Journal of Oral Biosciences最新文献

Objectives: Osteoporosis is a systemic bone metabolism disorder characterized by decreased bone mass and strength. Osteoclasts (OCs) are giant multinucleated cells that regulate bone homeostasis by degrading bone matrix. Excessive OC differentiation and activity can lead to serious bone metabolic disorders including osteoporosis. Current treatments, including antiresorptive drugs, exert considerable adverse effects, including jaw osteonecrosis. Herbal medicines, such as Ninjinyoeito (NYT), may also offer efficacy, but with fewer adverse effects. In this study, we investigated NYT's effects on osteoclastogenesis.

Methods: Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were performed to examine NYT's effects on OC differentiation and function. OC-related gene expression at mRNA and protein levels was investigated to confirm NYT's inhibitory action against osteoclastogenesis. We also demonstrated involvement of signaling pathways mediated by IκBα and mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38] and showed nuclear translocation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and nuclear factor kappa B (NF-κB) p65 during osteoclastogenesis.

Results: TRAP staining and bone resorption assays confirmed that NYT significantly inhibited OC differentiation and function. Western blot and RT-PCR results showed that NYT ameliorated osteoclastogenesis by suppressing mRNA and protein level expression of OC-related genes. Moreover, blots and immunocytochemistry (ICC) data clarified that NYT abrogates signaling pathways mediated by IκBα and MAPK (ERK, JNK, p38), and demonstrated nuclear translocation of NFATc1 and NF-κB p65 during OC differentiation.

Conclusions: These findings suggest NYT is an alternative therapeutic candidate for treating osteoporosis.

Objective: Rhythmic jaw muscle activities (RJMAs) occur during rapid eye movement (REM) sleep in humans and animals even though motoneurons are inhibited. The present study compared the characteristics of jaw muscle activities induced by electrical microstimulations of the corticobulbar tract (CT) during REM sleep with those during wakefulness and non-REM sleep.

Methods: Eleven guinea pigs were surgically prepared for polygraphic recordings with the implantation of a stimulating electrode. Long- and short-train repetitive electrical microstimulations were applied to the CT under freely moving conditions. The response rate, latency, burst amplitude, and cycle length in the digastric muscle were calculated and cortical and cardiac activities were quantified.

Results: Long-train microstimulations induced RJMAs in the digastric muscle followed by masseter muscle activity during wakefulness and non-REM sleep and only induced rhythmic digastric muscle activity during REM sleep. The response rate of RJMAs and the burst amplitude of digastric muscles were significantly lower during REM sleep than during wakefulness and non-REM sleep. However, response latency did not significantly differ between REM sleep and wakefulness. Transient cortical and cardiac changes were associated with RJMAs induced during non-REM sleep, but not during REM sleep. Short-train microstimulations induced a short-latency digastric response, the amplitude of which was significantly lower during REM sleep than during non-REM sleep and wakefulness.

Conclusions: These results suggest that the masticatory CPG was activated by electrical CT stimulations independently of the motoneuron inhibitory system during REM sleep.

Objective: Potassium nitrate (KNO₃) suppresses nociception induced by dental hypersensitivity (HYS). We aimed to examine the effects of KNO₃ on the neural activity of the trigeminal spinal subnucleus caudalis (Vc) in HYS model rats.

Methods: KNO₃ or vehicle was applied to the exposed dentin of HYS rats for 3 days. c-Fos expression and neuronal activity in the Vc after acetone treatment for cold stimulation were examined to evaluate the effects of KNO₃ application on dentin.

Results: The number of c-Fos-immunoreactive cells in the Vc was lower in the group that received KNO₃ (KNO₃ group) than in the group that received vehicle (control group). Spike firing of Vc neurons in response to cold stimulation of the dentin was recorded before and after KNO₃ application to the cavity, and the increased neural activity was effectively suppressed by KNO₃ application. Scanning electron microscopy revealed that the dentin tubules were not occluded by deposits in any of the groups.

Conclusions: KNO₃-induced suppression of Vc neuronal activity does not involve physical occlusion of the dentin tubules but likely involves suppression of Aδ or C-fiber activities in the tooth pulp, resulting in the suppression of Vc neuronal activities.

Objective: To study the effects of losartan, an angiotensin II receptor blocker, in the SCC4 and SCC25 human tongue squamous cell carcinoma cell lines.

Methods: Cell proliferation was measured by MTS/PMS activity and trypan blue exclusion assays. The levels of the cell proliferation marker, cyclin D1, were analyzed by western blotting. Apoptosis was assessed by caspase-3 activation and Annexin V-FITC/propidium iodide double staining. Activation of epidermal growth factor receptor (EGFR) and ERK1/2 was validated by western blotting.

Results: Moderate concentrations of losartan enhanced the proliferation of SCC4 and SCC25 cells. However, high losartan concentrations induced apoptosis in SCC4 cells. Losartan activated the EGFR/ERK1/2/cyclin D1 signaling axis, which in turn promoted cell proliferation. Afatinib (EGFR inhibitor) and U0126 (ERK1/2 inhibitor) abolished losartan-induced cell proliferation. In contrast, UC2288 (p21 inhibitor) enhanced it.

Conclusions: Losartan exhibited dual effects on tongue squamous cell carcinoma cells. Moderate losartan concentrations facilitated cell proliferation, whereas high concentrations induced cytotoxicity in tongue carcinoma cells.

Objectives: Interleukin (IL)-2 production by mouse spleen cells stimulated with an anti-CD3 antibody is significantly enhanced by caffeic acid phenethyl ester (CAPE), a major constituent of Chinese propolis (CP). In this study, we evaluated the functional significance of IL-2 in CAPE-treated activated spleen cells.

Methods: Mouse spleen cells were stimulated with an anti-CD3 monoclonal antibody in the presence of CAPE. Cytokine production was examined using an enzyme-linked immunosorbent assay (ELISA). Messenger RNA level expression was examined via reverse transcription quantitative polymerase chain reaction (RT-PCR). IL-2 function was assessed using IL-2 and a neutralizing antibody. Spleen cell subsets were identified and characterized using flow cytometry.

Results: CAPE treatment of anti-CD3 antibody-stimulated spleen cells reduced IFN-γ production, then enhanced IL-2 production, followed by enhancement of IL-4 and IL-10 production. The Th2 cytokine production enhancing effects of CAPE were completely abolished by addition of an anti-IL-2 neutralizing antibody. In the absence of CAPE, exogenously added IL-2 could enhance IL-4 production to a lesser degree, but did not stimulate IL-10 production, in stimulated spleen cells. Interestingly, CAPE significantly reduced the proportions of CD4⁺ and CD8⁺ cells, and increased those of CD4^-CD8^- cells among anti-CD3 stimulated spleen cells, in the presence or absence of anti-IL-2 neutralizing antibody treatment.

Conclusions: CAPE reduced IFN-γ production, then enhanced IL-4 and IL-10 production via the activity of specifically elevated IL-2 in stimulated spleen cells. CAPE exerted these effects in a CD4^- CD8^- cell specific manner.

Objectives: Signal transducer and activator of transcription 3 (STAT3) is one of the key proliferation mechanism-related proteins that helps in oral squamous cell carcinoma (OSCC) progression. Immune evasion by STAT3 is mediated by the JAK2/STAT3/PDL1 signaling axis. Based on previous findings, we hypothesized that STAT3-binding partners participate in the inhibition of anti-tumor activity in OSCC.

Methods: A 3D cancer-immune co-culture model was constructed using oral cancer cell lines SCC4, SCC9, SCC25, and CAL27 and normal oral cell line OKF6. The cells were co-cultured with natural killer (NK-92) and Jurkat cells. The target protein STAT3 was chosen based on SWATH data, and co-immunoprecipitation (Co-IP)-based proteomics was conducted. The Co-IP LC-MS/MS output was analyzed to determine the protein interaction network, gene ontology, pathway analysis, and protein cluster annotation.

Results: STAT3 in oral cancer cell lines interacts with the epidermal growth factor receptor (EGFR) and other proteins that participate in proliferation and immune mechanisms. Proteome analysis showed that some STAT3-binding proteins found in this study are known immune system regulators.

Conclusion: Overall, STAT3 interactive proteins regulate the immune system in oral squamous cell carcinoma cells.

Objectives: This study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms.

Methods: The role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632.

Results: Treatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 indicated that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance.

Conclusions: The TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

Objectives: Porphyromonas gulae is a major causative agent of periodontal disease in companion animals that possesses various virulence factors, including fimbriae, lipopolysaccharides, and proteases. P. gulae fimbriae are classified into three genotypes (A, B, and C) based on their nucleotide sequences. Type C fimbrial isolates have been reported to be more virulent than other fimA types, suggesting that different fimA types may aid in the regulation of periodontal pathogenesis. Detailed findings regarding the ability of P. gulae to form biofilms have yet to be reported. Here, we investigated the contributions of fimbrial genotypes in P. gulae biofilm formation.

Methods: P. gulae and P. gingivalis biofilms were generated on plates and analyzed using confocal laser microscopy. Additionally, the biofilms formed were assessed by staining with crystal violet. Furthermore, the physical strength of P. gulae biofilms was examined by ultrasonication.

Results: Biofilms formed by P. gulae type C were denser than those formed by types A and B. Moreover, the amount of biofilm formed by type C strains was significantly greater than that formed by type A and B strains, which was similar to the biofilms formed by P. gingivalis with type II fimbriae. Additionally, the physical strength of the type C biofilm was significantly greater than that of the other strains.

Conclusions: These results suggest that FimA variation may coordinate for biofilm formation. This is the first report on the observation and characterization of P. gulae biofilm formation.

Objectives: Porphyromonas gingivalis is a pathogenic bacterium that causes periodontitis and dental pulp infection. Autophagy is a potential mechanism involved in inflammatory disease. This study established an in vitro model of P. gingivalis intracellular infection in human dental pulp fibroblasts (HDPFs) to investigate the effects of live P. gingivalis on HDPFs.

Methods: Morphological and quantification techniques such as fluorescence microscopy, transmission electron microscopy (TEM), indirect immunofluorescence analysis, enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (PCR), and western blotting were used in this study.

Results: After cell invasion, P. gingivalis is mainly localized in the cytoplasm and lysosomes. Additionally, P. gingivalis activates autophagy in HDPFs by upregulating the expression of autophagy-related gene Beclin-1, activate autophagy-related gene12 (ATG12), and microtubule-associated protein light chain 3 (LC3). Furthermore, the invasion of P. gingivalis leads to increased phosphorylation of PI3K, Akt, and mTOR with the addition of rapamycin, whereas the addition of wortmannin decreased phosphorylation. This invasion of P. gingivalis, also causes an inflammatory response, leading to the upregulation of IL-1β, IL-6, and TNF-α. Rapamycin helps decrease levels of pro-inflammatory cytokines, but the addition of wortmannin increases them. These results show that the invasion of P. gingivalis can cause excessive inflammation and promote the autophagy of HDPFs, which is regulated by PI3K/Akt/mTOR.

Conclusions: P. gingivalis escapes the immune system by inducing autophagy in the host cells, causing excessive inflammation. P. gingivalis regulates autophagy in HDPFs through the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway.

Objectives: Details about salivary gland tumor histogenesis remain unknown. Here, we established a newly generated murine salivary gland tumor model that could overexpress pleomorphic adenoma gene 1 (PLAG1) and attempted to clarify the events that occur during the early phase of salivary gland tumor histogenesis.

Methods: Salivary gland tumors were generated using murine models (Sox9IRES-CreERT2; ROSA26-PLAG1). Lineage tracing of Sox9-expressing cells was performed using Sox9IRES-CreERT2; ROSA26-tdTomato mice, which were generated by crossing Sox9^CreERT2/- and ROSA26-tdTomato mice (expressing the tdTomato fluorescent protein). Organ-cultured embryonic salivary glands from the murine model were morphologically analyzed, and mRNA sequencing was conducted two days after tumor induction for gene enrichment and functional annotation analysis.

Results: Salivary gland tumors exhibited epithelial features with acinar-like structures because of gene rearrangements in the luminal cells. Structural disturbances in the duct-acinar unit of the salivary gland were observed and cancer-related pathways were enriched among the differentially upregulated genes in the early phase of tumor induction in an organ-cultured embryonic salivary gland tumor model.

Conclusions: The newly generated murine salivary gland tumor model may show that the tumorization of luminal stem/progenitor cells can result in the development of salivary gland tumors comprising only luminal cells.