整蛋白型肠内营养制剂改善脓毒症肠损伤代谢紊乱的机制研究

Mechanism of intact-protein enteral nutrition formula improving intestinal injury and metabolic disorders in sepsis

摘要:

目的 利用代谢组学方法研究脓毒症中整蛋白营养制剂引起的代谢紊乱，为脓毒症的临床治疗提供了新的理论和实验基础。方法 8~12周龄雄性小鼠随机分为3组：假手术组（Sham组）、脓毒症组（CLP组）和脓毒症+整蛋白组（CLP+IPEN组），每组6只小鼠。CLP组采用盲肠结扎和穿刺诱导脓毒症，而Sham组仅行剖腹手术，未行结扎穿刺。CLP+IPEN组术后接受额外的整蛋白肠内营养制剂。每天监测体质量变化7 d，并在建模和喂养3 d后采集样本。染色观察回肠组织病理学变化，实时荧光定量聚合酶链式反应（quantitative real-time PCR）检测不同目的基因mRNA的表达。根据特定的标准，筛选整蛋白营养制剂治疗脓毒症的差异代谢物。结果 CLP组与CLP+IPEN组进行比较，发现有15种差异代谢物。在CLP+IPEN组中，3-甲基-1，3，5-戊三醇（dimethyl 3-hydroxy-3-methylpentane-1，5-dioate）、丙二酸（malonic acid）、苯酰胺（l-Serine，n-methoxycarbonyl-methyl ester）等5种代谢物的含量比CLP组有所增加（P<0.05）。在整个实验过程中，3组小鼠的体质量均逐渐减轻，其中CLP组在4 d的体质量减轻最为明显（P<0.05）。这表明，整蛋白营养制剂可以减轻脓毒症小鼠的体质量减轻。CLP+IPEN组的Chiu评分明显低于CLP组（P<0.05），提示肠黏膜损伤明显减少。CLP组和Sham组中闭合蛋白（occludin）、紧密连接蛋白1（zonula occludens-1，ZO-1）和结肠黏蛋白（recombinant mucin 2，MUC2）的表达均升高，提示脓毒症导致肠道屏障功能受损。与CLP组相比，CLP+IPEN组中Occludin、ZO-1和MUC2的表达明显降低（P<0.05）。结论 本研究通过代谢组学方法深入研究整蛋白营养制剂中脓毒症引起的代谢紊乱。整蛋白肠内营养制剂可以通过调节亚油酸代谢、不饱和脂肪酸生物合成、脂肪酸生物合成和细胞色素P450物质的代谢来改善脓毒症相关肠道损伤的代谢紊乱。此外，这些制剂在增强肠道屏障功能、减轻小鼠体质量减轻和减轻肠道损伤严重程度方面具有潜力，从而为增强脓毒症治疗功效奠定了基础。

Abstract:

Objective To investigate the metabolic disorders induced by intact-protein enteral nutrition formula in sepsis through metabolomics methods，and to provide new theoretical and experimental bases for the clinical treatment of sepsis.Methods Male mice，aged 8-12 weeks，were randomly divided into sham-operation group（Sham group），sepsis group（CLP），and sepsis+intact-protein enteral nutrition group（CLP+IPEN group），with 6 mice in each group. The mice in the CLP group were treated with cecal ligation and puncture to induce sepsis，while those in the Sham group were given laparotomy alone without ligation and puncture. The mice in the CLP+IPEN group received additional intact-protein enteral nutrition formula after surgery. Daily weight changes were monitored for 7 days，and samples were collected after 3 days of modeling and feeding. Staining was used to observe the histopathological changes of the ileum，and quantitative real-time PCR was used to measure the expression of different proteins. Differentially expressed metabolites in the treatment of sepsis with intact-protein enteral nutrition formula were identified based on specific criteria.Results There were 15 differentially expressed metabolites between the CLP group and the CLP+IPEN group. Compared with the CLP group，the CLP+IPEN group had significant increases in the content of five metabolites including dimethyl 3-hydroxy-3-methylpentane-1，5-dioate，malonic acid，and L-Serine，N-（methoxycarbonyl）-methyl ester（P<0.05）. Throughout the experiment，all three groups of mice showed a gradual reduction in body weight，and the CLP group showed the most significant weight loss on day 4（P<0.05），suggesting that intact-protein enteral nutrition formula could alleviate weight loss in mice with sepsis. The CLP+IPEN group had a significantly lower Chiu score than the CLP group（P<0.05），indicating a notable reduction in intestinal mucosal injury. Both the CLP group and the Sham group had significant increases in the expression of occludin，zonula occludens-1（ZO-1），and MUC2，suggesting that sepsis caused impairment of intestinal barrier function. Compared with the CLP group，the CLP+IPEN group had significant reductions in the expression of occludin，ZO-1，and MUC2（P<0.05）.Conclusion This study investigates the metabolic disorders induced by intact-protein enteral nutrition formula in sepsis through metabolomics methods，and the results show that intact-protein enteral nutrition formula can alleviate metabolic disorders in sepsis-related intestinal injury by regulating linoleic acid metabolism，biosynthesis of unsaturated fatty acids，biosynthesis of fatty acids，and metabolism of cytochrome P450 substances. In addition，such formulas have the potential in enhancing intestinal barrier function，mitigating weight loss in mice，and reducing the severity of intestinal injury，thereby laying a foundation for strengthening the efficacy of sepsis treatment.