- 學位論文
葉酸營養狀況對自體免疫小鼠組織L-isoaspartate 含量和免疫反應的影響
Folate status affects tissue L-isoaspartate contents and immune responses in autoimmune mice
摘要
摘要 自體蛋白質經後修飾作用生成 L-isoaspartate (isoAsp)，可能會改變蛋白質的弁遄B活性，和免疫耐受性。蛋白質甲基轉移酵素 (protein L-isoaspartyl methyltransferase, PIMT) 可進行此種修復，而葉酸在其中可能扮演一個輔因子幫助修復 isoAsp 。本實驗旨在探討自體免疫鼠和正常鼠，葉酸營養狀況對 isoAsp堆積的影響。實驗 (一)，以鼠齡10週大的 NZB/W F1 和 BALB/c 雌鼠，隨機分為四組，分別餵食以 AIN-76 為基礎、葉酸含量不同的飲食。各組葉酸餵食劑量分別為0 mg/kg diet (FD 組)、2 mg/kg diet (F1 組)、4 mg/kg diet (F2 組)，和 10 mg/kg diet (F5 組)，進行實驗達5個月。實驗 (二)，以鼠齡 6週大的 MRL/lpr 和 BALB/c 雌鼠，隨機分為四組，各組葉酸餵食劑量分別為 0 mg/kg diet (FD 組)、2 mg/kg diet (F1 組)、10 mg/kg diet (F5 組) 和 20 mg/kg diet (F5 組)，進行實驗10週，觀察飲食葉酸的影響。實驗結果顯示血漿、血球、肝臟和腎臟中葉酸濃度會隨飲食葉酸攝取量增加而顯著增加，在腦和脾臟則無顯著差異。葉酸的補充可以降低組織脂質過氧化 TBARS 值的量。在實驗 (一) NZB/W F1 鼠葉酸補充會顯著增加脾臟 isoAsp 含量，且腎臟 isoAsp 含量和尿蛋白成正相關 (p=0.0436)。在排除葉酸缺乏組後，腎臟 isoAsp 含量和抗雙股或單股去氧核糖核酸自體抗體亦成正相關 (p=0.0137; p=0.0472)。在實驗 (二) MRL/lpr 鼠腎臟 isoAsp 含量也顯著堆積，血漿葉酸含量和抗雙股去氧核糖核酸自體抗體成正相關 (p=0.0308)，且在排除葉酸缺乏組後，血漿葉酸含量和腎臟 isoAsp 含量亦成正相關 (p<0.0001)。此外，自體免疫鼠給予葉酸補充，會增加 B 細胞增生、自體抗體生成、有較高的 PGE2 分泌並有加重腎炎情形。IsoAsp 含量在年老的小鼠含量較高。補充葉酸會增加 isoAsp堆積，可能會加重自體免疫疾病。缺乏葉酸isoAsp會增加，但是會降低免疫反應，故對於自體免疫病程是有益的，相較於補充葉酸，可延長其生命期。補充葉酸所造成的自體免疫鼠和正常鼠 isoAsp 含量增加的機制，仍待進一步探討。
並列摘要
Abstract Self-proteins undergo spontaneous posttranslational modifications, such as isoaspartyl form (IsoAsp), may alter the protein function, activity and alter the maintenance of peripheral immune tolerance. The enzyme protein L-isoaspartyl methyltransferase (PIMT) is involved in the repair of various proteins and folate may act as a cofactor to help repair IsoAsp site. This study investigated the effect of folate status and L-isoaspartate accumulation on autoimmune-prone mice and BALB/c mice. In exp1, ten-week-old female NZB/W F1 and BALB/c mice were divided into four groups fed an AIN 76-based diet containing either 0 (FD), 2 (F1), 4 (F2), or 10 (F5) mg folic acid/kg diet for 5 months. In exp2, six-week-old female MRL/lpr mice and BALB/c mice were divided into four groups fed an AIN 76-based diet containing either 0 (FD), 2 (F1), 10 (F5), or 20 (F10) mg folic acid/kg diet for 10 weeks to investigate the effects of dietary folate. The results showed that the folate concentrations in plasma, RBC, liver, and kidney significantly increased with increment of dietary folate, but no changes in brain and spleen. Supplementation with folate was found to have a preventive effect on the elevation of the TBARS values in tissues. In exp1 and exp2, we found that folate supplementation did not eliminate IsoAsp contents in mice. In exp1, the isoAsp content of NZB/W F1 mice accumulated with folate supplementation significantly in spleen, and isoAsp content in kidney was positive correlation with proteinuria (p=0.0436). IsoAsp content in kidney was positive correlation with anti-ds DNA autoantibodies and anti-ss DNA autoantibodies (p=0.0137; p=0.0472) when FD group was excluded. In exp2, the isoAsp content of MRL/lpr mice accumulated significantly in kidney, and folate concentration in plasma was positive correlation with anti-ds DNA autoantibodies (p=0.0308), and positive correlation with isoAsp content in kidney (p<0.0001) when FD group was excluded. In addition, autoimmune-prone mice fed folate supplement diet increased B cell proliferation, autoantibodies production, had higher PGE2 and enhanced lupus nephritis. The levels of isoAsp contents were higher in the older mice. Folate supplementation increased IsoAsp accumulation and may aggravate autoimmune disease. The mice fed folate deficient diet also had higher IsoAsp contents but lower immune response which may benefit autoimmune progress and had longer lifespan compared to the folate supplement group. Further studies are needed to clarify the mechanisms how folate supplementation increased isoAsp contents in both autoimmune and normal mice.