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
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 Ar g ，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 (PH A) 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 Ar g 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。
Effects of arginine supplementation on nutrient met abolism, antioxidant capacity and immune respons
e in burned mice
The purpose of this study was to explore the effect of arginine (Arg) supplementation on nutrients metabolism and immune re sponse in burned mice. There were 4 experiments in this study. In Exp. one, sixty male BALB/c mice were assigned to two gr oups 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 o n the 3 consecutive days after burn with 10 mice on each respective day. The antioxidant enzyme activities and lipid peroxidat ion products in liver, lung and kidney were analyzed to compare the oxidative damage of the organs between the 2 groups. Mi ce 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 phytohemag glutinin (PHA) with different concentrations to investigate the secretion of cytokines. The purpose of Exp.3 was to compare t he 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. Blo od were withdrawn at week 0 for baseline measurement, and 1, 4, 7 weeks for analysis the titers of antibody specific for P. aer uginosa. The results showed that antioxidant enzymes glutathione peroxidase (GSHPx) and superoxide dismutase (SOD) activ ities in the tissues were tend to be lower in the Arg group than the Gly group on the different days after burn. Liver lipid perox idation products were significantly lower in the Arg group at day 1 and 2 after burn than the Gly group. Also, lipid peroxidati on products were lower in kidney homogenates in the Arg group than the Gly group. Results in Exp.2 demonstrated that interl eukin (IL)-10 concentrations in PHA-stimulated splenocyte of the Arg group were significantly higher than the Gly group. Int erferon- in LPS-stimulated splenocyte was also significantly higher in the Arg group than the Gly group. However, no differ ences in IL-2 and IL-4 concentrations were observed in splenocyte between the 2 groups. In addition, the concentration of pro inflammatory factor TNF- secreted by LPS stimulated peritoneal macrophages was also no difference between these two gro ups. About the survival rate of burned mice after P. aeruginosa infection, there was no significant difference between the 2 gro ups 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 tit er 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 suppleme ntation enhances the production of specific antibody, and may also have beneficial effects on some cytokines secretion from st imulated splenocyte in vitro.
