| 摘要: | 病菌感染、胃酸或異物性吸入，均會產生急性肺損傷(Acute lung injury, ALI)的情況，嚴重者甚會引起急性呼吸窘迫症候群(Acute respiratory distress syndrome, ADRS)。麩醯胺（Glutamine, GLN）是體內含量最高的胺基酸，重大傷病時為條件性必須胺基酸，許多動物與臨床試驗均顯示補充GLN可有效改善病程與預後，本研究以氣管給予小鼠HCl，模擬人類胃酸逆流的吸入性肺炎，選用脂多醣(lipopolysaccharide, LPS) 代替病菌感染，目的在於探討GLN對於HCl及LPS誘導急性肺損傷是否具有保護效應。
採用6週齡BALB/c品系雌鼠76隻，採用2 × 2 實驗設計，雙因子為GLN與ALI。小鼠隨機分兩組，控制組(15% 新鮮油, C)、GLN組(15% 新鮮油+ 4.17% GLN, G)，飼料成分依照AIN-93G 配方調整脂質比例為15%高油飼料，飼料與飲水皆採自由攝取(ad libitum)。飼養10天後，除正常組(C、G)外，ALI組(C、G)分別由氣管注入(intractracheal instillation) HCl (0.2N, 2 mL/kg BW) 及LPS (5 mg/kg BW)，3小時後犧牲，採集血液、肺臟、肺泡沖洗液（Broncho alveolar lavage fluid, BALF），分析血清全身性發炎指標、肺臟組織、BALF中發炎指標、氧化相關指標與相關mRNA基因表現。
由Two-way ANOVA 統計得知，ALI誘導效應方面，小鼠心臟、肺臟、肝臟及脾臟之相對重量有顯著增加；抗氧化酵素catalase、GR、GST、Se-GPx顯著降低；免疫與發炎系統結果，血清IL-6顯著增加，肺臟組織IL-1β、IL-6、TNF-α濃度顯著增加、IL-10顯著降低，Myeloperoxidase (MPO)活性明顯增加。添加GLN的效應方面，得知脾臟重量及相對重量有降低趨勢；GR、Se-GPx酵素活性有趨勢增加；免疫與發炎系統，添加GLN使小鼠血清IL-6顯著下降，肺臟組織IL-1β、IL-6、TNF-α濃度顯著下降，MPO活性無顯著差異；BALF之IL-1β、IL-10和RAGE (Receptor for advanced glycation end products) 均顯著降低，IL-6、TNF-α無顯著差異。相關分子之mRNA表現結果，COX-2、IL-6、IL-1β、NOX1顯著降低，IFN-γ顯著增加；存活率結果，添加GLN能提高ALI小鼠之存活率。RAGE標的於第一型肺泡上皮細胞，在ALI 模式中其濃度顯著提高會加重肺水腫病況，進一步使促發炎細胞激素與助氧分子的產生，因此推測短期添加GLN可降低小鼠ALI肺部損傷程度。
結論：氣管滴入HCl 與LPS 誘發小鼠急性肺損傷的動物模式中，飼料補充GLN 10天，確實可降低血清及肺臟組織促發炎相關細胞激素含量，明顯降低肺部損傷指標RAGE，而且提高存活率。
Glutamine (GLN) is known to be a conditionally essential amino acid during critical illness and injury. GLN has been shown to improve the outcome in clinical and experimental sepsis. However, mechanisms of GLN remain unknow. The purpose of this study was to determine the GLN's protective effects on acid and LPS-induced aute lung injury (ALI) in mice.
We used 2×2 factorial design. The two factors were identified as GLN and ALI. 6-wk-old female BALB/c mice (n=76) were used and divided into two groups, including C group (15% fresh soybean oil) and G group (15% fresh soybean oil + 4.17% GLN). After the 10 days feeding period, half of mice were induced ALI by intratracheal administration of HCl (0.2N, 2 mL/kg BW) and LPS (5 mg/kg BW). All animals were sacrificed at 3 h after intratracheal instillation acid and LPS. Blood, bronchoalveolar lavage fluid (BALF) and lung were collected and assessed for associated parameters, such as inflammatory cytokines, antioxidant defense system and the gene expression of related moleculars.
The main effect of ALI from the two-way ANOVA results, we found that relative lung weight were significantly increased, the activities of catalase, glutathione reductase (GR), glutathione S-transferase (GST) and glutathione peroxidase (Se-GPx) were all significantly decreased in ALI-induced mice. ALI-treated mice caused a significant increase on the levels of IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) activity in the lung. These results showed that acid and LPS administration resulted in higher inflammatory cytokines production and lower antioxidative enzymes activities. In the GLN group, levels of IL-6, IL-1β and TNF-α were all significantly
decreased, but did not affect the increased activities of MPO in the lung. The GLN group had lower IL-1β, IL-10 and Receptor for advanced glycation end-products (RAGE) in the BALF. RAGE is a novel marker of alveolar epithelial type I cell injury and is elevated in the pulmonary edema fluid with ALI. The mRNA abundances of COX-2, IL-6, IL-1β and NOX1 all significantly decreased by GLN treatment in ALI-induced mice, there was a significant increase in IFN-γ mRNA in the lung. The livability of mice showed that GLN improved survival. Therefore, we speculated that GLN could attenuates lung injury induced by acid and LPS administration.
In conclusion, our results indicated that GLN caused a decrease in inflammatory cytokines and RAGE. We suggested that GLN had a protective effect on HCl- and LPS-induced ALI in mice. |
