Study on temporal change characteristics of energy and material metabolism in burned mice
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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每日体重变化量、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。对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的代谢物进行比较。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、摄食量总体比较,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）,仅伤后1~7 d的呼吸熵在夜晚显著降低（P<0.05）,伤后1、3 d夜晚的碳水化合物氧化率均明显降低（P<0.05）,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,脂肪酸的生物合成与降解,异亮氨酸、色氨酸等氨基酸的生物合成与代谢以及糖酵解/糖异生通路均与REE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。烧伤小鼠的高代谢状态可持续至伤后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 change patterns of energy and material metabolism in burned mice. Methods This study was an experimental study. Sixteen male C57BL/6N mice aged 8-10 weeks were assigned using a random number table to sham injury group (n=8) treated with sham injury and burn group (n=8) treated with burn injury. From days 1 to 14 after injury, the daily water consumption, food intake, activity level, 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 after injury, cumulative water consumption, food intake, and activity level from days 1 to 14 after injury, energy expenditure and nighttime carbohydrate and fat oxidation rates on days 1, 3, 7, 10, and 14 after injury were calculated. Forty-eight male C57BL/6N mice aged 8-10 weeks were assigned using a random number table with 8 mice being selected for sham injury, and the remaining 40 mice selected for burn injury. Cardiac blood was collected with plasma obtained from burned mice at days 1, 3, 7, 10, and 14 after injury and from sham-injured mice at 1 day after injury. Liquid chromatography-mass spectrometry was employed to identify plasma metabolites (hereinafter referred to as metabolites) and determine their temporal dynamics. Metabolites from sham-injured mice at 1 day after injury were compared with those from burned mice at the same time point, and metabolites from burned mice at days 1, 3, 7, 10, and 14 after injury were compared. 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 There were no statistically significant differences in the daily and cumulative water consumption and food intake between the sham injury group and burn group of mice from days 1 to 14 after injury (P>0.05). Compared with those in sham injury group, the body weight changes of mice in burn group were significantly reduced from days 2 to 14 after injury (P<0.05), the cumulative daytime and nighttime activity level were significantly reduced from days 1 to 14 after injury (P<0.05), the daily activity level lost normal circadian rhythms from days 1 to 14 after injury, the energy expenditure was significantly reduced on day 1 (P<0.05) but was significantly increased on days 7, 10, and 14 after injury (P<0.05), the REE was significantly increased from days 4 to 14 after injury (P<0.05), the respiratory entropy was significantly reduced only during nighttime from days 1 to 7 after injury (P<0.05), the carbohydrate oxidation rates were significantly reduced at night on days 1 and 3 after injury (P<0.05), and the fat oxidation rates were significantly increased at night on days 1, 3, and 7 after injury (P<0.05). A total of 450 metabolites were identified in plasma samples from burned and sham-injured mice. Among these, 253 metabolites exhibited specific temporal patterns and could be clustered into 5 distinct patterns. Eighty-five metabolites of burned mice exhibited statistically significant changes following injury (P<0.05). There were forty metabolites significantly related with REE (with variable importance in projection >1.0), with the top metabolites including linoleic acid, oleic acid, glucose, etc. KEGG pathway enrichment analysis revealed that the biosynthesis and degradation of fatty acid, the biosynthesis and metabolism of amino acid (e.g., isoleucine and tryptophan), as well as the glycolysis/gluconeogenesis pathway were significantly associated with REE (P<0.05). Conclusions Both burn injury treatment and after injury time significantly affect the energy metabolism patterns and material metabolism characteristics in mice. The hypermetabolic state of burned mice persists up to 14 days after injury, during which multiple fatty acid, amino acid, and glucose metabolic pathways are associated with hypermetabolism. -
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=4.184 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；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；横轴上数字与血浆代谢物对应关系为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牛磺鹅去氧胆酸、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
图 4 假伤和烧伤小鼠血浆代谢物与静息能量消耗的正交偏最小二乘（OPLS）回归分析及血浆代谢物的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,即与因变量（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,即与因变量（静息能量消耗）最相关的潜在变量方向,反映其对因变量的预测能力；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中变量投影重要性反映该血浆代谢物在解释静息能量消耗中的重要性；横轴上数字与血浆代谢物对应关系为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相对丰度时序变化比较（
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各指标比较所得,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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