茯苓运化颗粒治疗2型糖尿病大鼠的非靶向代谢组学分析

Non-targeted Metabolomics Analysis of Fuling Yunhua Granules in Treatment of Type 2 Diabetes Mellitus Rats

摘要:

目的 基于非靶向代谢组学技术，分析茯苓运化颗粒对2型糖尿病（T2DM）大鼠血清内源性差异代谢物的调控作用，明确茯苓运化颗粒发挥改善T2DM作用的代谢调控途径。方法 70只SD级大鼠，雌雄各半，随机分为空白组、模型组、茯苓运化颗粒高、中、低剂量组（以生药量计，20.70、10.35、5.18 g·kg^-1）及阳性药组（盐酸吡格列酮片8.1 mg·kg^-1）。除空白组外，其余各组均以高糖高脂饲料喂养联合腹腔注射链脲佐菌素（STZ）构建T2DM大鼠模型。造模成功后，给药组灌胃给予相应药物治疗，空白组及模型组灌胃等体积生理盐水，1次/d，共给药28 d。给药期间检测各组大鼠空腹血糖、糖化血红蛋白A1c（GHbA1c）水平，给药结束后采用苏木素-伊红（HE）染色观察大鼠胰腺组织的病理形态变化。采用超高效液相色谱-线性离子阱-静电场轨道阱高分辨质谱法（UPLC-LTQ-Orbitrap MS）检测大鼠血清内源性代谢物水平，通过主成分分析（PCA）和正交偏最小二乘法-判别分析（OPLS-DA）处理数据。利用人类代谢组数据库（HMDB）和京都基因与基因组百科全书（KEGG）鉴定差异代谢物，筛选变量重要性投影（VIP）值>1，P<0.05，差异倍数（FC）<0.6或FC>1的差异代谢物，并通过MetaboAnalyst 5.0对筛选的差异代谢物进行代谢通路富集分析。通过受试者工作特征（ROC）曲线对筛选得到的差异代谢物进行诊断评价。结果 与空白组比较，模型组大鼠空腹血糖水平显著升高（P<0.01），GHbA1c含量有增高趋势，但差异无统计学意义，大鼠胰腺组织明显损伤，胰岛数量减少，胰岛β细胞明显减少、萎缩、肿大；与模型组比较，茯苓运化颗粒高剂量组和阳性药组大鼠给药2周后，空腹血糖明显降低（P<0.05，P<0.01），茯苓运化颗粒高剂量组大鼠GHbA1c含量明显降低（P<0.05），茯苓运化颗粒高、中、低剂量组大鼠胰腺组织病变减轻。非靶向代谢组学结果显示，与空白组比较，模型组共有46个差异代谢物发生明显变化；通路富集分析发现，T2DM主要影响了大鼠体内初级胆汁酸生物合成、D-氨基酸代谢、类固醇激素生物合成、甘油磷脂代谢等生物过程。与模型组比较，茯苓运化颗粒高剂量组中8个差异代谢物的水平明显回调，通路富集分析发现，主要涉及D-氨基酸代谢，视黄醇的新陈代谢，甘氨酸、丝氨酸和苏氨酸的代谢，色氨酸代谢等代谢途径。ROC曲线进一步分析发现，11-顺视黄醇、D-哌啶酸、D-丝氨酸、硫酸对甲酚4个特征性差异标志物对于茯苓运化颗粒治疗T2DM具有较高的诊断价值。结论 茯苓运化颗粒可改善T2DM大鼠症状，其机制可能与调节差异代谢物，调控氨基酸类代谢和视黄醇代谢等通路相关。

Abstract:

Objective Based on non-targeted metabolomics， to analyze the regulation of endogenous differential metabolites in serum of type 2 diabetes mellitus（T2DM） rats by Fuling Yunhua granules， and to clarify the metabolic pathways through which this granules exerted its effect on improving T2DM.Method Seventy SD rats， half male and half female， were randomly divided into the control group， model group， and high， medium， low dose groups of Fuling Yunhua granules（20.70， 10.35， 5.18 g·kg^-1 in raw drug amount） and the positive drug group（pioglitazone hydrochloride tablets， 8.1 mg·kg^-1）. Except for the control group， other groups were fed with high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin（STZ） to establish a T2DM rat model. After successful modeling， the treatment groups were administered the corresponding drugs by gavage， and the control group and model group were treated with an equal volume of saline by gavage， once/d， for 28 d. Fasting blood glucose（FBG） and glycosylated hemoglobin A1c（GHbA1c） levels were measured in all groups of rats during the administration period， and hematoxylin-eosin（HE） staining was used to observe the pathomorphological changes in the pancreatic tissues of rats at the end of the administration period. The endogenous metabolite levels in rat serum were detected by ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-LTQ-Orbitrap MS）， and the data were processed using principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）. Differential metabolites were identified by the Human Metabolome Database（HMDB） and the Kyoto Encyclopedia of Genes and Genomes（KEGG）， and screened for differential metabolites with variable importance in the projection（VIP） value>1， P<0.05， and fold change（FC）<0.6 or FC>1. And the metabolic pathway enrichment analysis of the screened differential metabolites was performed by MetaboAnalyst 5.0， then the screened differential metabolites were diagnosed and evaluated by the receiver operating characteristic（ROC） curves.Result Compared with the control group， the FBG level of rats in the model group increased significantly（P<0.01）， the GHbA1c content tended to increase， but the difference was not statistically significant， and the pancreatic tissue of rats was obviously damaged， the number of pancreatic islets decreased， and the pancreatic β-cells were obviously reduced， atrophied and enlarged. Compared with the model group， the FBG levels of rats in the high dose group of Fuling Yunhua granules and the positive drug group were significantly reduced after 2 weeks of administration（P<0.05， P<0.01）， the GHbA1c content of rats in the high dose group of Fuling Yunhua granules was significantly reduced（P<0.05）， and the pancreatic tissue lesions of rats in the different dose groups of Fuling Yunhua granules were reduced. The results of non-targeted metabolomics showed that 46 differential metabolites were significantly changed in the model group compared with the blank group. Pathway enrichment analysis found that T2DM mainly affected biological processes including biosynthesis of primary bile acid， D-amino acid metabolism， steroid hormone biosynthesis， and glycerophospholipid metabolism in rats. Compared with the model group， the levels of 8 differential metabolites in the high dose group of Fuling Yunhua granules were significantly adjusted， and the pathway enrichment analysis found that D-amino acid metabolism， retinol metabolism， glycine， serine and threonine metabolism， tryptophan metabolism and other metabolic pathways were mainly involved. ROC curves further analysis revealed that the four characteristic differential markers of 11-cis-retinol， D-piperidinic acid， D-serine， and p-cresol sulfate had high diagnostic value for the treatment of T2DM with Fuling Yunhua granules.Conclusion Fuling Yunhua granules can improve the symptoms of T2DM rats by regulating the amino acid metabolic and retinol metabolic pathways through the modulation of endogenous differential metabolites.