Normal Buccal Mucosa Specimens Research Articles

Elevation of S100A4 Expression in Buccal Mucosal Fibroblasts by Arecoline: Involvement in the Pathogenesis of Oral Submucous Fibrosis

BackgroundS100A4, a member of the calcium-binding proteins, is dramatically elevated in a variety of fibrotic diseases. Areca quid chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). OSF has been considered as a pre-cancerous condition of oral mucosa. The aim of this study was to determine the critical role of S100A4 expression in the pathogenesis of OSF both in vitro and in vivo.Methodology/Principal FindingThirty OSF tissues from areca quid chewers and ten normal buccal mucosa samples without areca quid chewing were analyzed by using immunohistochemistry for S100A4 expression in vivo. Collagen gel contraction capability and expression of tissue inhibitor of metalloproteinases 1 (TIMP1)/MMP9 in arecoline-stimulated BMFs with S100A4 knockdown was presented in vitro. Initially, S100A4 expression was higher in areca quid chewing-associated OSF specimens than normal buccal mucosa specimens (p = 0.001). Arecoline, a major areca nut alkaloid, led to dose- and time-dependent elevation of S100A4 expression in normal buccal mucosa fibroblasts BMFs (p<0.05). The additions of pharmacological agents rapamycin (mTOR inhibitor), PD98059 (ERK inhibitor), and Bay117082 (NF-κB inhibitor) were found to inhibit arecoline-induced S100A4 expression (p<0.05) in BMFs. Down-regulation of S100A4 by lentiviral infection significantly reversed arecoline-induced collagen gel contraction and TIMP1/MMP9 expression.Conclusion/SignificanceThese results suggest that S100A4 expression is significantly up-regulated in OSF specimens. Arecoline-induced S100A4 expression was down-regulated by rapamycin, PD98059, and Bay117082. Targeting S100A4 might be a potential therapeutic target for OSF through TIMP1/MMP9 down-regulation.

Elevated expression of NF-κB in oral submucous fibrosis – Evidence for NF-κB induction by safrole in human buccal mucosal fibroblasts

Nuclear factor-kappa B (NF-kappaB) is considered to be important in many inflammatory and immune responses. The aim of this study was to compare NF-kappaB expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induction of NF-kappaB expression. Seventeen OSF and six normal buccal mucosa specimens were examined by immunohistochemistry. Primary human buccal mucosal fibroblasts (BMFs) were established and challenged with safrole, a major polyphenolic compound in the influorescence of Piper betel, by cytotoxicity and western blot assays. Furthermore, glutathione precursor N-acetyl-L-cysteine (NAC), extracellular signal-regulated protein kinase (ERK) inhibitor PD98059, cyclooxygenase-2 (COX-2) inhibitor NS-398, dexamethasone, and cyclosporin A were added to find the possible mechanism. NF-kappaB expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. Safrole was cytotoxic to BMFs in a dose-dependent manner (p<0.05). Western blot demonstrated highly elevated NF-kappaB protein expression in BMFs stimulated by safrole (p<0.05). In addition, pretreatment with pharmacological agents markedly inhibited the safrole induced-NF-kappaB expression (p<0.05). The result suggests that chewing areca quid may activate NF-kappaB expression that may be involved in the pathogenesis of OSF. NF-kappaB expression induced by safrole in fibroblasts may be mediated by ERK activation and COX-2 signal transduction pathway.

Raised keratinocyte growth factor‐1 expression in oral submucous fibrosis in vivo and upregulated by arecoline in human buccal mucosal fibroblasts in vitro

Keratinocyte growth factor-1 (KGF-1) is the seventh member of the fibroblast growth factor family. KGF-1 is produced by mesenchymal cells such as fibroblasts and upregulated in a variety of hyperplastic tissues. Currently, there is limited information about the regulation of KGF-1 expression in areca quid-associated oral submucous fibrosis (OSF). The aim of the study was to compare KGF-1 expression in normal human buccal mucosa and OSF specimens and further to explore the potential mechanism that may lead to induce KGF-1 expression. The expression of KGF-1 from fibroblasts cultured from OSF and normal buccal mucosa were using reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, arecoline, a major areca nut alkaloid, was challenged to normal buccal mucosa fibroblasts (BMFs) to elucidate whether KGF-1 expression could affect by arecoline. Furthermore, 25 OSF specimens and six normal buccal mucosa specimens were examined by immunohistochemistry. Fibroblasts derived from OSF were found to exhibit higher KGF-1 expression than BMFs both in mRNA and protein levels (P < 0.05). In addition, upregulation of KGF-1 mRNA gene and protein expression were found in BMFs stimulated by arecoline (P < 0.05). From the results of immunohistochemistry, KGF-1 expression was significantly higher in OSF specimens and expressed mainly by fibroblasts, endothelial cells, inflammatory cells, and epithelial cells. Taken together, these results suggest that KGF-1 expression is significantly upregulated in OSF tissues from areca quid chewers and arecoline may be responsible for the enhanced KGF-1 expression in vivo.