应用代谢组学方法研究肠内给予丙氨酰谷氨酰胺对大鼠肠缺血再灌注损伤的保护作用

Enteral administration of alanyl-glutamine against intestine ischemia reperfusion injury in rats:Metabonomic analysis

目的代谢组学(metabonomics)是生物学研究的新的平台,它可以通过发现生物标本中代谢物质谱的变化进而从机体整体或特定器官代谢状态的角度去评估营养干预措施的作用。丙氨酰谷氨酰胺(alanyl-glutamine,Ala-Gln)是临床上常用的人工合成的含谷氨酰胺(glutamine,Gln)的二肽,在创伤修复过程中发挥重要的作用。文中利用代谢组学的方法观察肠内给予Ala-Gln对Sprague-Dawley(SD)大鼠肠缺血再灌注损伤(intestine ischemia reperfusion injury,IIRI)模型门静脉血代谢谱的影响,并同时探讨其与小肠组织学和局部炎症反应的关系。方法 32只SD大鼠分为假手术(sham operation,S)组(n=8),IIRI组(n=8),Ala-Gln组(n=8)和丙氨酸(alanine,Ala)组(n=8)。除S组外的其余大鼠均通过结扎肠系膜上动脉(superior mesenteric artery,SMA)给予60 min的缺血过程。Ala-Gln组和Ala组从缺血前3 d至再灌注72 h后(肠缺血当天除外)分别通过灌胃给予Ala-Gln(0.6 g.kg-1.d-1)或Ala(0.6g.kg-1.d-1),再灌注72h后取门静脉血上气相色谱质谱联机(gas chromatography coupled with mass spectrometry,GC/MS)进行代谢物质的分离和识别。代谢组数据通过SIMCA-P+12.0软件进行偏最小二乘法(partial least squares,PLS)分析,得出得分图、载荷图及各种物质的变量重要性因子(variable importancein the projection,VIP)值。同时进行小肠病理学检查及门静脉血肿瘤坏死因子(tumor necrosis factor,TNF)-α和白介素(Inter-leukins,IL)-1、6、10浓度检测。结果 PLS分析的得分图能够较明显地区别各组标本。根据VIP值和载荷图,研究发现3种有意义的差异物质分别是葡萄糖(VIP值3.36008)、尿素(VIP值2.81043)和胆固醇(VIP值1.00429)。Ala-Gln能明显缓解IIRI后门静脉血葡萄糖(P<0.01)丰度的降低以及尿素(P<0.01)和胆固醇(P<0.01)丰度的升高。Ala-Gln能部分改善IIRI所致的小肠组织学改变并缓解肠门静脉血TNF-α、IL-1、IL-6和IL-10的变化。而Ala对该IIRI模型并未表现出保护作用。结论肠内给予Ala-Gln有利于维持IIRI后大鼠肠道组织的代谢稳定,该作用与Ala-Gln对肠黏膜完整性的保护和对肠道炎症反应的抑制有关。

Objective Metabonomics is a new platform for bioresearches,which can be applied to the assessment of the effects of nutritional interventions from the viewpoint of metabolic status of the whole body or special organs via manifested changes of the metabolite spectrum in biofluids.Alanyl-glutamine（Ala-Gln）,an artificially synthetic dipeptide containing glutamine（Gln）,plays an important role in traumatic recovery.This study aimed to observe the effect of enteral administration of Ala-Gln on the metabolite profile of the portal vein in SD rats after intestine ischemia reperfusion injury（IIRI） using metabonomic analysis,and to investigate its relationships with intestinal histology and local inflammatory response.Methods Thirty-two SD rats were equally divided into a sham operation（SO） group,an IIRI group,an Ala-Gln group and and alanine（Ala） group.All the rats but those in the SO group were subjected to 60 minutes of intestine ischemia by ligating the superior mesenteric artery（SMA）.Those in the Ala-Gln and Ala groups were administered orally with Ala-Gln（0.6 gkg-1·d-1） and Ala（0.6 gkg-1·d-1）,respectively,from the 3rd day prior to the ischemia procedure to the 72h reperfusion period,except on the day of ischemia.Blood was harvested from the portal vein following reperfusion.Metabolites in the plasma were separated and identified by gas chromatography coupled with mass spectrometry（GC/MS）.Metabolome data were processed via partial least squares（PLS） analysis using the SIMCA-P＋12.0 software.The score plot,loading plot and variable importance in projection（VIP） value of metabolites were obtained.Histological alterations of the intestine and concentrations of tumor necrosis factor（TNF）-α and interleukins（IL）-1,6 and 10 in the portal blood were observed simultaneously.Results Samples clustering of different groups was shown clearly in the score plot.According to the VIP value and loading plot of PLS analysis,we found 3 metabolites of most significance： glucose（VIP value 3.36008）,urea（VIP value 2.81043） and cholesterol（VIP value 1.00429）.Ala-Gln could relieve the reduction of glucose abundance（P0.01） and the rise of urea（P0.01） and cholesterol abundance（P0.01） in the portal blood after IIRI.Ala-Gln could partially ameliorate the histological changes of the intestine and relieve the concentration alteration of TNF-α,IL-1,IL-6 and IL-10 in the portal blood after IIRI.However,Ala exerted no protective effect on this IIRI model.Conclusion Enteral administration of Ala-Gln helps enhance the metabolic stability of the rat intestine after IIRI,which is associated with its protective effect on the integrity of intestinal mucosal morphology and inhibitory effect on the inflammatory response.