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摘要:
目的 探究烧伤小鼠能量与物质代谢随伤后时间变化的规律。 方法 该研究为实验研究。取16只8~10周龄雄性C57BL/6N小鼠,按照随机数字表法分为行假伤处理的假伤组和行烫伤（以下称烧伤）处理的烧伤组,每组8只。伤后1~14 d,采用小动物代谢监测系统动态监测小鼠每日饮水量、摄食量、活动量、能量消耗、静息能量消耗（REE）等指标并计算小鼠伤后1~14 d每天体重变化量、呼吸熵,伤后1~14 d累积饮水量、摄食量、活动量,伤后1、3、7、10、14 d能量消耗及夜晚的碳水化合物与脂肪氧化率。取48只8~10周龄雄性C57BL/6N小鼠,按照随机数字表法选取8只做假伤处理,剩余40只做烧伤处理。采集烧伤小鼠伤后1、3、7、10、14 d及假伤小鼠伤后1 d心脏血液并获取血浆。采用液相质谱分析鉴定血浆代谢物（以下简称代谢物）的种类,并确定其随时间变化的规律。将假伤小鼠与烧伤小鼠伤后1 d的代谢物进行比较,同时对烧伤小鼠伤后1、3、7、10、14 d的代谢物进行分析。进一步分析筛选出的代谢物与REE的相关性,并对相关代谢物进行京都基因与基因组百科全书（KEGG）通路富集分析。 结果 烧伤组与假伤组小鼠伤后1~14 d每日及累积饮水量、摄食量比较,差异均无统计学意义（P>0.05）。与假伤组相比,烧伤组小鼠伤后2~14 d体重变化量均显著降低（P<0.05）,伤后1~14 d白天及夜晚累积活动量均显著降低（P<0.05）,伤后1~14 d每日活动量失去正常昼夜节律,伤后1 d能量消耗显著降低（P<0.05）但伤后7、10、14 d能量消耗均显著升高（P<0.05）,伤后4~14 d的REE均显著升高（P<0.05）,伤后1~7 d的呼吸熵在夜晚显著降低（P<0.05）,烧伤组小鼠伤后1、3 d夜晚的碳水化合物氧化率均明显降低（P<0.05）,烧伤组小鼠伤后1、3、7 d夜晚的脂肪氧化率均明显升高（P<0.05）。从假伤和烧伤小鼠血浆样本中共鉴定出450种代谢物,其中253种代谢物表现出特定的时间变化规律,可聚类为5种不同的模式。烧伤小鼠伤后有85种代谢物发生了有统计学意义的变化（P<0.05）。与REE显著相关的代谢物共有40种（变量投影重要性>1.0）,其中排序靠前的代谢物包括亚油酸、油酸、葡萄糖等,且以脂肪酸类为主。KEGG通路富集分析显示,脂肪酸的合成与降解,异亮氨酸、色氨酸等氨基酸的合成与代谢以及糖酵解/糖异生通路与REE的显著关联（P<0.05）。 结论 烧伤处理及伤后时间均能显著影响小鼠的能量代谢模式及物质代谢特征。小鼠的高代谢状态可持续至烧伤后14 d,在此过程中多种脂肪酸、氨基酸及葡萄糖代谢通路与高代谢存在关联。 Abstract:Objective To investigate the temporal dynamics of energy metabolism and material metabolism in burned mice. Methods This study was an experimental study. Sixteen male C57BL/6N mice aged 8-10 weeks were randomly assigned using a random number table to sham injury group (n=8) treated with sham injury and burn group (n=8) treated with scald injury. From days 1 to 14 post-burn, the daily water intake, food intake, activity levels, energy expenditure, and resting energy expenditure (REE) of mice were dynamically monitored using a small animal metabolic monitoring system. Daily body weight changes and respiratory entropy from days 1 to 14 post-burn, cumulative water intake, food intake, and activity levels from days 1 to 14 post-burn, respiratory entropy from days 1 to 7 post-burn, energy expenditure on days 1, 3, 7, 10, and 14 post-burn, and nighttime carbohydrate and fat oxidation rates on days 1, 3, 7, 10, and 14 post-burn were calculated. Forty-eight male C57BL/6N mice aged 8-10 weeks were randomly assigned using a random number table: 8 mice received sham injury, and the remaining 40 mice underwent scald injury. Cardiac blood samples were collected from burn mice at 1, 3, 7, 10, and 14 days post-burn and from sham mice at 1 day post-burn. Plasma was obtained by centrifugation. Liquid chromatography-mass spectrometry was employed to identify plasma metabolites (hereinafter referred to as metabolites) and determine their temporal dynamics. Metabolites from sham mice at 1 day post-burn were compared with those from burn-injured mice at the same time point, and metabolites from burn-injured mice at 1, 3, 7, 10, and 14 days post-burn were analyzed. Additionally, the correlation between the selected metabolites and REE was analyzed, and the relevant metabolites underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Results Compared with those in sham group, the daily and cumulative water intake and food intake of mice in burn group from days 1 to 14 post-burn showed no statistically significant differences overall (P>0.05); the body weight changes of mice in burn group were significantly reduced from days 2 to 14 post-burn (P<0.05); cumulative daytime and nighttime activity levels of mice in burn group were significantly reduced from days 1 to 14 post-burn (P<0.05); daily activity levels of mice in burn group lost normal circadian rhythms from days 1 to 14 post-burn; energy expenditure of mice in burn group was significantly reduced on day 1 post-burn (P<0.05) and significantly increased on days 7, 10, and 14 post-burn (P<0.05); the REE of mice in burn group was significantly increased from days 4 to 14 post-burn (P<0.05); respiratory entropy of mice in burn group was significantly reduced during nighttime from days 1 to 7 post-burn (P<0.05), while no significant difference was observed during daytime (P>0.05); carbohydrate oxidation rates of mice in burn group were significantly reduced at night on days 1 and 3 post-burn (P<0.05); fat oxidation rates of mice in burn group were significantly increased at night on days 1, 3, and 7 post-burn (P<0.05). A total of 450 metabolites were identified in plasma samples from mice. Among these, 253 metabolites exhibited specific temporal patterns and could be clustered into five distinct patterns. Eighty-five metabolites of burn-injured mice exhibited statistically significant changes following injury (P<0.05). Forty metabolites showed significant association with REE, with the top metabolites including linoleic acid, oleic acid, glucose, etc. KEGG pathway enrichment analysis revealed significant associations between REE and pathways including fatty acid synthesis and degradation, amino acid (e.g., isoleucine, tryptophan) synthesis and metabolism, and glycolysis/gluconeogenesis (P<0.05). Conclusions Both burn injury and time significantly influence energy metabolism patterns and material metabolism characteristics in mice. The hypermetabolic state persisted up to 14 days post-burn, with multiple fatty acid, amino acid, and glucose metabolic pathways associated with this elevated metabolism. -
Key words:
- Burns /
- Metabolomics /
- Energy Metabolism /
- Hypermetabolism /
- Material Metabolism /
- Metabolic Cage
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参考文献
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图 1 2组小鼠伤后1~14 d的饮水量、摄食量、体重变化量及活动量变化（样本数为8）。1A. 每日饮水量[M(Q1,Q3)],分组因素主效应,F<0.01,P=0.955；时间因素主效应,F=7.78,P<0.001；二者交互作用,F=1.30,P=0.220；1B.每日摄食量[M(Q1,Q3)],分组因素主效应,F=0.30,P=0.596；时间因素主效应,F=25.14,P<0.001；二者交互作用,F=0.99,P=0.463；1C.累积饮水量[M(Q1,Q3)],P>0.999；1D.累积摄食量[M(Q1,Q3)],P>0.999；1E.每日体重变化量（
注：假伤组、烧伤组小鼠背部分别浸入37 ℃温水、90 ℃热水造成假伤、烧伤；图1G中灰色背景表示夜晚,白色背景表示白天；活动量用打断红外线次数表示
图 2 2组小鼠伤后各时间点的能量代谢变化（样本数为8）。2A.伤后1~14 d白天和夜晚的能量消耗（
注：假伤组、烧伤组小鼠背部分别浸入37 ℃温水、90 ℃热水造成假伤、烧伤；图2A、2D中灰色背景表示夜晚,白色背景表示白天；1 kcal=4.18 kJ
图 3 假伤和烧伤小鼠血浆代谢物的时序性变化特征
注：0、1、2、3、4指5种不同模式；纵轴上的s1指假伤后1 d,1、3、7、10、14为烧伤后时间；血浆代谢物：1甘氨酸、2 N-乙酰甘氨酸、3亚精胺、4岩藻糖、5己酰甘氨酸、6果糖、7辛酰甘氨酸、8生物素、9棕榈油酸、10 13,14-二氢-15-酮前列腺素 A2、11二十二碳六烯酸乙酯、12脱氧胆酸、13胆酸、14 β-鼠胆酸、15牛磺鹅脱氧胆酸、16丙氨酸、17乙酰乙酸甲酯、18异喹啉、19喹啉、20顺式-4-羟基-D-脯氨酸、21葫芦巴碱、22 水杨酸、23水苏碱、24蛋氨酸、25氢化肉桂酸、26肉碱、27马尿酸、28 5-羟基吲哚-3-乙酸、29肉桂酰甘氨酸、30脯氨亮氨酸、31胞壁酸、32黄豆苷元、33胆红素、34巴豆酸、35琥珀酸半醛、36 3-羟基丁酸、37延胡索酸、38琥珀酸、39苹果酸、40 α-酮戊二酸、41谷氨酰胺、42 N-异戊酰甘氨酸、43紫苏酸、44 3-甲基组氨酸、45 N-乙酰基-D-别异亮氨酸、46 N-甲酰甲硫氨酸、47柠檬酸、48吲哚-3-硫酸、49肉豆蔻酸、50胞苷、51十六碳烯酸、52己酰肉碱、53脱落酸、54腺苷、55反式-10-庚碳烯酸、56亚油酸、57二十碳二烯酸、58二十碳烯酸、59花生酸、60二十二碳五烯酸、61肾上腺酸、62二十二碳三烯酸、63二十二烯酸、64芥酸酰胺、65二十二碳六烯酸甲酯、66神经酸、67棕榈酰肉碱、68胸腺嘧啶、69异亮氨酸、70乙酰胆碱、71谷氨酸、72黄嘌呤、73 N-乙酰基-L-亮氨酸、74 12,13-diHOME、75棕榈酰乙醇酰胺、76维甲酸、77二十碳五烯酸、78 N-乙酰神经氨酸、79癸酰肉碱、80丙酮酸、81 3-烯丙基-2-羟基苯甲酸、82乙氧基喹啉、83胱氨酸、84还原型谷胱甘肽、85氧化型谷胱甘肽；不同颜色反映丰度的高低；以假伤后1 d数据为基准,对烧伤后各时间点数据进行归一化处理
图 4 烧伤和假伤小鼠血浆代谢物与静息能量消耗的正交偏最小二乘（OPLS）回归分析及京都基因与基因组百科全书通路富集分析。4A.OPLS评分图,R2X=0.723,R2Y=0.835,Q2=0.68；假伤后1 d的样本数为7,烧伤后1、3、7、10、14 d的样本数依次为5、5、6、5、8；4B.OPLS模型的200次置换检验图；4C.变量投影重要性评分排序前22的血浆代谢物；4D.京都基因与基因组百科全书通路富集分析
注：图4A中1.137 44是纵坐标轴缩放系数,to[1]表示第一正交得分,即与应变量（静息能量消耗）无关的组内变化,反映干扰或批次效应等无关变异；1.008 23指横坐标轴的缩放系数,t[1]表示第一预测得分,即与应变量（静息能量消耗）最相关的潜在变量方向,反映其对应变量的预测能力；图4B中R²指决定系数,Q²指交叉验证决定系数；相关系数表示置换分类与真实分类之间的相关性；图4C中变量投影重要性评分反映该血浆代谢物在解释静息能量消耗中的重要性；血浆代谢物：1亚油酸、2油酸、3葡萄糖、4棕榈酸、5棕榈油酸、6异亮氨酸、7硬脂酸、8 3-吲哚硫酸、9 3-羟基丁酸、10肉豆蔻酸、11花生四烯酸、12色氨酸、13乳酸、14缬氨酸、15乙酰基β-甲基胆碱、16二十二碳六烯酸、17肉碱、18顺-11-二十碳烯酸、19磷酸、20胆碱、21丙酮酸、22甜菜碱
Table 1. 假伤和烧伤小鼠5种时序性变化特征模式中各2种代表性血浆代谢物时序变化比较（样本数≥5,
模式类型 血浆代谢物 假伤后1 d 烧伤后1 d 烧伤后3 d 烧伤后7 d 烧伤后10 d 烧伤后14 d t值 P值 F值 P1值 模式0 甘氨酸 1.00±0.15 0.38±0.17 0.39±0.26 0.24±0.13 0.84±0.12 0.93±0.20 6.65 <0.001 16.46 <0.001 牛磺鹅去氧胆酸 1.00±0.45 0.34±0.15 0.38±0.16 0.50±0.26 0.66±0.23 0.62±0.27 3.56 0.008 0.94 0.415 模式1 丙氨酸 1.00±0.23 1.63±0.23 0.92±0.52 1.16±0.46 0.45±0.08 0.53±0.22 4.72 <0.001 13.08 0.003 马尿酸 1.00±0.43 0.59±0.30 2.46±0.75 2.88±1.65 0.57±0.26 1.66±0.57 1.95 0.080 5.33 0.046 模式2 谷氨酰胺 1.00±0.16 1.50±0.25 0.96±0.13 1.01±0.16 0.89±0.17 0.87±0.10 4.24 0.002 13.26 0.001 3-甲基组氨酸 1.00±0.08 1.46±0.27 0.83±0.18 0.61±0.18 0.72±0.21 0.78±0.25 3.71 0.016 15.05 <0.001 模式3 谷氨酸 1.00±0.21 2.21±0.46 1.64±0.35 1.71±0.43 1.08±0.18 1.42±0.28 6.08 <0.001 9.26 0.022 12,13-diHOME 1.00±0.62 4.83±1.07 3.15±1.45 0.68±0.20 2.69±1.04 2.51±1.02 7.18 <0.001 10.72 0.004 模式4 还原型谷胱甘肽 1.00±0.62 2.86±0.75 3.66±0.86 3.46±1.15 1.94±0.80 2.15±0.73 4.54 0.002 5.94 0.018 氧化型谷胱甘肽 1.00±0.46 3.53±0.32 4.02±0.89 4.10±1.15 2.24±0.63 2.71±1.36 10.71 <0.001 3.79 0.056 注：假伤后1 d的样本数为7,烧伤后1、3、7、10、14 d的样本数依次为5、5、6、5、8；以假伤后1 d数据为基准,对烧伤后各时间点数据进行归一化处理；t值、P值为烧伤后1 d与假伤后1 d指标比较所得,F值、P1值为烧伤后1、3、7、10、14 d各指标总体比较所得 
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