探討果寡醣調節D-半乳糖注射Balb/c雄鼠模式之週邊單核球免疫反應及腸道生理

Effects of fructo-oligosaccharide on the immune function of peripheral blood monocytes and intestinal physiology in D-galactose-treated Balb/c mice

徐瑋婷(Wei-Ting Hsu)

指導教授 ： 陳曉鈴

中山醫學大學/健康管理學院/營養學研究所/碩士(2010年)

摘要

目的:本研究有兩個目的。第一是探討以D-半乳糖注射Balb/c雄鼠模式是否成功模擬自然老化對於週邊免疫作用以及腸道功能指標的影響。第二是探討補充果寡醣以及維生素E是否降低D-半乳糖誘發老化對上述指標的效應。
材料方法：12週齡(每組n=10)的Balb/c雄鼠，隨機分組至自然老化組 (NA)、Vehicle控制組 (Control)、DG老化組 (DG)、DG果寡醣組 (DG + FO)和DG維生素E組 (DG + Vit E)。Control組接受皮下注射0.3 mL的0.9%生理食鹽水；各DG組則注射10% D-半乳糖生理食鹽水，51天後犧牲。NA組小鼠不接受注射，於47週齡大犧牲。實驗期間NA、Control和DG組均給予標準小鼠飼料，果寡醣及維生素E飼料則每公斤各添加50 g及2 g的活性成份。犧牲前收集3天糞便，經禁食24小時後以CO2窒息動物，抽取右心房血以分離週邊血液單核球(Peripheral blood monocyte, PBMC)，並採集腸道組織及盲腸內容物以進行後續分析。
結果：結果顯示DG老化小鼠和NA小鼠有相似的老化免疫反應，PBMC之自然殺手細胞活性、對E. coli之噬菌作用皆以DG組最低。補充果寡醣不但使DG效應消失，且提升自然殺手細胞和噬菌作用之活性至比Control及DG + Vit E組更高。PBMC與內毒素脂多醣共培養1天後TNF-α、IL-1β和IL-6分泌量方面，DG和NA組皆較Control組高，但在補充果寡醣及維生素E後可顯著降低TNF-α及IL-1β分泌量；IL-10的分泌量在NA組最低，DG果寡醣組之分泌量則高於Control 組。在腸道絨毛型態方面，初步發現NA和DG組之12指腸與空腸之絨毛高度皆較Control組低，肌肉層也較薄，在補充果寡醣後可改善此現象。小腸黏膜細胞雙醣酶活性方面，初步發現NA組具有較低之麥芽醣酶活性，DG處理無法顯著降低任何黏膜雙醣酶活性，補充果寡醣亦無調整其活性的作用。
盲腸乙酸含量在DG和NA組皆較Control低，但補充果寡醣後有提升的趨勢但無統計上的顯著效應。糞便Bifidobacterium spp. 菌數在DG和NA組較Control組低，但補充果寡醣及α-tocopherol後可增加其含量； Clostridium spp. 菌數在DG組較其他各組高。
結論：本研究結果顯示D-半乳糖可成功誘發Balb/c雄鼠老化相關的週邊單核球免疫反應，降低其非特異性免疫反應並增加發炎反應，並且顯著降低小腸前端絨毛長度及肌肉層厚度、降低盲腸乙酸濃度及糞便bifidobacteria菌數。補充果寡醣後對於週邊單核球免疫反應、小腸前端絨毛長度及肌肉層厚度、和腸道菌相有正向的影響。

關鍵字

老化 ； D-半乳糖 ； 自然殺手細胞 ； 噬菌作用 ； 短鏈脂肪酸 ； Bifidobacterium spp. ； Clostridium spp. ； 雙醣酶腸道型態 ； 細胞激素

並列摘要

Objectives: The aims of study were two folds. The first aim was to determine whether chronic administration of D-galactose (DG) into male Balb/c mice induced aging-related alterations in the peripheral immunity and indices of intestinal function. The second aim was to determine effects of fructo-oligosaccharide (FO) and a positive control,α-tocopherol, on the DG-induced alterations.
Materials and Methods: Twelve-week-old Balb/c mice (n=10/group) were divided into five groups: natural aging (NA), vehicle control (control), D-galactose (DG), DG + FO and DG + Vit E. The control and DG-injected groups were subcutaneously administered with 0.3 mL of 0.9% saline vehicle and 10% (w/v) DG, respectively, for 51 days. The NA group was sacrificed at 47 weeks old without any injection. NA, control and DG groups were fed a standard rodent chow. The DG + FO and DG + Vit E diet was supplemented with 50 g active component of FO and 2 gα-tocopherol per kg diet, respectively. All except NA groups were sacrificed on day 52 before a 24-h fast. Feces were collected three continuous days before sacrifice. Mice were asphyxiated with CO2. The peripheral blood monocyte (PBMC) prepared from blood, intestine and cecum content were collected for further analysis.
Result: Results indicated that NA and DG groups exerted similar immune response. The NK cell and phagocytosis activities of PBMC were the lowest in the DG group, and the greatest in the DG + FO group. After coincubated with lipopolysaccharide for 1 day, PBMC from the NA and DG groups secreted greater amounts of proinflammatory cytokines TNF-α, IL-1βand IL-6；FO andα-tocopherol supplementation significantly reduced the TNF-αand IL-1β
secretion. The secretion of anti-inflammatory cytokines IL-10 was the lowest in the NA group and the greatest in the DG + FO group. The villous heights and of muscular thickness in the duodenum and jejunum were reduced in the NA and DG groups, which was improved with FO. The mucosal maltase activity was reduced in the NA group, but not the DG group. FO did not further enhance this enzyme activity. Cecal acetate concentrations were lower in the NA and DG groups as compared with that in the control; addition of FO tended to, but not significantly, increases the level as compared that in the DG group. Fecal bifidobacteria count was reduced in the DG and NA group as compared with the control group. Both FO and α-tocopherol enhanced the bifidobacteria count in the DG-induced aging mice.
Conclusion: This study indicated that DG induced aging-related alteration in immune response and indices of intestinal dysfunction in Balb/c mice. Supplementation of FO normalized the alterations in the NK cell activity, phagocytosis activity and cytokine profile of PBMC. In addition, FO improved the villous height, muscular thickness in the upper small intestine and colonic microflora profile in the DG-induced aging mice.

並列關鍵字

D-galactose ； aging ； natural killers cell ； phagocytosis ； cytokines ； Bifidobacterium. ； Clostridium. ； short chain fatty acid ； maltase ； intestinal morphology

參考文獻

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