葉酸營養狀況對高油飲食之實驗性自體免疫腦脊髓炎小鼠與T淋巴球活性的影響

Abstract

多發性硬化症是一種中樞神經系統中去髓鞘化的神經發炎性疾病，屬於自體免疫疾病，盛行率有逐年提高的趨勢。多發性硬化症常用的動物模式為實驗性自體免疫腦脊髓炎(EAE)，其中Th1與Th17是誘發疾病主要的T細胞，而調節性T細胞被認為對病程發展有保護的功能。葉酸為人體必需的水溶性維生素，缺乏時會影響免疫細胞的活化與增生。本實驗首先用EL4小鼠T細胞株來探討不同葉酸營養狀態對T細胞活性的影響，接著利用實驗性自體免疫腦脊髓炎小鼠來探討葉酸營養狀況對多發性硬化症的影響。 細胞實驗以不同葉酸濃度之培養基培養EL4小鼠T細胞株三天後，以PMA/ ionomycin刺激3至24小時，測量上清液IL-2與IL-4濃度、收集細胞測IL-2 與其轉錄因子的mRNA及NF-B表現量。結果顯示，葉酸缺乏時會顯著降低細胞生長，但促進IL-2的分泌卻不影響IL-4的分泌、促進NFB與NFAT5 mRNA的表現。表示在葉酸缺乏下可能會透過NFB與NFAT5來影響EL4 T細胞株的IL-2表現。 動物實驗選用4週齡C57BL/6公鼠，隨機分4組，分別餵食不同葉酸含量的AIN-93G高油飼料(油脂佔54%總熱量)，葉酸含量為0 mg/kg diet (Hf0組)、2 mg/kg diet (Hf1組)、20 mg/kg diet (Hf10組)，和短期(誘發前三周)改餵食高油飼料的NF/HF組。餵食10週後以MOG35-55誘發EAE，期間觀察病情與體重，於誘發後15天犧牲，分析腦、脊隨、脾臟與腿部淋巴結T細胞的比率，脾臟與腿部淋巴結細胞分泌細胞激素的濃度，測量腦部細胞激素mRNA表現量。結果顯示，Hf0組淋巴結細胞分泌較多的IL-2、IFN-、IL-6與IL-17，脾臟細胞分泌較多的IL-2與較少的IL-10和較低的增生能力。腦部促發炎激素的mRNA表現量較高，但浸潤於中樞神經系統中的Th1、Th17與調節型T細胞較少。Hf10組淋巴結細胞分泌較少的IFN-、IL-10，脾臟細胞分泌較多的IL-2、IFN-與較少的IL-10。腦部促發炎激素的mRNA表現量較高，有較多的調節型T細胞浸潤入中樞神經系統內，但卻有較嚴重的病情分數指標。NF/HF組脾臟細胞分泌較多的IL-6與IL-17，分泌較少的IL-10。腦部促發炎激素的mRNA表現量較高，但浸潤於中樞神經系統中的Th1、Th17與調節型T細胞較少。綜合以上，可知在高油飲食EAE模式下，葉酸的缺乏會造成較嚴重的周邊及腦部發炎反應，但有較少的Th1、Th17與調節型T細胞浸潤入中樞神經系統內，故葉酸缺乏對病情的影響並不顯著，然而葉酸的補充會造成較嚴重的EAE病情與腦部發炎反應，還須進一步的實驗來做驗證。

Multiple sclerosis (MS) is the prototypical inflammatory demyelinating disease of the central nervous system (CNS). Experimental Autoimmune Encephalomyelitis (EAE), which is a common animal model of Multiple sclerosis, is mediated by myelin specific CD4+ helper T cells. Th1 as well as Th17 phenotype is the major T helper cells in EAE pathogenesis. It is considered that regulatory T cell plays a protective role in pathogenic process. Recent studies showed that folate deficiency may enhance the inflammatory response of macrophages. Besides, folate can also reduces the activiton and proliferation of immune cell. In this study, we used EL4 T lymphocyte to investigate the effects of folate status on immune activation in vivo, and an animal model was used to investigate the effects of folate status on EAE-induced mice, which were fed with high fat diet. The EL4 T cell line was cultured in different folate status medium. The data showed that folate deficiency significantly increased IL-2 secretion and its mRNA expression. Moreover, the IL-2 transcription factor NFAT5 and NFκB were upregulated. The results showed that folate deficiency may promote higher IL-2 secretion via activating NFκB and NFAT5 pathway. The male C57BL6/J mice were divided into four groups fed an AIN-96G high fat diet (54% of total calorie) which containing either 0 (Hf0), 2 (Hf1), 20 (Hf10) mg folate per kg diet. Anthor group was fed AIN-96G hight fat diet only for 3 weeks before EAE induced (NF/HF).After 10 weeks, use MOG35-55 to induced EAE mode, and sacrifice at 15 day after EAE induced.The data showed that the C57BL/6 male mice, which were fed folate-deficient high fat diet or short-period high fat diet, had serious peripheral and brain inflammation. There were less Th1, Th17, and regulatory T cells that infiltrated into CNS of these mice. However, there is no sign of serious clinical symptoms on EAE. In comparison, the mice fed with folate-supplement high fat diet had more serious clinical symptoms on EAE and brain inflammation. Besides, there were more regulatory T cells infiltrated into CNS.