NODI Signal Pathway Stimulation By IE-DAP Regulates Cigarette Smoking-induced Suppression On Defense Ability Of Oral Mucosa Epithelial Cell

Chapter 1 Influence of iE-DAP on NOD1 Signal Pathway in Oral Mucosa Epithelial CellObjectivesOral Mucosa Epithelial Cell is regarded as the first line of defence against invading pathogens in the oral cavity.NOD1 signaling pathway plays an important role in oral innate immunity system.The crucial molecules of NOD1 signaling pathway consist of NOD1,RIP2,p-NF-κB.The peptidoglycan derivatives iE-DAP is one of the key fragment recognized by NOD1.Some decent researches have confirmed that iE-DAP can stimulate NOD1 pathway in different cell types and regulate the secretion of cytokines.Our aim is to explore the influence of iE-DAP on some key proteins and downstream cytokines of NOD1 pathway in oral mucosa epithelial cell.MethodsLeuk-1 cells were cultured in vitro and stimulated by iE-DAP of different doses for 24h.Cell viability was measured by MTT assay.The protein expression of NOD1,RIP2 and p-NF-κB in NOD1 signaling pathway was tested by western blot.Cytokines IL-1β IL-6,IL-8,IL-10,TNF-α,IFN-γ and hBD-1,-2,-3 were investigated by ELISA,while mRNA expression of those transcription factors was detected by qRT-PCR.Results1.iE-DAP had no significant influences on cell viability until the dose went up to 1000μg/ml for 24h;2.iE-DAP increased the NOD1 expression in Leuk-1 cells in a dose-dependent manner.And 50μg/mL iE-DAP up-regulated expression levels of RIP2 and p-NF-κB significantly;3.iE-DAP up-regulated expressions of IL-1β IL-6,IL-10,TNF-α,IFN-γ and hBD-2,participating in innate immunity and defensive response of Leuk-1 cells.ConclusionsNOD1 agonist iE-DAP plays an important role in regulating innate immune system of Leuk-1 cells through enhancing the expression of key proteins in NOD1 pathway and regulating the secretion of its downstream cytokines.Chapter 2 iE-DAP Regulates Cigarette Smoking-Induced Suppression on Defense Ability of Oral Mucosa Epithelial CellObjectivesCigarette smoking has been associated with an increased incidence of suppression on oral innate system and regarded as one of the risk factors for oral mucosa diseases like oral leukoplakia(OLK).Our previous study showed that CSE could suppress the expression of NOD1 pathway and down-regulated hBD-1 and hBD-3,which is important in immune defense.These data indicated that CSE inhibited oral mucosa immunity through NOD1 pathway.This study explained how iE-DAP regulate suppression of smoking on defense ability of oral mucosa epithelial cell.MethodsLeuk-1 cells were cultured in vitro and stimulated by CSE of different doses.MTT assay was used to measure cell viability.We used iE-DAP to stimulate cell for 24h after exposed to CSE for 24h.Western blot analysis was used to detect the changes in relative proteins expression of NOD1 pathway.qRT-PCR was performed to determine the mRNA levels of IL-1β,IL-6,IL-8,IL-10,TNF-α,IFN-γ and hBD-1,-2,-3.These cytokines production in culture supernatants was detected by ELISA assay.Results1.CSE treatment for 24h did not decrease cell viability until its concentration up to 32%;2.iE-DAP could reverse the CSE-induced down-regulation of NOD1 expression,and weaken the effects of CSE on the expression of RIP2 and p-NF-κB.3.iE-DAP reversed the effects of CSE on the cytokines and hBDs expression in Leuk-1 cells.ConclusionsNOD1 agonist iE-DAP regulated the effects of CSE on innate immunity and defensive ability of oral mucosal epithelial cells through reversing the CSE-induced suppression of NOD1 pathway and downstream cytokines.