| Objective:In order to explore how the relative contents of amino acids and metabolic pathways regulate the variation of pharmacological phenotypes dependent on the dose alteration of DanHong (DH) injection, and trying to find some dose dependent metabolic mechanism from the micro level.Methods:Thirty-six SD rats were divided into6groups, including4DH dose groups (1ml/kg,2.5ml/kg,5ml/kg and10ml/kg) and2control groups (vehicle and sham). After middle cerebral artery occlusion (MCAO), the neurological deficit score, as well as the body and brain weights of all rats were observed for3days, and the volume of cerebral ischemia was measured after decapitation. The serum levels of20free amino acids were determined by HPLC (High Performance Liquid Chromatography) and then transformed by a relative quantitative analysis of amino acids in serum metabolic spectrum. Moreover, multivariate statistical analysis methods PCA (Principal Component Analysis) and PLS-DA (Partial Least Squares Discriminant Analysis), MetPa (Metabolomics Pathway Analysis) and MetaboAnalyst were also employed to analyze these amino acids biological data sets.Results:A negative correlation between the DH doses and the volume of cerebral ischemia was observed (R2=0.8105). All DH doses increased the body weight, and reduced the volume of cerebral ischemia, brain weight and neurological deficit score. Compared with the vehicle group,5,6,7and7non-overlapping metabolites were identified in each of the4dose groups, respectively; and8overlapping metabolites were shared among the4dose groups. Meanwhile, the serum levels of glutamic acid, aspartic acid and serine tended to increase as the doses were escalated. All different metabolites in metabolic pathways were analyzed by MetPa and MetaboAnalyst,compared with the vehicle group,3,2,2and5non over lapping metabolic pathways were found in each of the4dose groups, respectively; and glycine, serine, threonine metabolism and histidine metabolism pathways were identified to be the overlapping ones among the4dose groups.Conclusion:1. DanHong injection can reduce the area of cerebral infarc-tion;2. This study establish a high performance liquid chroma tegraphy (HPLC) optimized condition with a much better separation of rat serum free amino acid successfully;3. There are much differences between MCAO rats serum free amino acid metabolism and normal rats; DanHong injection can adjust the rat serum amino acid metabolic profile gradually to the normal group;4.Overlapping and non-overlapping metabolites and metabolic pathways hierarchically reveal the metabolic mechanism of the dose-dependent neuroprotective effect of DH injection. The method of amino acid metabolomics may be considered as a novel approach to explore the systematic pharmacological mechanism of reversing complex disease. |
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