二甲双胍对老年脓毒症急性肺损伤大鼠肠道代谢组学的影响

The effect of metformin on intestinal metabolomics in aged septic acute lung injury rats

目的探索二甲双胍对老年脓毒症大鼠肠道代谢物的影响及对肺脏损伤的保护作用。方法SD大鼠在郑州大学动物实验中心进行饲养,将14只老年SD大鼠随机分为三组:假手术组(age-Sham,AgS组,n=4)、盲肠结扎穿孔诱导脓毒症组(age-Cecal ligation and puncture,AgCLP组,n=5)、CLP处理1 h后灌胃二甲双胍(100 mg/kg)处理组(age-Metformin,AgMET组,n=5),并于造模后24 h收集各组大鼠粪便和肺组织。采用液相色谱-串联质谱非靶向代谢组学分析粪便中的代谢物组成及组间差异。两组间的代谢物和各组间代谢变化的可视化结果分别用正交偏最小二乘判别分析、热图和主成分分析图进行展示。代谢物的通路分析使用MetaboAnalyst 3.0软件基于KEGG数据库和人类代谢组数据库进行分析。结果大鼠肺组织中的CCL4(F=203.00,P<0.001)、CXCL1(F=65.69,P<0.001)、IL-6(F=38.94,P<0.002)、TNF-α(F=14.85,P=0.005)的表达水平在组间比较均差异有统计学意义(均P<0.05)。然而,CCL2在AgCLP组和AgMET组间差异无统计学意义。此外,与AgS组相比,AgCLP组的7-甲基黄嘌呤、N-花生四烯酰甘氨酸、Manoalide等17个代谢物相对强度显著升高;而安替比林、Gly-Phe、伏立诺他等9个代谢物则明显降低,二甲双胍治疗后可逆转上述代谢物的变化。相关性分析发现,炎性指标IL-6和TNF-α水平与7-甲基黄嘌呤、N-花生四烯酰甘氨酸等代谢物相对强度呈正相关,而与安替比林、Gly-Phe呈负相关;CCL4和CXCL1与Manoalide呈正相关,而与伏立诺他呈负相关。结论本研究结果表明,二甲双胍改善了脓毒症急性肺损伤并调节了宿主肠道菌群代谢物,这可能为老年脓毒症急性肺损伤提供潜在的有效治疗手段。

Objective To observe the effect of metformin on intestinal metabolites and its protective effect on lung injury in an elderly sepsis rat.Methods SD rats were fed at the Animal Laboratory Center of Zhengzhou University,fourteen elderly SD rats were randomly divided into three groups:sham surgery(age-Sham,AgS group,n=4),cecal ligation and perforation induced sepsis(ageCecal ligation and puncture,AgCLP group,n=5),and oral administration of metformin(100 mg/kg)after 1 h of CLP treatment(age-Metformin,AgMET group,n=5).Collected rat feces 24 h after modeling,and analyzed the composition and inter group differences of metabolites in the feces using liquid chromatography tandem mass spectrometry non targeted metabolomics.Collected rat lung tissues and detected the expression levels of inflammation related genes and pathological changes in the tissue.The visualization of metabolic changes between groups were presented using orthogonal partial least squares discriminant analysis,heatmaps,and unsupervised principal component analysis,respectively.MetaboAnalyst 3.0 was used to evaluate the Pathway analysis of metabolites,and this software was based on the KEGG database and the human metabolome database.Results The expressions of CCL4(F=203.00,P<0.001),CXCL1(F=65.69,P<0.001),IL-6(F=38.94,P<0.002),TNF-α(F=14.85,P=0.005)between two groups of rats were significantly different(all P<0.05).However,there was no significant difference in CCL2 expression between AgCLP group and AgMET group.Furthermore,compared with the AgS group,the relative intensities of 17 metabolites such as 7-methylxanthine,N-Arachidonylglycine and Manolide in AgCLP group were significantly increased,whereas the 9 metabolites such as Phenazone,GlyPhe and Valyproline were significantly decreased,and metformin treatment could reverse these changes of the above metabolites.Correlation analysis showed that the IL-6 and TNF-αlevels were positively correlated with the relative strength of 7-Methylxanthine,N-Arachidonylglycine and other metabolites,but negatively correlated with the Phenazone and Gly-Phe.CCL4 and CXCL1 were positively correlated with Manolide,but negatively correlated with Valyproline.Conclusion The results of this study showed that metformin improved sepsis induced acute lung injury and regulates the host intestinal metabolites,which might provide a potential and effective treatment for elderly sepsis induced acute lung injury.