Research Of Proteome Differential Expression Of LC In Rat Model Of Liver Depression Syndrome

Research background:Syndrome differentiation and treatment are the characteristic and the essence of the Traditional Chinese Medicine.It emphasizes the personalized thinking of TCM."Syndrome" is a unique concept of TCM, it refers to the pathological generalization of the location, etiology, nature and the progression of diseases.Syndrome is a state of our body caused by internal and external factors, it is changing with the development of disease.Syndrome is conclusion about the current essence of the disease.So, the study of nature of disease is the central content of the modernization of TCM.In recent years,with the draft of the human genome has been completed,the focus of research is moving beyond genomics to proteomics.Protein has been playing an increasingly important role in the development of disease.This makes Proteomics quite valuable in the study of human diseases.As a new subject,proteins research the composition and of protein and its dynamic variety from the whole to grasp the whole situation of the physiological and biochemical process in the disease.Proteomics is an new research field that focuses on a whole set of proteins in a cell or a tissue of an organism.lt explains and clarifies the basic rules of the formation and development of Syndrome.It has overall and dynamic characters,and this is its core idea and it has its own theory and technology traits.From these points,Proteomics is very similar to the TCM theoretical system of the overall concept and individualization.This kind of research idea coincides with the characters of TCM.So this idea has obvious characters of Chinese medicine science.Liver depression syndrome is one of the common diseases which is caused by the dysfunction of liver and with the characters of depression,choking sensation in chest and irritable.Clinical epidemiology survey showed that liver diseases in the the viscera account for40%of the overall disease.Liver depression syndrome is the core syndrome of the liver disease and play an important role in the pathogenesis of the liver disease.Therefore,the nature of liver depression syndrome is always the hot topic in the area of Combine traditional Chinese and western medicine.There have been more and more researches about nature of the liver depression syndrome in recent years.Some preliminary research suggests that liver depression syndrome has modern pathophysiology basis and partly explain the scientificalness of TCM theory.We have achieved some stage achievements.Some researches indicate that Liver depression syndrome is likely to result from regulating function disorder of high nervous centre induced by negative psychic stress in which pathological changes of multi-system including nerve,endocrine,circulation,digestion,immunity,feeling and motion etc are involved.In recent years,the study of HPA and LC-NE axis has been developed rapidly.We seminar previously discovered liver depression syndrome can cause the up regulation of TH and c-fos in LC.This proved the relationship between LC and liver depression syndrome.Liver depression syndrome is one of the classic syndromes of liver disease of TCM.The research about its prescription and syndrome is always the most important aspect.The TCM Syndrome research has two feartures.One is to establish corresponding treatment through the result of syndrome differentiation,and the other is to infer the mechanism and symptoms of the disease from the composition and effect of the prescription.In theory,every prescription has its own applicable syndromes and only when the effect of prescription is appropriate for the mechanism of the disease,effect of the prescription can achieve optimal.Through this characteristic,we can establish the pre-determined animal disease models and then interfere them with the pre-determined prescription.After that, we select some abnormal indicators related to the disease and reaearch the animal models that are interfered to estimate whether the disease models are our pre-determined or not.At the same time,in the circumstance that the validation of the animal disease model is good,we observe and study some abnormal indicators and make sure the prescription we select is effect for this disease.Therefore, we hope to provide further evidence for the correlation of liver depression syndrome and LC and lay a solid foundation for the further study of the nature of liver depression syndrome by research into LC according to the methods of proteomics.Meanwhile, we selected some proteins and used xiaoyaosan to disprove it.Fluoxtine was selected as the positive-controlled medicine for its classic anti-depression effect.Objective:To set up the rat model of liver depression syndrome using the method of separation and chronic unpredictable mild stress to find the differentia of general condition and behaviorististics and from the point of proteomics,find the differentially expressed protein in the rat model of liver depressiom.And select the protein which we are interested in to verify.To discover the proteins related to liver depression syndrome from the reaearch of rat model and lay the foundation for the microcosmic differentiation indicatoers system of syndrome.At the same time, to provide technical and theoretical support for the comprehensive investigation on the related proteins from the point of clinical diseases.To explain the essence of liver depression syndrome and to study the clinical effect and mechanism of xiaoyaosan treating the it by comparing the differential expression proteins in the group of xiaoyaosan,fluoxetine and the model group.Methods:This study established the rat model of liver depression syndrome by the method of separation and chronic unpredictable mild stress(CUMS).We used two dimensional gel electrophoresis(2-DE)to sepatate the LC proteins,combining with matrix assisted laser desorption time-of-flight mass spectrometry(MALDI-OF-MS),to analyse the differential expression proteins in rat LC between the model group and the normal group.1. Established the rat model of liver depression syndrome by the method of separation and CUMS.We established two times of the rat model.The first time is for detection and identification to the differential proteins, the second time is for validation.We used the method of separation and CUMS according to the related literature reports. Eighteen female SD rats with SPF level and were into three groups according to a completely random method.Those were normal control group,liver depression syndrome model group,xiaoyaosan group and fluoxetine group.In each group, there were six rats.(in the second time,there were72rats and were randomly divided into four groups.) Except normal group,rats of other groups were feed separately and were induced by a group of CUMS2times every day for three weeks.Xiaoyaosan group was combined with the interferon of xioayaosan on the basis of the model rats and fluoxetine group with fluoxetine intervention.At the same time, the normal group was feed freely and collectively.2. The extraction of protein sample and sample mix.Grinded the LC organization using the liqid nitrogen and lysed the LC cells at a low tempreture.We extracted proteins from LC and made protein quantification by the method of BCA.And mixed the LC regnization in the same group according to the weight.3.2-DE separation of LC.We introduced the methods of salt brige and low voltage desalination on the basis of conventional separation technology of proteins to future optimize this technology.The samples were pair-matched.We made the2-DE separation for all the groups and repeated for three times.4. Gel staining and image analysis.A new kind of colloidal dye technology was used on the gel we obtained from the2-DE separation.On the one hand,this method can improve the sensitivity of gel staining,on the other hand,it can reduce the interference in the later mass-spectrometric detection.After dyeing and dehydration,the gels were scanned by optical density transmission scanners and the images were got. We used the software of PDquest7.0to analyze the images of gels and got the quantity of each protein expression,soelectric point and the molecular weight of proteins and so on.The protein spot whose expression has more than two times difference was defined to differentially-expressed protein,the results were automatically generated by the software and then were verified manually.5. The mass spectrometry identification and database confirm of differentially expressed protein spots.To the differentially expressed protein spots which we received,we applied MALDI-TOF-MS to make the detection of peptide mass figerint(PMF).We cut out the protein spots first and then got the PMF of every protein spot by the steps of water-washing, decolorizing, reduction and alkylation, digestion, extraction, sample and mass spectrometry.After these, we searched major biological and protein-related database by the software of Mascot, to find the proteins which are matched with the PMF we have got and know about the related information.6. CLIA to test T3, T4and TSH levels of serum in different groups.7. HE stain to observe morphological changes of LC.8. Immunohistochemical method to detect the expression changes of the protein we choose in LC and analyze the results by Imageproplus6.0software.9. Western blot method to detect the expression changes of the protein we choose in LC.10. RT-PCR technology to detect the expression changes of total RNA of the gene we choose in LC.11. Statistical analysis.The experimental results are expressed by x±s. SPSS13.0software was applied for it.ANOVA of repeated measures was applied for the weight changes.For the other indicators, the mean comparison of multiple samples used One Way ANOVA to analyse.When the variance was homogeneous, Least-significant difference (LSD) was applied for multiple comparison and when the variance was heteroscedastic, Tamhane’s T2was applied for multiple comparison and the value of P≤0.05would mean significant difference.Results:1. We made the rat model of liver depression syndrome succeedly.During the3weeks we interfered them by CUMS procedures,the model rats first were easily angery and then displayed the typical appearance such as low activity,slow reaction,reduces food and drink.Rats from xiaoyaosan group and fluoxtein group also showed some above abnormalities,but they were not obvious.There were no difference between the two medicine groups.Rats in normal group lived well.The rats’weight increase was significantly different between groups(P=0.017).It showed rats in model group grow slowest,while rats in xiaoyaosan group and fluoxetine group grow more than them.Fluid consumption test reavealed that the model group lose appetite for sweet water after3weeks.Statistics showed that there were differences between the three groups(P=0.000).The open field test reavealed that the erect times and travelling grids both reduced in model group and had statistical differences(P=0.000),xiaoyaosan group and fluoxetine group were better than it,but still have a gap with the normal group(P=0.000).The results showed that separation and CUMS could eatablish the model of liver depression syndrome succeedly and simulate clinical symptoms of liver depression syndrome such as interest loss and anhedonia.2.2-DE was used to separate the whole proteins of LC and the ideal2-DE maps were obtained.Tweenty one differential protein spots were checked out in the three groups(normal, model and xiaoyaosan group).3. We found seventeen differential proteins by the mass spectrometry identification and database confirm of differentially expressed protein spots.4. T3and T4levels in serum was highest in the model group and lowest in the normal group, the levels in xiaoyaosan group and fluoxetine group were between the two groups (P=0.000).TSH levels of serum in all the groups were not statistically significant (P=0.740).5. HE staining showed no obvious morphological change of locus coeruleus in all of groups. 6. Expression results of Immunohistochemical detection of LC showed that the number and mean density of Laxetin,Sirt2and TTR positive cells in model group were lower than others(P<0.05).7. Expression results of Weatern Blot showed that the expression of Laxetin, Sirt2and TTR were all lower than others(P<0.05).8. The expression of LaxetinmRNA, Sirt2mRNA and TTRmRNA by RT-PCR was observed that all were reduced in model group while xiaoyaosan group and fluoxetine group were higher than it.Conclusion:We eatablished the rat model of liver depression syndrome by separation and CUMS and researched the LC proteomics.21differential proteins related to liver depression syndrome were obtained,among them17proteins could be matched with the information in database to determine their names.Then,we selected the proteins of Latexin,Sirt2and TTR from the17proteins to further verified.Among the3proteins,2-DE results suggested that the expression of Latexin and TTR were reduced in model group and Sirt2was up-regulated.Then IHC,WB and RT-PCR technologys were used to prove that the expression of Laxetin,Sirt2and TTR was reduced in liver depression syndrome and xiaoyaosan,fluoxetine could improve this condition. The results revealed the three proteins involved in the process of liver depression syndrome.Xiaoyaosan and fluoxetine play the same role in it and have no differences. This result suggests that the expression of proteins identified by2-DE is just a prompt and is not necessarily a100%exact match.So we used three other methods to further verify the expression of the three proteins to increase the credibility.The results provided theoretical basis for the further research of the molecular mechanisms of liver depression syndrome, and make the essence study of liver depression syndrome have a further development.But the direct relationship between the three proteins and liver depression syndrome has not yet validated and elaborated. This remains to be further research.