短鏈脂肪酸與嬰兒異位性皮膚炎及高脂飲食小鼠腸道免疫之相關性探討

沈瑩柔

doi:10.6342/NTU201703241

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短链脂肪酸与婴儿异位性皮肤炎及高脂饮食小鼠肠道免疫之相关性探讨

Study on the association of short chain fatty acids with infant atopic dermatitis and intestinal immunity of high-fat diet fed mice

沈莹柔 , 硕士　　导师：林璧凤　　

繁体中文 DOI： 10.6342/NTU201703241

异位性皮肤炎；脂肪酸；短链脂肪酸；母乳；肠道免疫；比菲德氏菌；益生菌；寒天寡醣； atopic dermatitis ； fatty acids ； short chain fatty acids ； breast milk ； intestinal immunity ； Bifidobacterium ； probiotics ； agaro-oligosaccharide

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摘要 │ 参考文献 (234) │ 国际计量

摘要〈TOP〉

短链脂肪酸为肠道菌代谢水溶性纤维的产物，与肠道免疫的关系日益受到重视，目前被认为具有抗发炎的作用，能改善气喘、肥胖、肠道发炎等疾病。故本研究探讨短链脂肪酸是否与过敏疾病和肠道免疫反应有关。分别检测异位性皮肤炎婴儿所哺喂的母乳，及给予高油饮食小鼠添加益生质寒天寡醣，探讨短链脂肪酸与免疫反应的相关性。第一部份实验样品於台大医院小儿科过敏门诊，收集6个月龄以下罹患异位性皮肤炎婴儿的母乳，分别测定短链脂肪酸 (C2~C4) 及中长链脂肪酸 (C8-C24) 含量，并对母亲进行1个月的饮食频率问卷调查。结果显示，异位性皮肤炎婴儿所摄取母乳的MUFAs、n-3 PUFAs、palmitic acid及arachidonic acid脂肪酸百分比较高，而capric acid和myristic acid较低，短链脂肪酸则无显着差异。第二部分以高油饮食小鼠模式，探讨短链脂肪酸和肠道菌组成对肠道免疫之影响。实验设计为给予5周龄C57BL/6J小鼠28% 高油饲料 (HF组，油脂热量为51%)，高油添加3%寒天寡醣 (HF-AOS组)、或添加109/day CFU市售益生菌粉末 (HF-PRO组)。喂饲8周後牺牲，分析脾脏、肠系膜淋巴结与皮耶式体的免疫指标，盲肠和粪便的短链脂肪酸以及粪便IgA含量。结果显示，高油饮食造成小鼠粪便比菲德氏菌属显着下降，而HF-PRO组添加益生菌确实显着增加粪便比菲德氏菌属的含量。HF-PRO组小鼠MLN的CD103+树突细胞比例也较高，PP细胞分泌IL-2显着较HF组低，脾脏细胞和MLN细胞IL-17分泌量趋势较HF组高，而脾脏细胞IL-2分泌量显着较HF组高。HF-AOS组母鼠宫旁脂显着最轻，血清和肝脏脂质含量显着最少，但在粪便中显着最多，显示寒天寡醣可能会抑制脂质吸收。HF-AOS组盲肠显着最重，粪便的肠杆菌科含量略高，盲肠及粪便的短链脂肪酸含量显着最低。MLN细胞分泌IFN-γ有增加的趋势，粪便IgA的浓度显着最高。另外大肠组织切片也观察到绒毛破损、黏液分泌量较少，推测本实验添加寒天寡醣的量偏高可能吸附肠道水份，造成黏膜细胞受损，肠道蠕动较快速，使得肠道菌无法利用寒天寡醣发酵生成短链脂肪酸，使肠道免疫趋向抵抗感染的发炎反应。综合以上，本研究发现母乳有较低比率的SFAs、较高比率的MUFAs及n-3 PUFAs可能与婴儿异位性皮肤炎相关，但未呈现母乳的短链脂肪酸含量与婴儿异位性皮肤炎有关。高油饮食小鼠添加寒天寡醣剂量可能过高，导致小鼠轻微腹泻且肠道处於较发炎的状态，而高油饮食添加益生菌，显着降低PP细胞IL-2的分泌量，但有促进MLN细胞分泌IL-17及脾脏细胞分泌IL-2和IL-17的趋势，意喻高油饮食下补充益生菌，改善肠道免疫的效果有限。

并列摘要〈TOP〉

The microbial metabolites short chain fatty acids (SCFAs) are fermented from soluble dietary fiber. Significant attention has been focused on the relationship between SCFAs and intestinal immunity. SCFAs was shown to have the anti-inflammatory effect, and could ameliorate asthma, obesity, intestinal inflammation. Thus, the aim of this study is to investigate the association of SCFAs with allergic disease and intestinal immunity. To investigate the effect of SCFAs on immune response, we analyzed breast milk from the infant with atopic dermatitis (AD), and related parameters from high-fat diet fed mice supplemented with prebiotics agaro-oligosaccharide. In the first part, we cooperated with the Department of the Pediatric at NTU hospital to collect breast milk from infants’ mothers to detect the C8-C24 fatty acids composition and short chain fatty acids (SCFAs) concentration. We also analyzed maternal diet by FFQ (food frequency questionnaire). The results showed that the percentages of MUFAs, n-3 PUFAs, palmitic acid and arachidonic acid of breast milk were higher, and capric acid and myristic acid were lower in the AD group than the health group. SCFAs concentration had no significant difference between two groups. In the second part, we investigated the effect of SCFAs and microbiota on intestinal immunity in high-fat diet fed mice. 5 week-old C57BL/6J mice were fed either 28% fat diet (HF group), HF containing 3% AOS (HF-AOS group) or 109/day CFU probiotics (HF-PRO group) for 8 weeks. Immune indicators of spleen, MLN, PP, fecal and cecal SCFAs, and fecal IgA were also detected. The results showed that fecal Bifidobacterium was decreased in high-fat diet fed mice, and probiotics increased fecal Bifidobacterium in the HF-PRO group. CD103+ dendritic cell percentage was increased in MLN, IL-2 secretion from PP was lower, IL-17 secretion from MLN was higher, and IL-2 and IL-17 secretion from splenocytes were higher in the HF-PRO group compared with the HF group. The parametrial white adipose from female mice was the lightest in the HF-AOS group. The lipid content in serum and liver were the least, but the lipid content was the highest in the HF-AOS group. These data showed that AOS might inhibit the absorption of lipid. The cecum was heaviest, fecal Enterobacteriaceae increased, and fecal and cecal SCFAs were decreased in the HF-AOS group. Fecal IgA and IFN-γ secreted from MLN cells were increased in the HF-AOS group. Evaluating colon histomorphology from H&E and PAS stained, the villous were damaged and with less mucin. We surmised that the mucus was damaged due to the high dose of AOS and its water containing ability. It also led to pro-inflammatory response in mice intestine with less SCFAs production. In conclusion, the lower percentage of SFAs and the higher percentage of MUFAs and n-3 PUFAs might be associated with infant AD. But there was no association between breast milk SCFAs and infant AD. Our results found out that 3% AOS in high-fat diet fed mice might lead to dysbiosis and inflammation in mice intestine, which might due to overdose. High-fat diet with probiotics could decrease IL-2 secretion from PP, but tend to promote IL-17 secretion in MLN and IL-2 and IL-17 secretion in spleen. Thus, the results indicated that the effect of probiotics on high-fat diet fed mice was limited.

参考文献 ( 234 ) 〈TOP〉

Adam, C. L., Gratz, S. W., Peinado, D. I., Thomson, L. M., Garden, K. E., Williams, P. A., et al. (2016). Effects of dietary fibre (pectin) and/or increased protein (casein or pea) on satiety, body weight, adiposity and caecal fermentation in high fat diet-induced obese rats. PLoS One, 11(5).
连结：
Adam, C. L., Williams, P. A., Dalby, M. J., Garden, K., Thomson, L. M., Richardson, A. J., et al. (2014). Different types of soluble fermentable dietary fibre decrease food intake, body weight gain and adiposity in young adult male rats. Nutrition & Metabolism, 11, 36.
连结：
Agrawal, R., Wisniewski, J. A., & Woodfolk, J. A. (2011). The role of regulatory T cells in atopic dermatitis. Current Problems in Dermatology, 41, 112-124.
连结：
Akdis, M., Verhagen, J., Taylor, A., Karamloo, F., Karagiannidis, C., Crameri, R., et al. (2004). Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. Journal of Experimental Medicine, 199(11), 1567-1575.
连结：
Andreas, N. J., Kampmann, B., & Mehring Le-Doare, K. (2015). Human breast milk: A review on its composition and bioactivity. Early Human Development, 91(11), 629-635.
连结：

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