Effects of Laurus nobilis Leaf Extract (LAURESH®) on Oral and Gut Microbiota Diversity in Mice.

BACKGROUND/AIM: The leaves of Laurus nobilis have been used for culinary purposes for many years and have recently been shown to have beneficial effects on human health by altering microbiota composition. However, the effects of L. nobilis on the diversity of microbiomes in the oral cavity and gut remain unknown. Therefore, in this study, we examined the effects of an extract of L. nobilis on the diversity of microbiomes in the oral cavity and gut in mice. MATERIALS AND METHODS: C57BL/6J mice were randomly divided into two groups and fed a standard diet (SD) and a standard diet containing 5% LAURESH®, a laurel extract (SDL). After 10 weeks, oral swabs and fecal samples were collected. The bacterial DNA extracted from the oral swabs and feces was used for microbiota analysis using 16S rRNA sequencing. The sequencing data were analyzed using the Quantitative Insights into Microbial Ecology 2 in the DADA2 pipeline and 16S rRNA database. RESULTS: The α-diversity of the oral microbiome was significantly greater in the SDL group than in the SD group. The ß-diversity of the oral microbiome was also significantly different between the groups. Moreover, the taxonomic abundance analysis showed that five bacteria in the gut were significantly different among the groups. Furthermore, the SDL diet increased the abundance of beneficial gut bacteria, such as Akkermansia sp. CONCLUSION: Increased diversity of the oral microbiome and proportion of Akkermansia sp. in the gut microbiome induced by L. nobilis consumption may benefit oral and gut health.

Transcriptomic analysis of the submandibular gland under psychological stress condition.

BACKGROUND: Psychological stress is associated with changes in salivary flow and composition. However, studies to show the effect of psychological stress on the transcriptome of the salivary gland are limited. This study aims to perform a transcriptomic analysis of the submandibular gland under psychological stress using a chronic restraint stress model of rats. METHODS: Sprague-Dawley rats were divided into stress groups and control groups. Psychological stress was induced in the stress group rats by enclosing them in a plastic tube for 4 h daily over 6 weeks. RNA sequencing was performed on RNA extracted from the submandibular gland. The differentially expressed genes were identified, and the genes of interest were further validated using qRT-PCR, immunofluorescence, and western blot. RESULTS: A comparison between control and stress groups showed 45 differentially expressed genes. The top five altered genes in RNA sequencing data showed similar gene expression in qRT-PCR validation. The most downregulated gene in the stress group, FosB, was a gene of interest and was further validated for its protein-level expression using immunofluorescence and western blot. The genesets for gene ontology cellular component, molecular function, and KEGG showed that pathways related to ribosome biosynthesis and function were downregulated in the stress group compared to the control. CONCLUSION: Psychological stress showed transcriptomic alteration in the submandibular gland. The findings may be important in understanding stress-related oral diseases.

Ingenuity pathway analysis of gingival epithelial cells stimulated with estradiol and progesterone.

OBJECTIVE: Periodontal disease is a risk factor for preterm delivery, and elevated female hormone levels during pregnancy promote hormone-dependent periodontopathogenic bacterial growth and gingivitis. Although the saliva of pregnant women contains female hormones at elevated levels, their effects on the gingiva are poorly understood. Therefore, in this study, we investigated the effects of estradiol and progesterone stimulation on gingival epithelial cells via ingenuity pathway analysis. METHODS: Human gingival epithelial progenitors were cultured in a CnT-Prime medium; 17ß-estradiol (E2) and progesterone (P4) were used as the reagents. Cells treated with dimethyl sulfoxide alone were used as the control group. Cells in the control and experimental groups were incubated for 12 h. RNA was extracted from the cultured cells, RNA-Seq was performed, and pathway analysis was conducted. RESULTS: Differentially expressed genes were detected for 699 (over 2-fold increase) and 348 (decrease) genes in group E2 and for 1448 (increase) and 924 (decrease) genes in group P4 compared with those in the control group (FDR <0.05, n = 4). The z-scores of the pathways suggest that E2 and P4 increased the activity of the wound healing signaling pathway. The activation of this pathway was higher in the E2 and P4 groups than that in the control group. CONCLUSIONS: The results of this study suggest that estradiol and progesterone may affect gingival homeostasis and wound healing.

Effects of prolonged stimulation with heated tobacco products (Ploom TECH+ ) on gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Heated tobacco products have recently become commercially available. These products, as well as combustible cigarettes, produce aerosols; the risk of various diseases associated with heated tobacco products may be the same or higher than that with combustible cigarettes. In this study, we examined the effect of Ploom TECH+ extract on gingival epithelial cells. METHODS: Tobacco leaves from Ploom TECH+ tobacco capsules and water were mixed and heated; the supernatant subsequently collected was the heated tobacco product (HTP; control: HTP not added). Normal human gingival epithelial progenitors were cultured alternately with or without HTP for a total of 1 month. Subsequently, RNA, DNA, and proteins were isolated from these samples and comprehensively analyzed using RNA sequencing (RNA-seq), reduced representation bisulfite sequencing (RRBS), and western blotting, respectively. RESULTS: RNA-seq revealed that 284 genes showed a twofold increase and 145 genes showed a twofold decrease in gene expression. A heat map showed genetic differences between the control and HTP groups. A principal component analysis plot showed a clear genetic distribution between the control and HTP. Gene Ontology (GO) analysis showed that genes related to seven GO terms, including cornification and keratinization, were induced by long-term HTP stimulation. By contrast, GO pathways with a significant decrease in component expression were not detected. RRBS revealed that CpG island methylation increased more than twofold in 158 genes and decreased to less than twofold in 171 genes. Methylation of these CpG islands was not correlated with changes in gene expression levels. HTP treatment increased S100A7 expression. CONCLUSION: Long-term HTP stimulation affected epithelial differentiation and keratinization of gingival epithelial cells. Thus, habitual use of Ploom TECH+ may be a risk factor for tobacco-related oral mucosal diseases.

Systemic Administration of Lipopolysaccharide from Porphyromonas gingivalis Decreases Neprilysin Expression in the Mouse Hippocampus.

BACKGROUND/AIM: Alzheimer's disease is the most common type of neurodegenerative disorder in elderly individuals worldwide. Increasing evidence suggests that periodontal diseases are involved in the pathogenesis of Alzheimer's disease, and an association between periodontitis and amyloid-ß deposition in elderly individuals has been demonstrated. The aim of the present study was to examine the effects of systemic administration of Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS) on neprilysin expression in the hippocampus of adult and senescence-accelerated mice. MATERIALS AND METHODS: PG-LPS diluted in saline was intraperitoneally administered to male C57BL/6J and senescence-accelerated mouse prone 8 (SAMP8) mice at a dose of 5 mg/kg every 3 days for 3 months. Both C57BL/6J and SAMP8 mice administered saline without PG-LPS comprised the control group. The mRNA expression levels of neprilysin and interleukin (IL)-10 were evaluated using the quantitative reverse transcriptase-polymerase chain reaction. The protein levels of neprilysin were assessed using western blotting. Sections of the brain tissues were immunohistochemically stained. RESULTS: The serum IL-10 concentration significantly increased in both mouse strains after stimulation with PG-LPS. Neprilysin expression at both mRNA and protein levels was significantly lower in the SAMP8 PG-LPS group than those in the SAMP8 control group; however, they did not differ in PG-LPS-treated or non-treated C57BL/6J mice. Additionally, the immunofluorescence intensity of neprilysin in the CA3 region of the hippocampus in PG-LPS-treated SAMP8 mice was significantly lower than that in control SAMP8 mice. CONCLUSION: Porphyromonas gingivalis may reduce the expression of neprilysin in elderly individuals and thus increase amyloid-ß deposition.

Effect of psychological stress on the oral-gut microbiota and the potential oral-gut-brain axis.

Psychological stress in a chronic course is implicated in various diseases, such as coronary artery disease, diabetes, ulcerative colitis, and psychosomatic pain disorders. Commensal microbiota in the host tissues interact with each other and maintain overall health. Oral and gut microbiomes are considered as the most ecologically rich and taxonomically diverse microbiota communities in humans. The effects of psychological stress on the gut microbiome have been well documented, and the interaction is commonly referred as the microbiota-gut-brain axis. Like the gut microbiome, the oral microbiome contributes to maintaining both local and systemic health. Although the effects of psychological stress on the oral microbiome have been studied, comprehensive knowledge about the oral-brain axis is lacking. The oral cavity and gut can communicate with each other through the microbiota. Three-way interactions within the oral-gut-brain microbiota might exist in patients with psychological stress and disorders. The effect of psychological stress on the gut and oral microbiomes, and the potential interactions within the oral-gut-brain axis are discussed in this review.

The effect of Porphyromonas gingivalis on the gut microbiome of mice in relation to aging.

BACKGROUND AND OBJECTIVE: The translocation of oral bacteria, including Porphyromonas gingivalis, to the gut has been shown to alter gut microbiome. However, the effect of P. gingivalis on gut microbiome in relation to aging has not been demonstrated. We hypothesize that P. gingivalis has more detrimental effect on gut environment with increased age. The objective of this study is to investigate the effect of P. gingivalis on gut environment using aged mice. MATERIALS AND METHODS: C57BL/6J mice aged 4 weeks (young) or 76 weeks (old) were divided into four groups: control-young, control-old, P. gingivalis-administered young, and P. gingivalis-administered old. P. gingivalis was orally administered thrice weekly for 5 weeks. At 30 days after the last P. gingivalis administration, 16S rRNA sequencing was performed to study the gut microbiome. The mRNA and protein expression of intestinal junctional barrier molecules and the levels of the inflammatory cytokines IL-1ß and TNF-α in the serum were evaluated. RESULTS: Significant differences in the gut microbiomes between the groups, in terms of taxonomic abundance, bacterial diversity, and predicted metagenome function, were observed. A significant reduction in the alpha diversity and in the abundance of beneficial bacteria, such as Akkermansia and Clostridiaceae, in the P. gingivalis-administered old mice was observed. The mRNA and protein levels of Claudin-1 and Claudin-2 in the intestine were significantly elevated, while E-cadherin was significantly downregulated in the P. gingivalis-administered old mice, as were the serum levels of IL-1ß and TNF-α. CONCLUSION: The effect of P. gingivalis on the gut environment is more pronounced in old mice than in young mice.

Induction of Periodontal Ligament-like Cells by Coculture of Dental Pulp Cells, Dedifferentiated Cells Generated from Epithelial Cell Rests of Malassez, and Umbilical Vein Endothelial Cells.

INTRODUCTION: Apart from the epithelial cell rests of Malassez (ERMs), dental pulp (DP) contains the same types of mesenchymal cells as the periodontal ligament (PDL). ERMs may affect the characteristics of the mesenchymal cells in the PDL. The aim of this study was to examine whether DP cells cultured with ERMs and human umbilical vein endothelial cells (HUVECs) could transform into PDL-like cells. METHODS: Progenitor-dedifferentiated into stem-like cells (Pro-DSLCs) were produced by the induction of ERMs with 5-Azacytidine and valproic acid. DP cells were cultured in mesenchymal stem cell medium for 1 week under the following conditions: DP cells alone (controls); PDL cells alone; coculture of DP cells and ERMs (DP + ERM) or Pro-DSLCs (DP + Pro-DSLC); and coculture of DP cells, HUVECs, and ERMs (DP + ERM + HUVEC) or Pro-DSLCs (DP + Pro-DSLC + HUVEC). Quantitative real-time reverse transcription polymerase chain reaction, quantitative methylation-specific polymerase chain reaction, and flow cytometry were performed. RESULTS: The expression levels of PDL-related markers Msx1, Msx2, Ncam1, Postn, and S100a4 and mesenchymal stem cell-positive markers Cd29, Cd90, and Cd105 were significantly higher in the PDL cells and DP + Pro-DSLC + HUVEC cultures than in the controls (P < .05). The DNA methylation levels of Msx1 and Cd29 in the PDL cells and the DP + Pro-DSLC + HUVEC culture were significantly lower than in the controls (P < .01). We found a significant increase in the number of cells stained with MSX1 (P < .05) and CD29 (P < .01) in the DP + Pro-DSLC + HUVEC culture than in the controls. CONCLUSIONS: Coculture of DP cells with Pro-DSLCs and HUVECs induced their transformation into PDL-like cells. This method may prove to be useful for periodontal regeneration via tissue engineering.

Effect of Systemic Administration of Amitriptyline on Oral Microbes in Rats.

BACKGROUND/AIM: Amitriptyline is a major tricyclic antidepressant that is also used to relieve chronic orofacial pain. Recently, alterations in gut flora due to various antidepressants have been demonstrated. However, it remains unknown how antidepressants affect the oral environment, including microbiota and innate immunity. The aim of this study was to investigate the effects of amitriptyline on oral microflora and antimicrobial peptides. MATERIALS AND METHODS: Sprague-Dawley rats were intraperitoneally injected with amitriptyline for 2 weeks. The DNA extracted from the oral swabs were used to perform 16SrRNA sequencing to evaluate the oral microbiome. Quantitative RT-PCR was performed to evaluate the mRNA levels of antimicrobial peptides in the buccal tissues. RESULTS: No significant differences in salivary flow rates were observed between the amitriptyline and control groups. Taxonomic analysis showed significant alterations in bacteria such as Corynebacterium, Rothia, and Porphyromonas due to amitriptyline administration. The beta diversity showed significant differences between the amitriptyline and control groups. Additionally, the predicted metagenome functions were significantly different between the two groups. The mRNA expression levels of antimicrobial peptides in the amitriptyline group were significantly higher as compared to controls. CONCLUSION: Systemic administration of amitriptyline may affect the oral environment, including oral microbes and innate immunity in the oral mucosa.

Proteomic and microbiota analyses of the oral cavity during psychological stress.

Psychological stress is associated with various oral diseases such as aphthous stomatitis, oral lichen planus, taste disturbances and glossodynia. However, the underlying mechanism is still unknown. The aim of this study was to determine the effect of psychological stress on salivary proteins and the oral microbiota in a rat model of chronic restraint stress. Six-week-old Sprague Dawley rats were subjected to restraint stress for four hours daily for 1 month. The behavior, weights of the adrenal glands, and serum corticosterone levels were evaluated as stress markers. Proteomic analysis of the saliva was performed using two-dimensional gel electrophoresis followed by mass spectrometry and Western blotting. Analysis of the oral microbiota was performed via 16S rRNA next-generation sequencing. The low mean body weights, lower number of entries and time spent in the open arm of elevated plus maze, high adrenal gland/body weight ratios, and high serum corticosterone levels confirmed the high levels of stress in the stress group of rats compared to the controls. Thirty-three protein spots were found to be significantly altered between the two groups. After silver staining, seven visible spots were subjected for mass spectrometry, and the expression levels of the two most significantly altered proteins, BPI fold containing family A member 2 and von Ebner's gland protein, were confirmed by Western blotting. 16S rRNA sequencing analysis revealed a significant reduction in alpha diversity in the stress group compared to the controls. The abundances of oral bacteria, such as Facklamia and Corynebacterium, were significantly altered between the two groups. Additionally, analysis with PICRUSt2 software predicted 37 different functional pathways to be altered between the groups. In conclusion, the present study identified altered salivary proteins and oral microbiota due to psychological stress. These findings might aid in understanding the pathogenesis of stress-related oral diseases.

Alterations in the oral microbiome of individuals with a healthy oral environment following COVID-19 vaccination.

BACKGROUND: Several reports suggest that the microbiome of the digestive system affects vaccine efficacy and that the severity of coronavirus disease (COVID-19) is associated with decreased diversity of the oral and/or intestinal microbiome. The present study examined the effects of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine on the oral microbiome. METHODS: Forty healthy Japanese oral healthcare personnel were recruited, and unstimulated saliva was collected before vaccination, after the 1st vaccination, and after the 2nd vaccination. Genomic DNA was extracted from saliva samples, and PCR amplicons of the 16S rRNA gene were analyzed using next-generation sequencing. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. In addition, alterations in microbial function were assessed using PICRUSt2. RESULTS: SARS-CoV-2 mRNA vaccination significantly increased oral bacterial diversity and significantly decreased the proportion of the genus Bacteroides. CONCLUSIONS: The SARS-CoV-2 mRNA vaccine alters the oral microbiome; accordingly, vaccination might have beneficial effects on oral health.

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis.

Gut dysbiosis induces 'leaky gut,' a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium which causes periodontal tissue breakdown, and often enters the systemic blood flow. Oral administration of P. gingivalis induced gut dysbiosis in mice model, but no systemic administration of P. gingivalis has been reported thus far. In the present study, we investigated the effect of P. gingivalis-derived lipopolysaccharide (Pg-LPS) on the intestinal flora of our established mouse model. Eight-week-old C57BL/6J mice were intraperitoneally administered Pg-LPS. Three months later, DNA was extracted from stool, and RNA from the small and large intestines. After euthanizing the mice, pathological sections of the intestinal tract were prepared and stained with hematoxylin and eosin (H&E). Tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 expression levels were evaluated using quantitative PCR. 16S rRNA gene PCR amplicon analysis data were acquired using NGS. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. Furthermore, alterations in microbial function were performed by PICRUSt2. No significant inflammatory changes were observed in the H&E. No significant differences in the mRNA levels of IL-1ß, IL-6, and TNF-α were observed between the groups. Pg-LPS administration decreased the abundance of Allobacterium in the gut. A predictive metagenomic analysis by PICRUSt2 and STAMP showed that 47 pathways increased and 17 pathways decreased after Pg-LPS administration. Systemic application of periodontal pathogens may cause changes in the intestinal flora which may affect the physiological functions of the intestinal tract.

Bee Pollen Diet Alters the Bacterial Flora and Antimicrobial Peptides in the Oral Cavities of Mice.

BACKGROUND: Bee pollen (BP) has a broad range of beneficial effects on health. The aim of this study was to examine the effect of BP on the oral environment, including the microbiome and antimicrobial peptides. METHODS: C57BL/6J mice were randomly divided into two groups: control and BP. The BP group was fed with a 5% BP diet for 1 month. Swabs from the oral and buccal mucosa and samples of the intestinal stool were collected. Genomic DNA was extracted and the microbiome was analyzed via 16S rRNA sequencing. RESULTS: BP inhibited the growth of P. gingivalis at a concentration of >2.5%. The metagenomic study showed that the abundance of genus Lactococcus was significantly elevated in the oral and intestinal microbiomes of the BP group when compared to those of the control group. Significant alterations in alpha and beta diversity were observed between the oral microbiomes of the two groups. The mRNA levels of beta-defensin-2 and -3 were significantly upregulated in the buccal mucosa of the BP group. CONCLUSION: A BP diet may have a beneficial effect on oral and systemic health by modulating the bacterial flora and antimicrobial peptides of the oral cavity. Further investigations are needed to clarify how a BP diet affects overall human health.

Direct reprogramming of epithelial cell rests of malassez into mesenchymal-like cells by epigenetic agents.

The DNA demethylating agent, 5-Azacytidine (5Aza), and histone deacetylase inhibitor, valproic acid (Vpa), can improve the reprogramming efficiencies of pluripotent cells. This study aimed to examine the roles of 5Aza and Vpa in the dedifferentiation of epithelial cell rests of Malassez (ERM) into stem-like cells. Additionally, the ability of stem-like cells to differentiate into mesenchymal cells was evaluated. ERM was cultured in embryonic stem cell medium (ESCM) with 1 µM of 5Aza, or 2 mM of Vpa, or a combination of 5Aza and Vpa. The cells stimulated with both 5Aza and Vpa were named as progenitor-dedifferentiated into stem-like cells (Pro-DSLCs). The Pro-DSLCs cultured in ESCM alone for another week were named as DSLCs. The stem cell markers were significantly higher in the DSLCs than the controls (no additions). The mRNA and protein levels of the endothelial, mesenchymal stem, and osteogenic cell markers were significantly higher in the Pro-DSLCs and DSLCs than the controls. The combination of a demethylating agent and a deacetylated inhibitor induced the dedifferentiation of ERM into DSLCs. The Pro-DSLCs derived from ERM can be directly reprogrammed into mesenchymal-like cells without dedifferentiation into stem-like cells. Isolated ERM treated with epigenetic agents may be used for periodontal regeneration.

Analysis of DNA methylation of E-cadherin and p16ink4a in oral lichen planus/oral lichenoid lesions.

OBJECTIVES: Epigenetic phenomena are changes in gene expression not involving the DNA sequence. DNA methylation is a major occurrence underlying epigenetic changes in human cells. Although aberrant DNA methylation is well documented in malignant lesions, limited information has been shown on the involvement of DNA methylation in oral lichen planus and oral lichenoid lesions (OLP). The present study aimed to investigate DNA methylation of E-cadherin and p16 in OLP, and compare the findings with those in non-inflamed gingiva (Non), radicular cyst (RC), and oral squamous cell carcinoma (SCC). MATERIALS AND METHODS: Paraffin-embedded surgical biopsy specimens were sliced, DNA was extracted, bisulfite treatment was applied, and methylation-specific polymerase chain reaction was performed. Immunohistochemistry was performed to observe the relative expression patterns of these genes. RESULTS: E-cadherin was hypermethylated in OLP (p < 0.01), SCC (p < 0.01), and RC (p < 0.05), when compared with Non; DNA hypermethylation was confirmed in OLP and SCC when compared to Non and RC. Hypermethylation of p16ink4a was observed only in SCC (p < 0.01). CONCLUSION: DNA methylation levels of E-cadherin and p16ink4a were significantly higher in OLP than in normal tissues, and may be associated with the pathogenesis and progression of the disease.

Aberrant expression of DUSP4 is a specific phenomenon in betel quid-related oral cancer.

Oral cancer due to betel quid chewing habit is very common in South Asian countries. We attempted to detect the presence of a novel gene in epithelial cells stimulated with arecoline, a main component of betel quid. Human gingival epithelial progenitors were cultured and treated with a 3-day alternating regimen with/without 50 µg/ml arecoline for 1 month. DNA microarray and methylation arrays were analyzed to identify the candidate genes. Immunohistochemical staining was performed in the tissue samples. Genome-wide analyses, quantitative reverse transcription PCR and quantitative methylation-specific PCR revealed DUSP4 as the most significant and promising gene. The methylation levels of DUSP4 were significantly higher in the betel quid-related oral squamous cell carcinoma (OSCC) than those in the non-related OSCC and controls (Mann-Whitney U test, p < 0.05). The number of DUSP4 immunopositive cells in betel quid-related OSCC was significantly higher than those from the non-chewing patients and the controls (p < 0.05). Hypermethylation of DUSP4 may be considered as a specific event in betel quid-related oral cancer.

P. gingivalis Lipopolysaccharide Stimulates the Upregulated Expression of the Pancreatic Cancer-Related Genes Regenerating Islet-Derived 3 A/G in Mouse Pancreas.

Although epidemiological studies have shown a relationship between periodontal disease and pancreatic cancer, the molecular mechanisms involved remain unclear. In this study, the effects of systemic administration of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on gene expression were comprehensively explored in mouse pancreas that did not demonstrate any signs of inflammation. PG-LPS was prepared in physiological saline and intraperitoneally administered to male mice at a concentration of 5 mg/kg every 3 days for 1 month. After extracting total RNA from the excised mice pancreas, a comprehensive DNA microarray analysis of gene expression was performed. Tissue specimens were also subjected to hematoxylin-eosin staining and immunohistochemistry using anti-regenerating islet-derived 3A and G (Reg3A/G) antibody. ImageJ software was used to quantify the area of Reg3A/G positive cells in pancreatic islets by binarizing image date followed by area extraction. The results were compared using Mann-Whitney U test. Data are presented as mean ± standard deviation (SD) with p < 0.05 considered as significant. Reg3G, a gene related to pancreatic cancer, was one of the 10 genes with the highest levels of expression in the pancreas stimulated with PG-LPS. The comprehensive analysis revealed a 73-fold increase in Reg3G expression level in the PG-LPS group when compared with the control group; in addition, the expression level of Reg3A was increased by 11-fold in the PG-LPS group. Image analysis showed that the ratio of Reg3A/G positive cells was higher in the PG-LPS group than the control. Immunostaining showed the presence of Reg3A/G-positive cells in the alpha-cell equivalent areas around the islets of Langerhans in the PG-LPS group. These results support the notion that periodontal disease may be a risk factor for pancreatic cancer.

Involvement of RNase 7 in the malignant potential of oral squamous cell carcinoma cells.

RNase 7 is involved in the innate immunity of the oral epithelium. Variations in the expression levels of RNase 7 have been reported in cutaneous squamous cell carcinoma, but not in oral squamous cell carcinoma (OSCC). The present study investigated the expression levels of RNase 7 in OSCC and its role in the malignant potential of these cells. The localization of RNase 7 in OSCC tissue sections was determined via immunohistochemistry. Positive staining for RNase 7 was observed around the epithelial pearls and spinous cells of the OSCC tissues. Four different types of OSCC cell lines (OSC­19, BSC­OF, SAS, and HSC­2) and a normal keratinocyte (HaCaT) were used. The mRNA and protein expression levels of RNase 7 were significantly higher in the OSCC cells compared to the HaCaT cells. Based on our hypothesis that high levels of RNase 7 expression may be involved in the malignant potential of OSCC cells, the effect of RNase 7 knockdown on both proliferation and invasion were evaluated by transfecting the cells with siRNA. Cell numbers, cell invasion, and MMP 9 expression levels were significantly higher in the siRNA­BSC­OF, ­SAS, and ­HSC­2 cells compared to the BSC­OF, SAS, and HSC­2 cells. The extent of differentiation of the siRNA­OSCC cells was examined using the differentiation and undifferentiation markers involucrin (INV) and K14, respectively. The expression level of K14 was significantly higher in the siRNA­OSCC cells compared to the OSCC cells. Alternatively, HSC­2 and SAS cells demonstrated higher expression levels of INV compared to the siRNA­HSC­2 and ­SAS cells. These findings indicate that RNase 7 may contribute to the suppression of the malignant potential of OSCC.

Chronic stress-induced elevation of IL-1ß in the saliva and submandibular glands of mice.

Psychological stress is involved in the development of various oral diseases. Alterations in the levels of cytokines in the saliva of patients with stress-related oral diseases have been reported. However, the inconsistencies in the results of these studies might be attributed to differences in the local and systemic factors in the oral cavities of the patients. We examined the effect of chronic stress on three major inflammatory cytokines Interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the saliva and salivary glands of chronically stressed mice. Six-week-old C57BL/6 J mice were randomly divided into a control and a stress group. The mice in stress group were exposed to 4 h of stress daily for 10 days and subsequently saliva, as well as the submandibular glands, were collected from both groups. The expression levels of cytokines in the saliva were examined by enzyme-linked immunosorbent assay. The submandibular glands were subjected to histopathological and mRNA expression analyses. IL-1ß was significantly elevated in saliva of the chronic stressed mice. Furthermore, the mRNA expression levels of both IL-1ß and IL-6 were significantly elevated in the submandibular gland of chronic stressed mice. IL-1ß may be a potential salivary biomarker in response to chronic stress in mice.

Curcumin inhibits the expression of proinflammatory mediators and MMP-9 in gingival epithelial cells stimulated for a prolonged period with lipopolysaccharides derived from Porphyromonas gingivalis.

Curcumin, a yellow phytochemical found in the rhizomes of Curcuma longa, has various biological effects, including anti-oxidant and anti-inflammatory activities. In the present study, we examined the effect of curcumin on the expression of inflammatory cytokines in human gingival epithelial progenitor cells (HGEPs) stimulated for a prolonged period with lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. The cells were alternately cultured with LPS and/or curcumin every 3 days for 18 days. The expression levels of TNF-α, IL-1ß, IL-6, TIMP-1, and MMP-9 in the HGEPs were evaluated by quantitative real-time polymerase chain reaction. Enzyme-linked immunosorbent assay was used to measure the concentrations of these five proteins in the supernatant and nuclear factor (NF)-κB in the nuclear extracts. Curcumin inhibited the mRNA expression levels of TNF-α, IL-1ß, IL-6, and MMP-9 in HGEPs treated with curcumin over a prolonged period. Similarly, the expression levels of IL-1ß, IL-6, and MMP-9 were decreased in the culture supernatants. NF-κB activity was also inhibited in the cells cultured with curcumin. In conclusion, these findings indicate that curcumin inhibits the expression of inflammatory cytokines and MMP-9 in primary gingival epithelial cells stimulated with P. gingivalis-derived LPS via NF-κB activation.