Comparative Proteomic Analysis Of Hippocampus Of Tryptophan Depletion Rats

BackgroundMajor Depression Disorder is a persistent low mood state. The coresymptoms of depression are lack of interest, loss of pleasure, accompaniedwith anxiety, guilt, suicidal behavior and sleeping disorders. Depressionhas become one of the most serious diseases to human health, the lifetimeprevalence rate of5%to17%. Yet, the pathogenic mechanisms ofdepression are still not clear, together with lack of objective diagnosticbiomarkers. As reports shown the clinical efficacy of the selectiveserotonin reuptake inhibitors indicated that depression is closely related tothe function of5-HT system. And also tryptophan depletion test alters5-HT amount and function in the peripheral and central nervous system byreducing tryptophan intaked. Ultimately, it turns out to be an effectivemeans to analyze5-HT system. In this setting, proteomic analysis of braintissue of tryptophan depletion rats has not been reported yet.ObjectiveOur research follows the idea of translational medicine research.Establishment of acute tryptophan depletion model, analysis of the relationbetween5-HT system and animal depression-like behavior, and the use ofcomparative proteomics combined with mass spectrometry technology onhippocampal proteins were identified, aimed at analyzing protein level changes of diseases and supporting for clinical diagnosis.MethodsOur research consists of three parts: the first part was to establish andevaluate acute tryptophan depletion model in rats; subsequently, the secondpart was to conduct comparative proteomics research of hippocampus inrats with acute tryptophan depletion.In the context male Sprague-Dawley rats, weight300g~350g, wererandomly divided into three groups after behavioral screening: acutetryptophan depletion model group (TRP-), amino acid mixture withtryptophan group (TRP+) and saline group (Saline).The sucrose preferencetest and the open field test were conducted on animals behavioral testing,and the high performance liquid chromatography were used for detectingplasma amino acids of rats.We used the comparative proteomics-based analysis of singlehippocampus sample of acute tryptophan depletion. Trichloroacetic acid-acetone precipitation method was utilized in protein extraction, thetwo-dimensional electrophoresis was employed for protein separation, andthe2-DE images were therefore analyzed by ImageMaster2D Platinum6.0software, MALDI-TOF-MS was used for identifying differentiallyexpressed proteins, and the mass spectrometry data were searched in theNCBInr database in order to obtain protein information.ResultsAfter acute tryptophan depletion, rats manifestated significantdecrease in sucrose preference and total distances compare with Saline andTRP+groups (P<0.01and P<0.05, respectively).Plasma amino acids weredetected with high performance liquid chromatography, and the amount ofplasma tryptophan unraveled significant decrease in TRP-group compareswith Saline and TRP+groups (P<0.001), the TRP/∑LNAA ratio also indicate dramatic decrease in TRP-group compares with Saline and TRP+groups (P<0.001).Subsequently, the2-DE images were analyzed by ImageMaster2DPlatinum6.0software, each two groups for comparison. Accordingly therewere88proteins existed significant differences (%Vol>1, P<0.05). In themidst of14proteins were identified with MALDI-TOF-MS. Of note, theycontain cytoskeleton proteins, neurodevelopmental related proteins, signaltransduction proteins, energy metabolism proteins, transcription andregulation related factors, as well as oxidative metabolism-related proteins,which are important for providing further insight into major depressiondisorder.ConclusionHPLC plasma amino acid data disclosed that the model is successfuldepletion of tryptophan in the peripheral blood, and the decreased TRP/ΣLNAA ratio indicate that the amount of tryptophan in the central nervoussystem is also significantly reduced, therefore, herein we speculated thatthe amount of5-HT generated by the tryptophan are also significantlydown-regulated. Given the evaluation of animal behavior, we are sureabout that acute tryptophan depletion test can simulate the humandepression emotions, loss of pleasure, reduced activity and other emotionscaused by5-TH system changes. For one thing, that model is simplified,safe, as well as reversible. Through analyzing the function of differentiallyexpressed proteins, we found they are important for studying majordepression disorder. In prospect, this study calls for further underlyingmechanism study.