Proteomics analysis of liver proteins from rats with spleen-deficiency syndrome induced by chronic improper diet consumption and fatigue

Objective:To investigate a proteomics analysis of liver proteins from rats with spleen-deficiency syndrome (SDS) induced by chronic improper diet consumption and fatigue.Methods:This study used a liver proteomic profiling method to identify differentially expressed proteins and altered pathways involved in SDS rats.Specifically,we collected liver samples from a control group and a group with SDS induced by chronic improper diet consumption and fatigue for 4 weeks.The pooled liver proteins in each group were labeled with 8-plex isobaric tags for relative and absolute quantitation reagents.The labeled control and SDS group samples were pooled together and separated by high-pH reverse-phase liquid chromatography.The differentially expressed proteins from the liver proteomes were analyzed to identify potential biomarkers of SDS.Differentially expressed proteins were selected in conjunction with gene ontology and ingenuity pathway analysis.Results:We identified 2176 protein clusters with more than two peptides in the SDS group,with 141 proteins quantified as differentially expressed proteins.Of these,75 proteins were up-regulated,and 66 were down-regulated.Three activated signaling pathways,the thrombin,CXCR4 and synaptic long term depression signaling pathways,and a large multi-protein complex within the network were revealed in the liver proteomic analysis of SDS rats.Conclusions:This is the first report of a differential liver proteome under SDS conditions.The results suggest that the liver proteome partially reflects the pathological changes involved in SDS.The findings provide important information for comprehensively understanding the mechanisms of dysfunction or injury in the liver at the molecular level as a result of SDS.Furthermore,they provide a novel understanding of the connotation of SDS in the fleld of traditional Chinese Medicine.

The Diagnostic Criteria of Blood-Stasis Syndrome:Considerations for Standardization of Pattern Identification

Pattern identification （PI）, also called Bian Zheng （辨证）, syndrome differentiation, pattem diagnosis, or pattem classification, is the basic principle and the key concept of Chinese medicine （CM）. The core of PI is CM syndrome, on which CM theory, therapeutic method, prescribing formula and the use of Chinese herbal medicine are basically based. PI, in fact, is another classification method anticipated to improve the clinical efficacy. How to make an exact PI seems to be very important for taking full advantage of PI in clinical practice. Therefore, the establishment of diagnostic criterion of pattern has been the prerequisite for the standardization of PI. In recent years, a lot of diagnostic criteria of different CM patterns have been formulated. Taking the diagnostic criteria for blood-stasis syndrome as a model, the methodologies and considerations in establishing a pattern diagnostic criterion were discussed in this paper, which might be of great reference value in future PI standardization research.

Expression of T-Helper 17 Cells and Signal Transducers in Patients with Psoriasis Vulgaris of Blood-Heat Syndrome and Blood-Stasis Syndrome

Objective： To investigate the levels of cytokines related to T-helper （Th） 17 cells in serum and signal transducers in the psoriatic lesions of patients with psoriasis vulgaris of blood-heat syndrome （BHS） and blood-stasis syndrome （BSS）. Methods： Sixty patients with psoriasis vulgaris were divided into the BHS and BSS groups according to the syndrome differentiation of Chinese medicine （CM）. Ten healthy subjects were considered as the control group. Cytokine levels of interleukin （IL）-17, IL-23 and IL-6 in serum were determined by enzyme-linked immunosorbent assay. Expression levels of signal transducer and activator of transcription 3 （STAT3）, p38-mitogen-activated protein kinase （MAPK） and STAT6 in the psoriatic lesions were determined using immunohistochemistry （IHC）, Western blot, and real-time quantitative reverse transcription polymerase chain reaction, respectively. Results： Production of IL-17, IL-23 and IL-6 in the BHS group and BSS group were significantly increased compared with those in the control group （P〈0.05）. Levels of IL-17 and IL-23 in the BHS group were higher than those in the BSS group （P〈0.05）. Compared with the control group, IHC positive expressions and protein expressions of STAT3 and p38-MAPK, and the STAT3 mRNA expressions in the BHS and BSS groups were significantly higher （P〈0.05 or P〈0.01）. The protein expression of STAT3 in the BHS group was significantly higher than that in the BSS group （P〈0.05）. Conclusions： Cytokines in serum and signal transducers in the psoriatic lesions alter with various CM syndromes of psoriasis. The results provide scientific basis for the treatment based on syndrome differentiation of CM in treating psoriasis vulgaris.

Relationship between Platelet Activation Related Factors and Polymorphism of Related Genes in Patients with Coronary Heart Disease of Blood-stasis Syndrome

Objective： To comparatively study the expressive conditions of platelet activation related factors （GPⅠb, GPⅡb-Ⅲa and GMP-140） in healthy subjects and patients with coronary heart disease （CHD） of blood-stasis （BS） or non-blood-stasis （non-BS） syndrome, and to analyze the relationship between the activities of various glycoproteins and the polymorphism of genes. Methods： With case control design adopted, patients with the CHD （40 of BS, 37 of non-BS） and 39 healthy subjects for control, all fitting to the inclusion criteria, were selected in this study. The number of affected coronary branches was recorded by the contrast examination. The mean fluorescence intensity （MFI） of GPⅠb, GPⅡb-Ⅲa, and GMP-140 （CD42b, CD61, CD62p） in patients and healthy persons was measured with flow cytometry, the polymorphism of HPA-3 gene was detected by Taqman probe technique and that of HPA-2 gene was determined by gene sequencing. Results： MFI of CD61 and CD62p was higher in the CHD patients than in the healthy control, which was also higher in patients of BS syndrome than in patients of non-BS syndrome （P〈0.05）; MFI of CD42b was lower in the CHD patients than in the healthy control （P〈0.05）, but showing insignificant difference between BS and non-BS syndrome （P〉0.05）; at the same time, no significant difference of all the above-mentioned three MFI could be found in patients with various numbers of affected coronary branches, neither in patients with different genotypes at GPⅡb HPA-3 and GPⅠb HPA-2 polymorphism loci （P〉0.05）. Conclusion： （1） The activities of GP Ⅱ b-Ⅲa and GMP-140 were obviously increased in the genesis and developing process of CHD and CHD of BS syndrome, and so they could be taken as one of the objective indexes for microscopic diagnosis of BS syndrome. （2） The level of GPⅠb was lower in CHD patients than in healthy persons, but it was not a sensitive indicator for BS syndrome of CHD. （3） Levels of GP Ⅱb-Ⅲa, GPⅠb and GMP-140 were not related with the number of affected coronary branches in CHD patients. （4） The changes in amino-acids expression induced by the two loci brought no significant influence on GPⅠb and GP Ⅱb-Ⅲa activities.

Differential Expression of MicroRNA in Endothelial Cells Incubated with Serum of Hypertension Patients with Blood-Stasis Syndrome

Objective： To screen out blood-stasis syndrome （BSS）-associated microRNA and therefore determine the possible target for treating hypertension. Methods： A high-energy sequencing method and digital gene expression sequencing theory were adopted to sequence microRNA （miRNA） and messenger RNA （mRNA）, and to determine differential expression in human umbilical vein endothelial cells incubated with serum samples from hypertension patients with or without BSS, and healthy controls. The results were confirmed using gene prediction software. Results： A total of 13 miRNAs and 11 mRNAs showed statistical difference both in the BSS/normal groups and BSS/non-BSS groups, respectively. Four pairs of target mRNNmiRNA were identified： FRMD4Nhsa-miR-34a, MAP3K14/hsa-miR-34a, PER1/hsa-miR-34a, and FGF2/hsa-miR-132. Conclusion： Four mRNNmiRNA pairs mentioned above seem to be involved in pathogenesis and maintenance of hypertension with BSS.

Gut microbiota and Chinese medicine syndrome: altered fecal microbiotas in spleen(Pi)-deficient patients

OBJECTIVE:To investigate the fecal microbiota changes in patients with spleen(Pi)-deficiency(SPD),a common Chinese medicine syndrome with digestive and absorptive disturbances and to provide insight into the relationship between Chinese medicine syndrome and gut dysbiosis.METHODS:Fecal microbiotas from the stool samples of 53 SPD patients and 35 healthy subjects were analyzed via 16 S r RNA gene polymerase chain reaction(PCR)-denaturation gradient gel electrophoresis(DGGE).SPD-related marker genes from20 SPD patients and 20 healthy subjects were identified through gene sequencing,while some genes were quantified using quantitative PCR(q PCR).Discriminant analysis was conducted using SPSS software,and the canonical discriminant function formula for Pi-deficiency was established.RESULTS:Alterations in microbiota diversity and composition between the SPD and healthy groups were demonstrated via 16 S r RNA gene PCR-DGGE combined with multivariate statistical analysis.Fecal microbiota changes were also observed among different SPD-subtype patients.Eight SPD-related markers were found,and putative species corresponding to these markers were identified through gene sequencing,which may have potential associations with the common digestive dysfunctions in SPD patients.q PCR methods were established for two of these markers,which were significantly altered in the SPD patients.The canonical discriminant function formula was calculated for SPD,and the validity rates of these markers were over 85%.CONCLUSION:Fecal microbiotas are altered in patients with SPD,which may provide insight for further studies on clinically diagnosing and treating SPD.The results may also provide data to gain a better understanding of Traditional Chinese Medicine syndrome and gut dysbiosis.

Circulating Levels of Inflammatory Cytokines in Patients with Psoriasis Vulgaris of Different Chinese Medicine Syndromes

Objective： To investigate whether the serum levels of inflammation-related cytokines might be different between the healthy individuals and the psoriatic patients diagnosed of three varied Chinese medicine （CM） syndromes [blood-stasis syndrome （BSS）, blood-dryness syndrome （BDS） and wind-heat syndrome （WHS）]. Methods： A total of 62 psoriatic patients were recruited and assigned to 3 groups according to their CM syndromes, including 27 patients of BSS, 21 of BDS and 14 of WHS. Another 20 sex- and age- matched healthy subjects were enrolled into the control group. Serum concentrations of multiple cytokines, including monocyte chemotactic protein-1 （MCP-1）, macrophage inflammatory protein-1α （MIP-1α）, soluble CD40 ligand （SCD40L）, tumor necrosis factor-α （TNF-α）, epidermal growth factor （EGF）, interleukin-8 （IL-8）, interleukin-17 （IL-17）, interferon γ inducible protein-10 （IP-10） and vascular endothelial growth factor （VEGF）, were measured by a multiplexed flow cytometric assay. Results： The circulating levels of MIP-1α, TNF-α, IL-8, and IP-10 were significantly increased in the psoriatic patients compared with the healthy controls （P〈0.01）. Male and female patients tended to have higher serum levels of MCP-1 and IP-10, respectively （P〈0.05）. Interestingly, compared with the control group, 6 out of the 9 cytokines （MCP-1, MIP-1α, TNF-α, EGF, IL-8 and IP-10） were substantially increased in the BSS group （P〈0.05 or P〈0.01）, whereas only MIP-1α and IL-8 levels were elevated in the BDS group （P〈0.05 or P〈0.01） concurrent with lowered concentrations of SCD40L and IL-17 （P〈0.05）. In the WHS group, MIP-1α was the only cytokine whose level was evidently increased （P〈0.01）, in contrast to IL-17 which was decreased as compared with the control （P〈0.05）. The psoriatic patients overall owned higher levels of MIP-1 a and IL-8 in the circulation which were comparable among the 3 groups of CM syndromes （P〈0.01）. In contrast, TNF-α level of the BSS group was the highest among the three （P〈0.01）, followed by the BDS and the WHS groups. Conclusions： The expression profiles of cytokines in the circulation might not be necessarily identical for psoriatic patients with different CM syndromes. Accordingly, the serum concentrations of certain cytokines could potentially be used as the ancillary indices for the clinical classification of psoriatic CM syndromes.

Differential Gene Expression Profiles in Coronary Heart Disease Patients of Blood Stasis Syndrome in Traditional Chinese Medicine and Clinical Role of Target Gene

Objective:To investigate the differential gene expression profiles in coronary heart disease(CHD) patients of blood-stasis syndrome(BSS) by oligonucleotide microarray technique,and the clinical significance of target gene.Methods:Subjects were assigned to CHD patients with BSS(n=8),CHD patients without BSS (n=8),and BSS patients without CHD(n=8) based on coronary angiography and the diagnostic criteria of BSS. The sex- and age-matched healthy volunteers(n=8) were enrolled as the control group.Venous blood s...

Study on the Relationship between Blood Stasis Syndrome and Clinical Pathology in 227 Patients with Primary Glomerular Disease

To investigate the relationship between the severity of Chinese medicine(CM) bloodstasis syndrome(BSS) with clinical features and renal lesion indexes of the primary glomerular disease. Methods:An epidemiological survey was conducted to collect the data of 227 patients diagnosed as chronic primary glomerular diseases,and their severity of BSS were scored three days before renal biopsies were performed.The following clinical indexes were analyzed:age,course of glomerular diseases,24-h urine protein ration(Up...

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining

OBJECTIVE： To construct a protein-protein interaction（PPI） network in hypertension patients with blood-stasis syndrome（BSS） by using digital gene expression（DGE） sequencing and database mining techniques.METHODS： DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were f iltered by comparing the expression levels between the different experimental groups. Then functional categories and e nriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery（DAVID） to select those in the enrichment pathways. I nterologous Interaction Database（I2D） was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identif ied by comparing the number of relationships among genes. Confi rmed by quantitative reverse transcription-polymerase chain reaction（q RTPCR）, gene ontology（GO） analysis was used to infer the functional annotations of the potential candidate genes for BSS.RESULTS： With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2 D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identifi ed：DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2 BE, H3F3 B, CEBPB, SAT1 and GADD45 A. Verif ied through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored.CONCLUSION： Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.

Differentially Expressed Proteins in Rat Hippocampus after Chronic Immobilization Stress and Intervention Using Xiao Yao San Decoction

Objective To identify differentially expressed proteins in the hippocampus of rats after chronic immobilization stress(CIS)using a proteomics approach,and to study the effect of the Xiao Yao San(XYS)decoction on differentially expressed proteins.Methods Twenty-four Sprague Dawley rats were randomly assigned to one of four groups of equal body weight:control(non-stress),7-day stress,21-day stress and21-day stress+XYS treatment groups.Two-dimensional gel electrophoresis(2-DE)was used to detect differences in protein expression in rat hippocampus.One differentially expressed protein was measured and verified by western blotting.Results Seventeen proteins showed differential expression.Among these,eight could be identified:glial fibrillary acidic protein-2(GFAP-2),tubulin alpha-1c,cytoplasmic muscle actin2,14-3-3protein,β-2a tubulin,phosphatidylethanolamine binding protein,synucleinαsyn3,and a low molecular weight(18kD)protein.Six of these proteins exhibited increased expression,one showed decreased expression,and the other protein,which comprised five subtypes,were either increased or decreased.These proteins are known to be involved in immunity,signal transduction,cell cycle control,apoptosis,regulation of enzyme activity,cytoskeleton structure,and synaptic plasticity.GFAP-2was further analyzed,and its differential expression confirmed by western blotting.Conclusion Some proteins are differentially expressed in the hippocampus of rats under chronic stress.The biological functions of these differentially expressed proteins are varied.Finally,the XYS decoction can significantly up-or down-regulate these protein expression levels.