營養素及飲食因子對腸道免疫系統的影響

腸道免疫細胞受到抗原刺激後，所引發的免疫反應調控著腸道微環境的平衡。葉酸 (folic acid)、山苦瓜 (bitter gourd)、苜蓿芽乙酸乙酯萃物 (ethyl acetate extracts of alfalfa sprout, AEA)、靈芝三萜類 (ganoderma tsugae)及奇異果 (kiwi)皆具有免疫調節的作用，然而這些食材對於腸道免疫的影響，少有研究發表，故本實驗分為三個部分探討飲食對於腸道少數細胞群及細胞激素分泌的影響。實驗第一部分給予BALB/c小鼠含葉酸或缺乏葉酸的飼料，於餵食實驗5、9、13週犧牲，取得皮耶氏體 (Peyer's patch, PP)、腸繫膜淋巴結 (mesenteric lymph nodes, MLN)及脾臟 (spleen)，分析naive T cell、CD103+CD11b+ DC及Treg cells細胞群比例，PP及MLN細胞以裂殖素 (mitogen)刺激後，分析T細胞相關細胞激素及促發炎細胞激素。飼料中缺乏葉酸導致細胞處於未分化形式，葉酸缺乏亦顯著降低調節型T細胞比例，影響最顯著的部位為PP，餵食週數到9週，也觀察到在MLN及脾臟naive T細胞顯著較多，由於大部分T細胞為naive cell，PP及MLN細胞激素分泌方面，葉酸缺乏組T細胞相關細胞激素分泌量顯著降低; 補充10倍葉酸能顯著降低PP及MLN發炎細胞激素的分泌，然而葉酸的補充，PP及MLN調節型細胞群比例顯著減少。第二部分使用C57BL/6J小鼠，以髓鞘寡突細胞醣蛋白 (myelin oligodendrocyte glycoprotein, MOG)誘發實驗性自體免疫性腦脊髓炎 (experimental autoimmune encephalomyelitis, EAE)，在含5% fat diet中添加山苦瓜凍乾粉餵食10週，或以管餵方式餵食不同劑量之苜蓿芽乙酸乙酯萃物13週後犧牲，取得PP、MLN及脾臟分析腸道免疫指標。結果顯示，山苦瓜組MLN調節型T細胞比例顯著增加，ConA刺激下，山苦瓜能降低MLN TNF-a的分泌，而MLN IL-17的分泌則是顯著增加。IFN-g在MLN AEA-L組分泌量顯著下降。第三部分使用C57BL/6J小鼠，以30% fat之高油飲食誘發肥胖，飼料中添加山苦瓜或不同劑量的三萜類化合物，於餵食實驗22週後犧牲;另以LPS致急性發炎模式，在高油飼料中添加奇異果，餵食10週後犧牲。結果顯示，ConA刺激下，BGP組MLN IL-10分泌量顯著增加，TNF-a、IL-6分泌顯著降低; 不論劑量高低，三萜類化合物顯著降低PP或MLN中調節型T細胞比例，TRE2能降低ConA刺激後，MLN發炎激素的分泌。奇異果能顯著增加PP及MLN細胞IL-10的分泌量。由實驗結果得知，葉酸缺乏顯著降低Foxp3+ Treg比例，不利免疫調節。葉酸的補充、山苦瓜、2倍劑量的三萜類能顯著降低腸道促發炎細胞激素TNF-a、IL-6的分泌，有利於發炎反應的調節，另外山苦瓜及奇異果顯著增加腸道免疫組織IL-10的分泌，山苦瓜亦能促進MLN Foxp3+ Treg細胞群的增加，對維持免疫反應的平衡是有益的。

關鍵字

皮耶氏體；腸繫膜淋巴結；葉酸；山苦瓜；苜蓿芽乙酸乙酯萃物；靈芝三萜類；奇異果；發炎；調節型T細胞

並列摘要

Background: Under stimulation of antigen, the immune cells’ response regulate the balance of intestinal microenvironment. Folic acid, bitter gourd, ethyl acetate extracts of alfalfa sprout, Ganoderma tsugae and kiwi have immunomodulatory effects, but there are few studies about the influence of dietary factor on gut immune system, so the present study is to and investigate the impact of diet on gut immune cell populations and the cytokine secretion in PP and MLN in three different experiments. Results: The first part of the experiment, female BALB/c mice feed a diet containing folic acid or deficiency of folic acid. Mice were sacrificed after 5, 9, 13 weeks period of feeding. Peyer’s patch (PP), mesenteric lymph nodes (MLN) and spleen were collected for analyzed naive T cell, CD103+CD11b+ DC and Treg cells’ cell population analysis by flow cytometry. After mitogen stimulation, PP and MLN cells’ supernatant were used to analyze T cell related- and pro-inflammatory cytokines. The results showed that folate deficiency causes cells in an undifferentiated form, folate deficiency also significantly reduced the proportion of regulatory T cells, the first impact of the site is the PP, After 9 weeks of feeding, more naive T cells in MLN and spleen were also observed. As most of T cells were naive cell. in the folic acid deficient group, Th1, Th2, Treg, Th17 cytokine secretion by PP and MLN significantly reduced; PP and MLN cell supplemented with ten-fold folic acid, pro-inflammatory cytokine secretion significantly reduced from ConA or LPS. However, stimulated PP and MLN cell of mice supplemented with ten-fold folic acid also resulted in significant reduction of Treg cell population in PP and MLN. The second part of the experiment, female C57BL/6J mice were immunized with myelin oligodendrocyte glycoprotein (MOG) to induced experimental autoimmune encephalomyelitis (EAE), fed a AIN-76 diet containing 5% of fat, supplemented with bitter gourd lyophilized powder feeding for 10 weeks, or tube feeding with different doses of alfalfa sprouts ethyl acetate extracts. After 13 weeks. PP, MLN and spleen were collected for analysis of immune response. The results showed that regulatory T cell ratio increased significantly in MLN in the bitter gourd (BGP) group. Bitter gourd significantly reduce MLN cells’ TNF-a secretion, but increased IL-17 secretion in MLN. Low doses of ethyl acetate extract of alfalfa sprouts (AEA-L) reduce MLN IFN-g secretion from ConA-stimulated. The third part of the experiment, male C57BL/6J mice fed a high fat diet containing 30% fat for induction of obesity. The high fat diet added with bitter gourd lyophilized powder or different doses of triterpenoids for 22 weeks feeding period. Another high fat diet-induced obese model injected with LPS for acute inflammation were fed the diet consisted of green kiwifruit lyophilized powder for 10 weeks. The results showed that IL-10 secretion increased significantly in MLN in the BGP group. By contraries, stimulated MLN cells of mice in BGP group, TNF-a and IL-6 secretion significantly reduced; regardless of the dose of triterpenoids, which significantly reduced regulatory T cell ratio in PP or MLN. TNF-a, IL-6 secretion reduced in MLN in Two-fold dose of triterpenoids (TRE2) group from ConA stimulation. Kiwifruit significantly increased IL-10 secretion from ConA-stimulated PP and MLN cells. Conclusion: In conclusion, folate deficiency significantly reduced the proportion of Foxp3+ Treg cells, which is unfavorable for immune regulation. Folic acid supplement, bitter gourd, and triterpenoids can significantly reduce intestinal proinflammatory cytokine TNF-a, IL-6 secretion. Bitter gourd and kiwifruit enhanced IL-10 secretion in both PP and MLN. Bitter gourd also promote the propotion of Foxp3+ Treg cell in MLN. These dietary factors are conducive to the regulation of inflammatory response.

並列關鍵字

mesenteric lymph nodes ； folic acid ； bitter gourd ； alfalfa sprouts ethyl acetate extracts ； Ganoderma triterpenoids ； kiwifruit ； inflammation ； regulatory T cells