| Summary: | 博士 === 臺灣大學 === 獸醫學研究所 === 98 === Areca quid chewing is a major risk factor associated with oral submucous fibrosis and oral cancer. Experimental evidence indicates that immune deterioration is closely associated with the pathophysiology of areca-associated oral diseases. In addition, the induction of oxidative stress and cell death has been shown to play a role in the cytotoxic and genotoxic effects induced by areca nut extracts (ANE) in oral cells and neutrophils. As T lymphocytes are one of the major immunocompetent cells present in the lesions of both OSF and oral cancer patients, it is hypothesized that T cell–mediated immune responses may be altered by ANE. The present studies investigated the immunomodulatory effect of ANE on T cell reactivity and the role of reactive oxygen species (ROS) in ANE-mediated effects in vitro. In addition, the immunomodulatory effect of ANE and polyphenol-enriched ANE (PANE) was examined in vivo. ANE induced a marked cytotoxic effect, and suppressed the production of IL-2 and IFN-γ by splenocytes, whereas the production of IL-4 was unaffected. The thiol antioxidant N-acetyl-L-cysteine (NAC) partially but significantly attenuated ANE-mediated cytotoxicity and suppression of IL-2 and IFN-γ production. In splenic T cells, ANE increased the cellular ROS levels, which was also attenuated by the presence of NAC. Concordantly, the cellular level of glutathione was diminished by ANE in splenic T cells pretreated with NAC. These results demonstrated that ANE markedly suppressed T-cell activation and Th1 cytokine production, which was mediated, at least in part, by the induction of oxidative stress.
ANE significantly enhanced splenocyte apoptosis. The depolarization of mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-9 were induced by ANE, indicating the activation of the mitochondrion-dependent apoptotic pathway. Moreover, an increased level in the intracellular ROS was detected in ANE-treated splenocytes undergoing apoptosis. NAC significantly attenuated ANE-mediated apoptosis, caspase-9 activation and ROS production but not mitochondrial membrane potential depolarization. These results demonstrated the pro-apoptotic effect of ANE in primary splenocytes, which was mediated by the activation of the mitochondrion-dependent pathway and oxidative stress. In addition, PANE and its fractionated oligomeric procyanidins from pentamers to decamers were active in inducing apoptosis. A marked diminishment in the level of intracellular thiols was revealed in splenocytes treated with pentamers to decamers. Pretreatment with NAC resulted in significant attenuation of both apoptosis and thiol diminishment induced by areca procyanidins. These results indicated that highly oligomeric procyanidins derived from areca nut induced a chain length-dependent pro-apoptotic effect in primary lymphocytes possibly via the diminishment of intracellular thiols.
Intraperitoneal administration of antigen-sensitized BALB/c mice with ANE or PANE significantly increased the spleen index and the splenic cellularity of immature myeloid CD11b+ cells. The population of CD11b+Gr-1+ cells in the spleen and peripheral blood was markedly enhanced by ANE and PANE. In addition, ANE administration significantly augmented the production of IL-10, and the mRNA expression of iNOS and arginase I by splenocytes and splenic CD11b+ cells stimulated with lipopolysaccharide. These results suggested that ANE administration to antigen-sensitized mice enhanced the development of CD11b+Gr-1+ cells that exhibited a functional profile of myeloid-derived suppressor cells (MDSC).
Taken together, this study demonstrated the direct immunomodulatory effect of ANE on the down-regulation of Th1 cytokines in vitro, induction of lymphocyte apoptosis in vitro and generation of MDSC in vivo. In addition, areca-derived procyanidins may be the potential candidates responsible for the ANE-mediated immunomodulatory effects. These results provide evidence to show that areca constituents may directly compromise the cell-mediated immunity which was previously reported to be down-regulated in areca quid chewers with oral precancer and cancer.
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