Mechanistic Study On Effects Of Smoking On Defensive Function Of Oral Mucosal Epithelial Cells

Chapter 1 Effects of smoking on defensive function of oral mucosal epithelial cellsObjective:Smoking can suppress defensive function of respiratory and gastrointestinal mucosa, leading to infection of pathogenic microorganisms. However, few studies on effects of smoking on defensive function of oral mucosa were focused. The objective of this study was to observe effects of smoking on defensive function of oral mucosal epithelial cells.Methods:The difference of expression levels of nucleotide-binding oligomerization domain 1 (NOD1), human beta defensins (hBDs) and Caspase-12 in normal oral mucosa and oral leukoplakia tissues between smokers and non-smokers were analyzed by immunohistochemical staining and image analysis. By using HE staining, fungi argyrophil staining and anti-Candida albicans antibody immunohistochemical staining, Candida albicans infection was determined in normal oral mucosa and oral leukoplakia tissues and the infection rates between the non-smoking group and smoking group were compared.Results:The epithelial expression levels of NODI, hBD-1 and hBD-3 in normal oral mucosa and oral leukoplakia of smoking group were significantly lower than those of non-smoking group. The epithelial expression levels of hBD-2 and Caspase-12 in normal oral mucosa and oral leukoplakia of smoking group were remarkably higher than those of non-smoking group. The infection rates of Candida albicans in the normal oral mucosa and oral leukoplakia of smoking group were greatly higher than those of non-smoking group.Conclusion:Smoking could suppress defensive function of oral mucosal epithelial cells and promote Candida albicans infection.Chapter 2 Role of Caspase-12 in effects of cigarette smoke extract on defensive function of oral mucosal epithelial cellsObjective:Caspase-12 plays a key role in the regulation of innate immune defensive response, weakening mucosal defense of intestinal tract. The objective of this study was to observe the effect of cigarette smoke extract (CSE) on Caspase-12 expression in oral mucosal epithelial cells and explore the role of Caspase-12 in effects of CSE on the defensive function of oral mucosal epithelial cells.Methods:After the treatment of immortalized oral mucosal epithelial cell line Leuk-1 with CSE, the secretion levels of hBD-1,-2 and -3 were detected by using ELISA. After being incubated with the culture supernatant of CSE-exposed Leuk-1 cells, Candida albicans was plated and cultured. The anti-Candida albicans activity of the supernatant was evaluated by the colony count. The effect of CSE on expression level of Caspase-12 was assayed by Western blot. Through the transfection of Caspase-12 siRNA into Leuk-1 cells, the silencing of Caspase-12 expression was induced. Following CSE treatment, the mRNA expression levels of hBD-1,-2 and -3 in Leuk-1 cells were detected by quantitative PCR and the releases of hBD-1,-2 and -3 were assayed by ELISA. After being incubated with the culture supernatant of CSE-exposed Leuk-1 cells, Candida albicans was plated and cultured. The anti-Candida albicans activity of the supernatant was evaluated by the colony count. All the differences between Caspase-12 siRNA group and control siRNA group were compared.Results:CSE treatment markedly reduced the releases of hBD-1 and hBD-3 by Leuk-1 cells, and increased the release of hBD-2. The activity of culture supernatant of Leuk-1 cells against Candida albicans was significantly attenuated after CSE treatment. CSE treatment significantly enhanced the protein expression of Caspase-12. The transfection of Caspase-12 siRNA induced Caspase-12 silencing at mRNA and protein levels. Caspase-12 silencing markedly reversed the inhibitory effect of CSE on the expression and releases of hBD-1 and hBD-3. Caspase-12 silencing remarkably augmented the induced effect of CSE on the expression and releases of hBD-2. Caspase-12 silencing significantly increased the anti-Candida albicans activity of supernatant of Leuk-1 cells following CSE treatment.Conclusion:CSE treatment could enhance Caspase-12 expression in oral mucosal epithelial cells. Caspase-12 plays a crucial role in the suppression of defensive function of oral mucosal epithelial cells and the increase of Candida albicans infection due to CSE.Chapter 3 Regulation of Caspase-12 in the effect of cigarette smoke extract on NOD1 expression in oral mucosal epithelial cellsObjective:NOD1 plays an important role in local defense and cell homeostasis of oral cavity. Previous studies showed that CSE affected the expression and releases of hBDs by regulating NOD1 signaling. The objective of this study was to observe the effect of CSE on NOD1 expression in oral mucosal epithelial cells, and to investigate the regulation of Caspase-12 in the effect of CSE on NODI expression in oral mucosal epithelial cells.Methods:The effect of CSE on the expression level of NOD1 in Leuk-1 cells was assayed by immunoblotting. The interaction relationship between Caspase-12 and NOD1 was verified by co-immunoprecipitation and immunofluorescence double staining. The effect of Caspase-12 silencing on NOD1 expression level was observed. After CSE treatment, the expression level of NODI was detected by immunoblotting and the differences between Caspase-12 siRNA group and control siRNA group were compared.Results:CSE treatment remarkably inhibited the protein expression of NOD1. Caspase-12 and NOD1 were colocalized in Leuk-1 cells. The protein interaction between Caspase-12 and NOD1 was confirmed. Caspase-12 silencing markedly raised the level of NOD1 protein expression. Caspase-12 silencing partially reversed the inhibitory effect of CSE on the protein expression of NOD1.Conclusion:CSE could inhibit NOD1 expression in oral mucosal epithelial cells, in which the negative regulation of Caspase-12 on NOD1 expression could be involved.