| Depression is a psychiatric disorder characterized by permanent anhedonia and sadness. The pathological mechanism of depression is not fully understood yet, but may involve changes in nervous system, immunological system and endocrine system. Fluoxetine is a clinically frequent antidepressant drugs. The study on the antidepressant mechanisms of fluoxetine was mainly centered on nervous system, while that on the effects of fluoxetine on the metabolic networks was considerably limited. Oleamide is a recently found endogenous fatty acid amide with antidepressant effect, but the antidepressant mechanisms have not been reported in detail. Metabonomics, based on analysis the global metabolite profiles in biological samples, provides a powerful approach to recognise and reflect the variation of metabolic networks under the condition of disease or drug action. It has been successfully applied to the investigation of pathological mechanism of disease and action mechanism of drugs.In this thesis, plasma and urinary metabonomic methods were developed by using 1H NMR, RP-UPLC-MS, HILIC-UPLC-MS and GC-FID techniques. The metabonomic methods were applied to the study on metabolic alterations in chronic unpredictable mild stress (CUMS) induced depressive model rats as well as fluoxetine or oleamide-treated rats. Pattern recognition was utilized to classify these samples and reveal the biomarkers associated to depression and antidepressant mechanism, which were helpful to research the pathological mechanism of depression and antidepressant mechanisms of fluoxetine and oleamide.1.1H NMR analysis of plasma metabolite profiles of depressive model rats pre-and post-antidepressant drugs administrationSprague-Dawley rats were exposed to chronic unpredictable mild stress for 10 weeks and a depressive model was established successfully. Fluoxetine or oleamide was administrated to the model rats for two weeks. A 1H NMR method was developed for metabolite profile analysis of plasma from control, model and drug treated rats. Pattern recognition was utilized to classify groups and reveal the biomarkers related to depression and antidepressant drugs. A clear separation between model group and control group was obtained. Drug treated goups were also separated from model group and much closer to control group. The metabolite profiles of model rats were characterized by the significant increase of glutamine, valine, choline, lactate and N-acetyl glycoproteins together with the significant decrease of glucose, acetoacetate and 3-hydroxybutyrate in plasma. These biochemical changes are related to the disturbance in glycometabolism, energy metabolism, amino acid metabolism, inflammatory reaction and choline metabolism, which would be related to the pathological mechanism of depression. The common antidepressant mechanisms of fluoxetine and oleamide involved regulation of glycometabolism, energy metabolism and amino acid metabolism. The differential antidepressant mechanisms of them were inhibition of inflammatory reaction by fluoxetine and regulation of the choline metabolism by oleamide.2. UPLC-MS analysis of plasma and urinary metabolite profiles of depressive model rats pre-and post-antidepressant drugs administrationRP-UPLC-MS and HILIC-UPLC-MS methods were developed for metabolite profile analysis of plasma and urine from control, model and drug treated rats. Significant differences in urinary and plasma metabolite profiles were observed between model group and other groups by pattern recognition analysis. Based on plasma metabolite profiles analysis by using two separation modes, the concentrations of C22:6 LPC, C20:4 LPC, cholic acid, phenylalanine, deoxycytidine, serine, carnitine and acetylcarnitine were significantly increased and those of C14:0 LPC, C16:0 LPC, tryptophan, uric acid,3-indolepropionic acid, creatine, betaine, proline and tyrosine were significantly decreased in model group compared with control group. These biochemical changes are related to the disorders of oxidative stress, inflammatory reaction, amino acid metabolism, cholic acid metabolism and gut microflora. Based on urinary metabolite profiles analysis by using two separation modes, model rats were characterized by the significantly increased concentrations of kynurenic acid, xanthurenic acid, phenylalanine, N2-succinyl-L-ornithine, hippuric acid, phenylacetylglycine, 1-methylhistamine, carnitine and N-methylnicotinic acid together with the significantly decreased concentrations of tryptophan, indoxyl sulfate, indole-3-acetate, citrate, a-ketoglutarate, creatinine, taurine, betaine, alloxan and N-acetylhistamine, which are related to the disorders of tryptophan, phenylalanine and histamine metabolism, inflammatory reaction, abnormal functions of taurine and betaine, energy metabolism and gut microflora. The antidepressant effect of fluoxetine and oleamide involved inhibition of inflammatory reaction and oxidative stress and regulation of amino acid, histamine, betaine and gut microflora metabolism. The abnormal levels of phenylalanine, praline, tryptophan, serine and tyrosine were also improved by fluoxetine, suggested the board regulation for amino acid metabolism by fluoxetine. Besides, the altered kynurenine metabolic pathway and the abnormal concentrations of taurine,γ-butyrobetaine, hypoxanthine, nicotinic acid/isonicotinic acid were repaired by fluoxetine, indicated the multitargets for the antidepressant action of fluoxetine. Polyunsaturated, monounsaturated and saturated LPCs together with gut microflora were extensively regulated by oleamide. The results demonstrated the complementarity of HILIC and RPLC in metabonomic study. Combination of two separation modes can monitor metabolites with different polarity in plasma and urine samples and reveal more biomarkers to obtain the more biological information, which is helpful to investigating the pathological mechanism of depression and the antidepressant effects of drugs.3. GC-FID analysis of plasma and urinary metabolite profiles of depressive model rats pre-and post-antidepressant drugs administrationGC-FID method was developed by the derivatization with ethyl chlorformate for metabolite profile analysis of plasma and urine from control, model and drug treated rats. Significant differences of urinary and plasma metabolite profiles were observed between model group and other groups by pattern recognition analysis. Based on plasma metabolite profile study, the concentrations of valine and leucine were significantly increased and those of alanine, proline, tyrosine, tryptophan, hexadecanoic acid, octadecanoic acid and linoleate were decreased in model rats, which indicated a disturbance in amino acid metabolism and fatty acid metabolism. The disturbed amino acid metabolism in model rats was improved by fluoxetine and the decreased fatty acid metabolism was up regulated by oleamide. In the urine of model rats, the concentrations of valine, hippuric acid and phenylalanine were significantly increased and those of tyrosine, tryptophan and 1,2,3-Propanetricarboxylic acid were significantly decreased, which suggested a disturbance in amino acid metabolism, gut microflora and tricarboxylic acid cycle. The altered urinary metabolites measured by GC-FID were entirely improved by fluoxetine. After administration of oleamide, the concentrations of valine and hippuric acid were decreased and the concentration of 1,2,3-Propanetricarboxylic acid was increased.4. UPLC-MS analysis of plasma neurotransmitter profiles of depressive model rats pre-and post-antidepressant drugs administrationPlasma neurotransmitter profiles were developed by using UPLC-MS. The plasma samples were derivatized with dansyl chloride and measured in ESI positive ion mode with multiple reaction monitoring. The method was applied to the study on neurotransmitter alterations in control, model and drug treated rats. The results demonstrated the deficiency of 5-hydroxytryptamine and norepinephrine together with the imbalance of glutamic acid andγ-aminobutyric acid in model rats. Compared with model rats, the concentrations of plasma 5-hydroxytryptamine,5-hydroxyindoleacetic acid andγ-aminobutyric acid were significantly increased after administration of fluoxetine and oleamide. The concentration of plasma norepinephrine was significantly increased and the balance of glutamic acid andγ-aminobutyric acid was regulated by fluoxetine, which were related to the antidepressant action of fluoxetine.In this thesis, metabonomic methods based on different analytical techniques were successfully developed by using 1H NMR, UPLC-MS and GC-FID. More information about metabolite variation was reflected by comprehensive application of thses analytical methods. The results indicated the significant differences in plasma and urinary metabolite profiles between model group and control group. The disturbed metabolic network in model group was improved by fluoxetine and oleamide to different extent respectively. The biomarkers related to depression disease and the antidepressant mechanism of fluoxetine or oleamide were found. The pathological mechanism of depression and antidepressant mechanisms of fluoxetine and oleamide were systematically elucidated. |
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