檳榔子萃取物抑制T細胞功能及促進骨髓衍生抑制性細胞生成之免疫調節作用

The immunomodulatory effect of areca nut extract on the functionality of T cells and the induction of myeloid-derived suppressor cells

王家琪(Chia-Chi Wang)

指導教授 ： 詹東榮

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.03001

摘要

流行病學研究顯示嚼食檳榔會提高罹患口腔黏膜下纖維化症及口腔癌的機率，且實驗證據指出病人免疫系統的惡化與嚼食檳榔所導致的口腔疾病病程的發展有重要的相關性。研究發現檳榔子萃取物能直接引起嗜中性球細胞及多種口腔細胞的死亡且增加細胞的氧化性傷害。由於T淋巴球細胞已被證實為主要浸潤於口腔癌前病變組織或口腔腫瘤的重要免疫細胞，因此推測此大量浸潤的T淋巴球可能會受到檳榔成分的直接作用而導致其媒介的免疫功能受到影響。本論文主要研究檳榔子萃取物對於T淋巴球活性及功能的影響，並進一步探討活性氧化物（ROS）於檳榔子萃取物的免疫調節作用所扮演的角色。此外更進一步研究檳榔子萃取物在活體動物中的免疫調節作用。實驗結果顯示檳榔子萃取物對於脾臟細胞具有明顯的毒性且會抑制介白素-2（interleukin-2）及干擾素-γ（interferon-γ）的分泌，而對於介白素-4的抑制作用則不明顯。上述檳榔子萃取物的抑制作用可以被抗氧化劑N-乙醯半胱胺酸（N-acetyl-L-cysteine以下簡稱NAC）部分且有效的回復。檳榔子萃取物會增加T淋巴球ROS的增加及降低細胞內穀胱甘肽 （glutathione以下簡稱GSH）的含量，顯示檳榔子萃取物可藉由增加細胞內產生ROS來抑制T淋巴球的活化及第一型T幫手淋巴球細胞激素的製造。
檳榔子萃取物明顯地增加脾臟細胞的凋亡、促進粒線體膜電位的去極化、釋放粒線體內cytochrome c、活化細胞內caspase-9 及增加凋亡細胞的ROS。除了粒線體膜電位的去極化完全無法被NAC所預防，NAC對於檳榔子萃取物所引起的上述作用都具有部分但顯著的回復效果。此研究結果指出檳榔子萃取物對於脾臟細胞的毒性作用與活化粒線體凋亡途徑有關，同時會導致細胞內ROS的增加。由於檳榔子萃取物含有豐富的多酚類，進一步研究檳榔子萃取物中的多酚類是否可以引起脾臟細胞的凋亡，結果發現多酚類含量較多的檳榔子萃取物（PANE）同樣具有導致脾臟細胞凋亡的效果，且進一步比較萃取物中不同聚合程度的前花青素（procyanidins），發現聚合程度大於五的pentamers即開始會引起脾臟細胞的凋亡，隨著前花青素聚合程度愈高引起細胞凋亡的程度也愈明顯。此外PANE也會導致細胞內GSH的下降，顯示細胞內氧化性傷害的增加，且細胞內GSH下降的程度也與前花青素聚合程度成正相關。此部分研究顯示檳榔子萃取物中高聚合程度的前花青素可能會導致細胞抗氧化物GSH的下降而引起細胞的凋亡。
活體動物的試驗中發現，腹腔注射檳榔子萃取物及PANE會導致脾臟的腫脹及增加一群CD11b+的骨髓衍生性細胞，此群細胞具有小鼠骨髓衍生性抑制性細胞（MDSC）的重要細胞表面標誌: CD11b及Gr-1。CD11b+Gr-1+ 的細胞族群在給予檳榔子萃取物的小鼠脾臟及血液中的比率有顯著的增加。進一步分析該群細胞的功能發現其抑制性細胞激素介白素-10（IL-10）的分泌量、第一型精胺酸酶（arginase-I）酵素的活性及表現第一型精胺酸酶及誘導型一氧化氮合成酶（inducible nitric oxide synthase；iNOS）基因的能力都有顯著地增加，顯示此群細胞具有骨髓衍生抑制性細胞（myeloid-derived suppressor cell；MDSC）典型的功能特性。
綜合本論文的研究結果，發現檳榔子萃取物具有直接調節細胞媒介免疫反應的作用，包含降低T淋巴球細胞激素的分泌、導致脾臟細胞的凋亡及增加骨髓衍生抑制性細胞的數量。而檳榔子萃取物中的前花青素於上述的免疫調節作用中扮演重要的角色。本論文結果有助於了解檳榔子的成分如何調節免疫細胞及導致細胞媒介免疫反應惡化的可能機制。

關鍵字

檳榔子萃取物 ； 前花青素 ； 免疫抑制 ； 細胞激素 ； 凋亡 ； 骨髓衍生抑制性細胞 ； 氧化性傷害

並列摘要

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.

並列關鍵字

areca nut extract ； procyanidins ； immunosuppression ； cytokines ； apoptosis ； myeloid-derived suppressor cells ； oxidative stress

參考文獻

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