Arginine添加對燒傷老鼠營養素代謝,抗氧化狀態及免疫功能之影響

Effects of arginine supplementation on nutrient metabolism, antioxidant capacity and immune response in burned mice

蔡蕙如(Hui-Ju Tsai)

指導教授 ： 葉松鈴 ； 商惠芳

臺北醫學大學/公共衛生暨營養學院/保健營養學研究所/碩士(2001年)

摘要

本研究之目的在探討飲食中添加Arg對燒傷老鼠營養素代謝、抗氧化狀態及免疫反應之影響，同時為深入暸解Arg對老鼠免疫反應之影響，我們亦探討給予綠膿桿菌疫苗後Arg之添加對燒傷後感染所造成之影響。實驗對象為雄性BALB/c小鼠，實驗共分成四個部分，實驗一將老鼠分成2組，每組各30隻：一組在飼料中添加佔總熱量2%的Arg，另一組以等氮量之Glycine (Gly)代之，兩組除添加之胺基酸組成不同外，其餘飼料成分均相同。飼料共餵食四週，於四週後以酒精引致體表30% 之燒傷，由於老鼠燒傷之後三天內是異化作用最嚴重的時期，故分別於燒傷後之1、2、3天各犧牲10隻以比較兩組在不同時間點營養素代謝及各器官遭受氧化傷害之情形。實驗二實驗動物之飼養、分組及燒傷方法同實驗一，為瞭解Arg對燒傷老鼠免疫細胞之影響，老鼠飼養四週之後引致燒傷，取出老鼠燒傷24小時後之腹腔巨噬細胞及脾臟細胞以不同濃度之mitogen：lipopolysaccharide (LPS)及phytohemagglutinin (PHA)刺激之，以測定其細胞激素的分泌量。實驗三實驗動物之飼養、分組及燒傷方法同實驗一，但在燒傷後於燒傷部位皮下接種綠膿桿菌以引致感染並觀察存活率之差異。實驗四同樣分成兩組，每組各10隻，飼料共餵食7週，在第0週時先採血做為基準值，並於皮下注射綠膿桿菌疫苗，接著於第4週時再追加一劑，在第7週結束時引致燒傷，於燒傷24小時後將老鼠犧牲，分析第0、4、7週及燒傷24小時後老鼠血中綠膿桿菌抗體產生量。實驗一之結果顯示，在glutathione peroxidase的活性方面，肝臟及肺臟在燒傷後第1天及第3天，腎臟在燒傷後第1、2天，Arg組顯著較Gly組為低。Superoxide dismutase活性方面，Arg組之肝臟在燒傷後第3天，肺臟在燒傷後第1、2天，腎臟在燒傷後第1、3天均顯著較Gly組為低。在過氧化物之生成量方面，肝臟在燒傷後第1天及第2天，腎臟則在燒傷後之第2天，Arg組過氧化物之生成量顯著較Gly組為低。實驗二之脾臟細胞以PHA刺激前及刺激後，Arg組的interleukin(IL)-10之分泌量顯著較Gly組為高， IL-4則兩組之間沒有差異，interferon-γ (IFN-γ)則以LPS 20ng及50ng刺激後Arg較Gly組為高。在巨噬細胞方面，我們偵測與發炎反應有關細胞激素TNF-?的變化，結果Arg組與Gly組間無顯著差異。實驗三之燒傷老鼠被綠膿桿菌感染後存活率方面兩組之間無明顯差異。實驗四之結果顯示，隨著實驗週數的增加，抗體的生成量也會增加，且燒傷之後會使抗體的產生量減少，Arg組之抗體生成量在第4、7週及燒傷後，顯著較Gly組為高。此研究之結果顯示，飲食中添加Arg可顯著降低燒傷引致之器官過氧化物的產生，同時器官中抗氧化酵素之活性亦較低，推測Arg添加應可使器官因燒傷引致之氧化傷害降低，同時添加Arg可促進燒傷老鼠體內特異性抗體的產生，並使其脾臟細胞和巨噬細胞在部分細胞激素的分泌上有較佳之反應。

關鍵字

燒傷 ； 精胺酸 ； 細胞激素 ； 抗體 ； 脂質過氧化

並列摘要

The purpose of this study was to explore the effect of arginine (Arg) supplementation on nutrients metabolism and immune response in burned mice. There were 4 experiments in this study. In Exp. one, sixty male BALB/c mice were assigned to two groups and fed with diets supplemented with either Arg (2% of total calorie) or an equivalent amount of nitrogen in the form of glycine (Gly). After 4 weeks, all mice were received 30% body surface area burn injury. Mice of each group were sacrificed on the 3 consecutive days after burn with 10 mice on each respective day. The antioxidant enzyme activities and lipid peroxidation products in liver, lung and kidney were analyzed to compare the oxidative damage of the organs between the 2 groups. Mice grouping, feeding and burning procedures were the same in Exp. 2, 3, 4. In Exp. 2, mice were sacrificed 24 hours after burn, and the peritoneal macrophage and spleen cells were harvested and stimulated by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) with different concentrations to investigate the secretion of cytokines. The purpose of Exp.3 was to compare the survival rate of burned mice after Pseudomonas aeruginosa infection between the 2 groups. Thirty mice were infected with P. aeruginosa after burn, and survival was recorded twice per day for 2 weeks. Mice in the Exp. 4 were fed experimental diets for 7 weeks. All mice were immunized with vaccine of Pseudomonas aeruginosa on the first day and boostered at week 4. Blood were withdrawn at week 0 for baseline measurement, and 1, 4, 7 weeks for analysis the titers of antibody specific for P. aeruginosa. The results showed that antioxidant enzymes glutathione peroxidase (GSHPx) and superoxide dismutase (SOD) activities in the tissues were tend to be lower in the Arg group than the Gly group on the different days after burn. Liver lipid peroxidation products were significantly lower in the Arg group at day 1 and 2 after burn than the Gly group. Also, lipid peroxidation products were lower in kidney homogenates in the Arg group than the Gly group. Results in Exp.2 demonstrated that interleukin (IL)-10 concentrations in PHA-stimulated splenocyte of the Arg group were significantly higher than the Gly group. Interferon-? in LPS-stimulated splenocyte was also significantly higher in the Arg group than the Gly group. However, no differences in IL-2 and IL-4 concentrations were observed in splenocyte between the 2 groups. In addition, the concentration of proinflammatory factor TNF-? secreted by LPS stimulated peritoneal macrophages was also no difference between these two groups. About the survival rate of burned mice after P. aeruginosa infection, there was no significant difference between the 2 groups in Exp. 3. Measurements in Exp.4 indicated that the antibody titer specific for P. aeruginosa was increased in accordance with the proceeding of the experiment, while the titer of antibody was significantly reduced after burn injury. The antibody titer at week 4 and 7 were significantly higher in the Arg group than the Gly group. In summary, the results in this study suggest that burned mice supplemented with Arg had lower oxidative stress than the Gly supplemented group. Besides, Arg supplementation enhances the production of specific antibody, and may also have beneficial effects on some cytokines secretion from stimulated splenocyte in vitro.

並列關鍵字

burn ； aiginine ； cytokine ； antibody ； lipid peroxidation

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