharides (LPS) pretreating on the pathogenicity of Porphyromonas gingivalis (P.gingivalis). The antibacterial effects of D-sEV were evaluated by measuring the growth, biofilm formation, gingipains, and type IX secretion system (T9SS) expression of P.gingivalis. And the influence of D-sEV on P.gingivalis adhesion, invasion, cytotoxicity, and host immune response was examined in gingival epithelial cells (GECs). Then P.gingivalis treated with D-sEV was applied to investigate the pathogenicity in experimental periodontitis of mice. It showed that both D-sEV and P.gingivalis LPS-pretreated D-sEV (L-D-sEV) could target P.gingivalis, inhibit their growth and biofilm formation, and hinder the attachment and invasion in GECs, therefore remarkably decreasing P.gingivalis cytotoxicity and the expression of IL-1β and IL-6 in GECs. In addition, they significantly reduced the expression of P.gingivalis virulence factors (gingipains and T9SS). In vivo, it showed that the bacteria in the gingiva were significantly decreased after sEV treatment. Meanwhile, less bone loss and fewer inflammatory cells infiltration and osteoclast formation in D-sEV and L-D-sEV groups. Both D-sEV and L-D-sEV were proven to inhibit the pathogenicity of P.gingivalis and thus prevented the development of periodontitis.

Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells.

Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This in vitro study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by Aggregatibacter actinomycetemcomitans. OBA-9 GECs were infected with A. actinomycetemcomitans strain JP2 at an MOI of 1:100 and/or co-infected with Lactobacillus acidophilus La5 (La5) or Lacticaseibacillus rhamnosus Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1β, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with A. actinomycetemcomitans or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of A. actinomycetemcomitans to GECs was 1.8 × 107 (± 1.2 × 106) cells/well in the mono-infection but reduced to 1.2 × 107 (± 1.5 × 106) in the co-infection with Lr32 and to 6 × 106 (± 1 × 106) in the co-infection with La5 (p < 0.05). GECs mono-infected with A. actinomycetemcomitans produced CXCL-8, GM-CSF, and IL-1β, and the co-infection with both probiotic strains altered this profile. While the co-infection of A. actinomycetemcomitans with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1β. The probiotics upregulated the expression of TLR2 and downregulated TLR4 in cells co-infected with A. actinomycetemcomitans. A. actinomycetemcomitans-induced the upregulation of NRLP3 was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene BCL-2 was upregulated, whereas the pro-apoptotic BAX was downregulated in cells co-infected with A. actinomycetemcomitans and the probiotics. Infection with A. actinomycetemcomitans induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in A. actinomycetemcomitans colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, L. acidophilus La5 properties suggest that this strain has a higher potential to control A. actinomycetemcomitans-associated periodontitis than L. rhamnosus Lr32.

The Regulatory Effect of Coaggregation Between Fusobacterium nucleatum and Streptococcus gordonii on the Synergistic Virulence to Human Gingival Epithelial Cells.

In subgingival plaque biofilms, Fusobacterium nucleatum is closely related to the occurrence and development of periodontitis. Streptococcus gordonii, as an accessory pathogen, can coaggregate with periodontal pathogens, facilitating the subgingival colonization of periodontal pathogens. Studies have shown that F. nucleatum can coaggregate with S. gordonii and colonize the subgingival plaque. However, most studies have focused on monocultures or coinfection of species and the potential impact of coaggregation between the two species on periodontal interactions to human gingival epithelial cells (hGECs) remains poorly understood. The present study explored the effect of coaggregation between F. nucleatum and S. gordonii on subgingival synergistic virulence to hGECs. The results showed that coaggregation inhibited the adhesion and invasion of F. nucleatum to hGECs compared with that in the F. nucleatum monoculture and coinfection group. Coaggregation and coinfection with F. nucleatum both enhanced S. gordonii adhesion to hGECs, but neither of the two groups affected S. gordonii invasion to hGECs compared with S. gordonii monoculture. The gene expression levels of TLR2 and TLR4 in hGECs in the coaggregation group were higher than those in the monoculture groups but lower than those in the coinfection group. Compared with coinfection, the coaggregation inhibited apoptosis of hGECs and promoted the secretion of the proinflammatory cytokines TNF-α and IL-6 by hGECs, showed a synergistic inflammatory effect, while coaggregation inhibited the secretion of the anti-inflammatory cytokine TGF-β1. Coaggregation enhanced the phosphorylation of p65, p38, and JNK proteins and therefore activated the NF-κB and MAPK signaling pathways. Pretreatment with a pathway antagonist/inhibitor decreased the phosphorylation levels of proteins and the secretion of TNF-α and IL-6. In conclusion, coaggregation inhibited the adhesion and invasion of F. nucleatum to hGECs. However, it enhanced the adhesion of S. gordonii to hGECs. Compared with coinfection, coaggregation inhibited the apoptosis of hGECs. The coaggregation coordinately promoted the secretion of TNF-α and IL-6 by hGECs through the TLR/NF-κB and TLR/MAPK signaling pathways while inhibiting the secretion of TGF-β1, thus aggravating the inflammatory response of hGECs.

PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose

BackgroundOxidative stress mediated by hyperglycemia damages cell-reparative processes such as mitophagy. Down-regulation of mitophagy is considered to be a susceptible factor for diabetes mellitus (DM) and its complications. However, the role of mitophagy in DM-associated periodontitis has not been fully elucidated. Apoptosis of human gingival epithelial cells (hGECs) is one of the representative events of DM-associated periodontitis. Thus, this study aimed to investigate PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy activated in the process of high glucose (HG)-induced hGECs apoptosis.MethodsFor dose–response studies, hGECs were incubated in different concentrations of glucose (5.5, 15, 25, and 50 mmol/L) for 48 h. Then, hGECs were challenged with 25 mmol/L glucose for 12 h and 48 h, respectively. Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL), caspase 9 and mitochondrial membrane potential (MMP). Subsequently, autophagy was evaluated by estimating P62, LC3 II mRNA levels, LC3 fluorescent puncta and LC3-II/I ratio. Meanwhile, the involvement of PINK1-mediated mitophagy was assessed by qRT-PCR, western blotting and immunofluorescence. Finally, hGECs were transfected with shPINK1 and analyzed by MMP, caspase 9 and annexin V-FITC apoptosis.ResultsThe number of TUNEL-positive cells and caspase 9 protein were significantly increased in cells challenged with HG (25 mmol/L) for 48 h (HG 48 h). MMP was impaired both at HG 12 h and HG 48 h, but the degree of depolarization was more serious at HG 48 h. The autophagy improved as the amount of LC3 II increased and p62 decreased in HG 12 h. During this process, HG 12 h treatment induced PINK1-mediated mitophagy. PINK1 silencing with HG 12 h resulted in MMP depolarization and cell apoptosis.ConclusionsThese results suggested that loss of the PINK1 gene may cause mitochondrial dysfunction and increase sensitivity to HG-induced apoptosis of hGECs at the early stage. PINK1 mediated mitophagy attenuates early apoptosis of gingival epithelial cells induced by high glucose.

Oral Microbiota-Host Interaction Mediated by Taste Receptors.

Taste receptors, originally identified in taste buds, function as the periphery receptors for taste stimuli and play an important role in food choice. Cohort studies have revealed that single nucleotide polymorphisms of taste receptors such as T1R1, T1R2, T2R38 are associated with susceptibility to oral diseases like dental caries. Recent studies have demonstrated the wide expression of taste receptors in various tissues, including intestinal epithelia, respiratory tract, and gingiva, with an emerging role of participating in the interaction between mucosa surface and microorganisms via monitoring a wide range of metabolites. On the one hand, individuals with different oral microbiomes exhibited varied taste sensitivity, suggesting a potential impact of the oral microbiota composition on taste receptor function. On the other hand, animal studies and in vitro studies have uncovered that a variety of oral cells expressing taste receptors such as gingival solitary chemosensory cells, gingival epithelial cells (GECs), and gingival fibroblasts can detect bacterial signals through bitter taste receptors to trigger host innate immune responses, thus regulating oral microbial homeostasis. This review focuses on how taste receptors, particularly bitter and sweet taste receptors, mediate the oral microbiota-host interaction as well as impact the occurrence and development of oral diseases. Further studies delineating the role of taste receptors in mediating oral microbiota-host interaction will advance our knowledge in oral ecological homeostasis establishment, providing a novel paradigm and treatment target for the better management of dental infectious diseases.