Acanthopanax senticosus ameliorates steatohepatitis through HNF4 alpha pathway activation in mice.

Non-alcoholic fatty liver disease is a common liver disease worldwide, and is associated with dysregulation of lipid metabolism, leading to inflammation and fibrosis. Acanthopanax senticosus Harms (ASH) is widely used in traditional medicine as an adaptogen food. We examined the effect of ASH on steatohepatitis using a high-fat diet mouse model. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet with ASH extract (ASHE). After 6 weeks, liver RNA transcriptome sequencing (RNA-Seq) was performed, followed by Ingenuity Pathway Analysis (IPA). Our findings revealed that mice fed a high-fat diet with 5% ASHE exhibited significantly reduced liver steatosis. These mice also demonstrated alleviated inflammation and reduced fibrosis in the liver. IPA of RNA-Seq indicated that hepatocyte nuclear factor 4 alpha (HNF4 alpha), a transcription factor, was the activated upstream regulator (P-value 0.00155, z score = 2.413) in the liver of ASHE-fed mice. Adenosine triphosphate binding cassette transporter 8 and carboxylesterase 2, downstream targets of HNF4 alpha pathway, were upregulated. Finally, ASHE-treated HepG2 cells exposed to palmitate exhibited significantly decreased lipid droplet contents. Our study provides that ASHE can activate HNF4 alpha pathway and promote fat secretion from hepatocytes, thereby serving as a prophylactic treatment for steatohepatitis in mice.

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.

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.

Increase in Bifidobacterium is a characteristic of the difference in the salivary microbiota of pregnant and non-pregnant women.

BACKGROUND: The establishment of symbiotic microbiota in pregnant women is important for both the mother and her offspring. Little is known about the salivary symbiotic bacteria in pregnancy, and analysis of composition of microbiome (ANCOM) is useful to detect small differences in the number of bacteria. The aim of this study was to investigate the differences in the salivary bacteria between healthy pregnant and non-pregnant women using ANCOM. METHODS: Unstimulated saliva samples were collected from 35 healthy pregnant women at 35 weeks gestation and 30 healthy non-pregnant women during menstruation. All participants underwent a periodontal examination. Estradiol and progesterone levels were examined by enzyme-linked immunosorbent assay. DNA extracted from the saliva was assessed by 16S ribosomal RNA amplicon sequencing and real-time PCR. RESULTS: Salivary estradiol and progesterone levels were significantly increased in pregnant women. The alpha and beta diversities were higher in pregnant women than in non-pregnant women. The largest effect size difference noted when the microbiota of the pregnant and non-pregnant women were analyzed was that for Bifidobacteriales. Levels of Bifidobacterium dentium, but not of Bifidobacterium adolescentis, were significantly increased in pregnant women, and the levels were significantly correlated with progesterone concentration. CONCLUSION: The results suggest that Bifidobacterium and progesterone levels are elevated in the saliva of healthy pregnant women compared with non-pregnant women.

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.

Analysis of the cells isolated from epithelial cell rests of Malassez through single-cell limiting dilution.

The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1-18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.

Acanthopanax senticosus Harms extract causes G0/G1 cell cycle arrest and autophagy via inhibition of Rubicon in human liver cancer cells.

Acanthopanax senticosus (Rupr. et Maxim) Harms (ASH), also known as Siberian ginseng or eleuthero, is a hardy shrub native to China, Korea, Russia and the northern region of Japan. ASH is used for the treatment of several diseases such as heart disease, hypertension, rheumatoid arthritis, allergies, chronic bronchitis, diabetes and cancer. In the present study, the inhibitory effect of the root extract of ASH (ASHE) on HuH­7 and HepG2 liver cancer cells was examined. ASHE suppressed liver cancer cell proliferation by inducing cell cycle arrest at the G0/G1 phase, as well as apoptosis, as indicated by the increased number of Annexin V and 7­AAD­positive cells. Furthermore, the expression of LC3­II, an autophagy marker, in these cells also increased post treatment with ASHE. LC3­II induction was further enhanced by co­treatment with chloroquine. Fluorescence and transmission electron micrographs of ASHE­treated liver cancer cells showed the presence of an increased number of autophagic vesicles. A decreased protein expression level of run domain Beclin­1­interacting and cysteine­rich domain­containing, an autophagy inhibitor, with no change in RUBCN mRNA expression was observed, indicating activation of the autophagosome­lysosome fusion step of autophagy. In conclusion, ASHE exerts cytostatic activity on liver cancer cells via both apoptosis and autophagy, and may serve as a potential therapeutic agent for management of liver cancer and autophagy­related diseases.

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.

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.

Alteration of fecal microbiota by fucoxanthin results in prevention of colorectal cancer in AOM/DSS mice.

Fucoxanthin (Fx), a marine carotenoid found in edible brown algae, is well known for having anticancer properties. The gut microbiota has been demonstrated as a hallmark for colorectal cancer progression in both humans and rodents. However, it remains unclear whether the gut microbiota is associated with the anticancer effect of Fx. We investigated the chemopreventive potency of Fx and its effect on gut microbiota in a mouse model of inflammation-associated colorectal cancer (by azoxymethane/dextran sulfate sodium treatment). Fx administration (30 mg/kg bw) during a 14 week period significantly inhibited the multiplicity of colorectal adenocarcinoma in mice. The number of apoptosis-like cleaved caspase-3high cells increased significantly in both colonic adenocarcinoma and mucosal crypts. Fx administration significantly suppressed Bacteroidlales (f_uc; g_uc) (0.3-fold) and Rikenellaceae (g_uc) (0.6-fold) and increased Lachnospiraceae (g_uc) (2.2-fold), compared with those of control mice. Oral administration of a fecal suspension obtained from Fx-treated mice, aimed to enhance Lachnospiraceae, suppress the number of colorectal adenocarcinomas in azoxymethane/dextran sulfate sodium-treated mice with a successful increase in Lachnospiraceae in the gut. Our findings suggested that an alteration in gut microbiota by dietary Fx might be an essential factor in the cancer chemopreventive effect of Fx in azoxymethane/dextran sulfate sodium-treated mice.

Alteration of oral flora in betel quid chewers in Sri Lanka.

BACKGROUND: Betel quid chewing is known as a crucial risk factor for oral diseases such as periodontal diseases, oral cancer, and precancerous lesions in Southeast Asian countries. Although abnormal oral bacterial flora may be linked to betel quid related-oral diseases such as oral cancer, precancerous lesions, and periodontal diseases, little information is available on alterations of their oral flora thus far. To identify these alterations, we analyzed the oral flora in betel quid chewers (BQC) and non-chewers (NC) in Sri Lanka. METHODS: Samples obtained from buccal swabs of BQC and NC were analyzed with a next generation sequencer. Data were processed and analyzed using the QIIME software package. Mann-Whitney U test and Permutational multivariate analysis of variance were used for statistical analyses. P values < 0.05 were considered to be statistically significant. RESULTS: In BQC, the proportion of periodontal pathogens including Actinomyces, Tannerella, and Prevotella was higher than that in NC (P < 0.05), while the proportion of cariogenic pathogens including Streptococcus, Lautropia, and Actinobacillus was lower than that in NC (P < 0.05). A statistically significant difference in Shannon index and PD Whole tree was observed between BQC and NC (P < 0.05). PCoA analysis detected different clusters in BQC and NC (P < 0.05). CONCLUSION: The results suggested that betel quid chewing significantly altered oral flora. Adequate oral health care may help prevent BQC from developing bacterial pathogen-related oral diseases.

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.

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.

DNA hypermethylation of sirtuin 1 (SIRT1) caused by betel quid chewing-a possible predictive biomarker for malignant transformation.

BACKGROUND: DNA hypermethylation of tumor suppressor genes is observed in precancerous lesions and oral cancer of individuals with the habits of betel quid (BQ) chewing. SIRT1 has been identified as playing a role in the maintenance of epithelial integrity, and its alteration is often related to carcinogenesis. However, the methylation and transcription status of SIRT1 in patients with BQ chewing-related oral cancer has not been investigated. We examined the methylation status of SIRT1 in paraffin-embedded tissue samples of oral squamous cell carcinoma (OSCC) obtained from BQ chewing and non-chewing patients and in tissue samples from healthy control subjects. In addition, we examined whether the hypermethylation of SIRT1 followed by its transcriptional downregulation in the human gingival epithelial cells could be caused by arecoline, a major component of BQ. Furthermore, we investigated the methylation status of SIRT1 in smear samples of macroscopically healthy buccal mucosa from subjects with a habit of BQ chewing. RESULTS: SIRT1 was significantly hypermethylated in tissue samples of OSCC from BQ chewers and non-chewers than in oral mucosa from healthy control subjects. Results also showed that the hypermethylation level of SIRT1 was significantly higher in OSCC of patients with BQ chewing habits than in those of non-chewing habits (p < 0.05). Our in vitro model showed that hypermethylation is followed by downregulation of the transcriptional level of SIRT1 (p < 0.05). The methylation levels of SIRT1 in the smear samples obtained from BQ chewing individuals were significantly higher than those in the samples obtained from individuals that did not chew BQ. The duration of BQ chewing habits was correlated positively to the frequency of SIRT1 hypermethylation (p < 0.05). CONCLUSIONS: Our results suggest that DNA hypermethylation of SIRT1 is involved in the occurrence of oral cancer in BQ chewing patients and that hypermethylation in the oral mucosa of BQ chewers could be a predictive marker for the occurrence of malignant transformation. This is the first report that showed DNA hypermethylation in clinically healthy oral epithelium of BQ chewers. Our study shows evidence that DNA hypermethylation may be an early event of oral carcinogenesis prior to observable clinical changes.

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.