| Research background and objectiveThe theory of "Spleen governing transportation and transformation"and "the spleen is the origin of the acquired constitution and source of qi-blood" in the Chinese medicine illustrates the spleen has a function in ingesta digest, absorb and transport, and provides energy and substance foundation for human growth, development and vital movement by metabolism of nutritive substance.Substance and energy metabolism is foundation of vital movement. It includes three stage of digest and absorb, intermdediate metabolism and excrete.Intermdediate metabolism is the main body of metabolism, and includes carbohydrate, lipide, protein, nucleic acid, trace element and energy metabolism.At present, many research results showed splenic asthenia animal existed the disorder of digest, absorb and nutrition metabolism.Thus, it is also similar between patho-manifestations of spleen and metabolism.In relativity research between splenasthenic syndrome and metabolism, it is a common method to determine biochemical indicator related to certain metabolism, but this method can not simultaneously study whole substance and energy metabolism for splenasthenic syndrome patients.Microarray technology is a omics resesrch method, and able to reflect organism all gene expression in certain state, which is very similar to the holism of Chinese medicine.So, at the beginning of this century, microarray has been extensive utilized to research animal model of splenasthenic syndrome.But because of a late beginning in China, microarray data analysis exist some problems, such as not pretreatment for microarray data, not statistics test for differentially expressed gene, and simple function annotation. These problems cause microarray data not to well explain disease mechanism.Follow the development of molecule biology and bioinformatics, some professional anlysis softwares raise the readout ability of microarray data. Moreover, many microarray data have been accumulated in recently years.So,it is more important to mine reported microarray data of splenasthenic syndrome by using bioinformics software than microarray experiment in study pathogenesy of splenasthenic syndrome.Based on the work foundation of gene expression profiling for splenasthenic syndrome with chronic superficial gastritis in our research groups, at the present study, microarray data of chronic superficial gastritis (below named chronic gastristis) splenasthenic-splenogastric hygropyrexia syndrome and splenasthenic syndrome-healthy volunteer, and Helicobacter pylori (H. pylori, Hp)-uninfected splenasthenic-Hp-infected splenogastric hygropyrexia syndrome were mined and bioinformatics analysis by BRB Array Tools and IPA software.For the former, differentially expressed genes related to substance and energy metabolism were analyzed to approach the correlation of splenasthenic syndrome and substance and energy metabolism, and the physiological function of spleen and pathogenesy of splenasthenic syndrome based on whole substances and energy metabolism level of organism; For the latter, differentially expressed genes were analyzed to approach pathogenic mechanism of Hp infection and understood genetic background of clinical outcome that Hp infection rate of splenogastric hygropyrexia syndrome with chronic gastritis was higher than splenasthenic syndrome, and provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.In addition, the microarray data from patients of syndrome or constitution of deficiency of QI and rat models of splenasthenic syndrome were also collected.Differentially expressed genes related to substance and energy metabolism from patients of splenasthenic syndrome, and that of rat models of splenasthenic syndrome and the peoples of syndrome or constitution of deficiency of QI were similarly compared to approach marked gene and metabolic pathway of splenasthenic syndrome, and provide some references for scientic annonation of basic theory of Chinese medicine spleen and differentiation of symptoms and classification of syndrome of splenasthenic syndrome, and provide some clue for pathophysiology research of splenasthenic syndrome.Materials and methods1.Materials(1)Microarray data originAll microarray raw data from chronic gastritis patients of splenasthenic-splenogastric hygropyrexia syndrome, splenasthenic syndrome-healthy volunteer and Hp-infected splenogastric hygropyrexia syndrome-Hp-uninfected splenasthenic syndrome were from early data studying pathogenesy of splenasthenic syndrome in our group, and some papers have been published based on these data. All differentially expressed genes from patients of syndrome or constitution of deficiency of QI including insufficiency of lung-QI, deficiency of both vital energy and yin, deficiency of vital energy, and rat models of splenasthenic syndrome including different modeling methods and tissue origin were from open publishing academic and degree papers.(2) Case origin of fluorescent quantitation PCR verification of differentially expressed genes from microarray data of splenasthenic-splenogastric hygropyrexia syndrome 1) Diagnostic and differentiation of symptoms and signs for classification of syndrome criteriaAll criteria of diagnose and classification symptons were from previosly criteria (WANG,Ying-fang, doctor thesis of Guangzhou university of Chinese medicine,2006),as followed:Diagnostic criteria of chronic gastritis refer to the Guide Principle of Clinical Research of Chinese Material Medica and New Drug.Differentiation of symptoms and signs criteria of splenasthenic syndrome Main sign:①body of the tongue light and big or has imprints of the teeth, moss thin and white;②appetite decrease;③abdominal distension;④loose stool or diarrhoea. Secondary sign:①emaciated;②weary and hypodynamia;③pulses extenuate.Decision:a) Main sign①necessary, b) Including the two of main sign②,③and④; Or including one of main sign②,③and④,and including over two of secondary sign.Differentiation of symptoms and signs criteria of splenogastric hygropyrexia syndrome Main sign:①coated tongue yellow freasy;②chest distress;③gastric cavity painful abdominal mass or gas pains;④poor appetite.Secondary sign:①bitter taste and sticky of mouth;②thirsty and few drink, or hot drink preference;③oose stool or including mucus;④nauseated;⑤body exhausted and debilitation;⑥pulses slow or slide.Decision:a) Main sign①necessary, b) Including the two of main sign②,③and④;or including one of main sign②,③and④,and including over two of secondary sign; or including over three of secondary sign.Diagnostic criteria of Hp infection Patients donot take medicine of antibiotics in recent one month.Both pathological section-staining and fast urease test were positive, which were Hp infected, otherwise were Hp uninfected.2) Receive and remove criteriaThe patients who meet criteria of chronic gastritis, splenasthenic syndrome and Hp uninfection or meet chronic gastritis, splenogastric hygropyrexia syndrome and Hp infection were received. The patients whose age less18or over65,pregnancy or breast-feed woman, hypersensitiveness constitution, with other digestive canal disease or serious primary disease and psychiosis, were removed.2.Methods(1)The analysis of differentially expressed genes related to substance and energy metabolism from patients of splenasthenic syndromeAll microarray raw data from chronic gastritis patients of splenasthenic-splenogastric hygropyrexia syndrome, splenasthenic syndrome-healthy volunteer and Hp-infected splenogastric hygropyrexia syndrome-Hp-uninfected splenasthenic syndrome were analyzed as follows:①Microarray raw data preprocessing and filtingThese gene spots, in which Cy5and Cy3signal intensity values exceeded200or one of them exceeded800,and the ratio of Cy5and Cy3was between0.1-10.0, were used to calculate the natural logarithm for their ratios of Cy5and Cy3(r=lnCy5/Cy3).Subsequently, the mean value (R) of all r value was obtained.All Cy3signal intensity value was corrected by product of Cy3and1/R to decrease systemic error of Cy5and Cy3fluorescently labeled system. All corrected Cy3and Cy5signal intensity values lessed200were substituted with200. The corrected Cy3and Cy5signal intensity values were uploaded to BRB Array Tools4.1.0Beta2software to analyze. Intensity filers excluded the spot in which both intensities are below200.If only one intensity was below200, this intensity was increased to200. A log base2transformation was applied to intensities before the arrays were normalized by using median over entire array. Gene filters were used to exclude corresponding spots for an entire gene from all arrays. Exclude a gene under any of the following conditions: minimum fold-change less than20%of expression data values have at least a1.5-fold change in either direction from the gene's median value and percent missing exceeded50%. Filtered data were annonated and further analyzed.②GO and KEGG gene sets analysisFiltered data were used to analysis of GO and KEGG. Healthy volunteers and patients of splenogastric hygropyrexia syndrome were acted as contrast groups, and splenic asthenia patients as experiment groups (In Hp analysis, contrast and experiment groups were exchanged).Statistics analysis were performed by paired t-test, LS/KS permutation test, and Efron-Tibshirani's GSA maxmean test,and P<0.005.③Class comparison of between groups of arraysFiltered data were used to class comparison between groups of arrays, which was used to identify differentially expressed gene among classes of samples.The patients of splenogastric hygropyrexia syndrome, healthy volunteer and Hp-uninfected splenasthenic syndrome were used for the baseline gene expression. Statistics analysis was performed by paired t-test with random variance model and the found gene list was determined by significance at0.05,0.01and0.001levels of univariate tests, respectively. Negatively reciprocal transformation was done for the genes with fold change of expression less than1. The genes which matched p<0.01or0.05or0.001and fold change exceeded2were defined as differentially expressed genes.UP-regulated and down-regulated genes were signed as "↑" and "↓"④efinition and analysis of genes related to substrance and energy metabolismThe differentially expressed genes containing the gene identifiers along with the corresponding fold changes were uploaded into the Ingenuity Pathways Analysis (IPA) website (version8.6)(www.ingenuity.com) to annotate bio-functions, construct and visualize molecular interaction networks. For only splenasthenic syndrome, the metabolism genes were definited by GO annonation and KEGG analysis of BRB Array Tools, biofunction classification and annonation of IPA website software to approach the correlation of splenasthenic syndrome and metabolism.For differentially expressed genes of Hp-uninfected splenasthenic syndrome,to approach pathogenic mechanism of Hp infection and provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.⑤Clustering analysis of differentially expressed genes related metabolismHierarchical clustering of genes and samples for differentially expressed genes were performed by BRB ArrayTools software.Analysis settings included center the genes, one minus correlation metric for genes or centered correlation for samples, and average linkage.(2) The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome, peoples of syndrome or constitution of deficiency of QI and rat models of splenasthenic syndrome①The similar comparison of genes of substance and energy metabolism from microarray data of between two patients of splenasthenic syndromeDifferentially expressed genes related metabolism from patients of splenasthenic syndrome-healthy volunteers and that of splenasthenic-splenogastric hygropyrexia syndrome were compared to analyze characteristic of gene expression profiling for splenasthenic syndrome, and filted its characteristic genes and pathways, and further approach the relationship of splenasthenic syndrome and metabolism.②The similar comparison of genes of substance and energy metabolism from patients of splenasthenic syndrome and that of peoples of syndrome or constitution of deficiency of QIDifferentially expressed genes from patients of syndrome or constitution of deficiency of QI were collected from publishing paper, and differentially expressed genes related to metabolism were definited according to above mentioned and selected to contrasting anlysis with that of patients of splenasthenic syndrome.The differentance of them were analyzed to decide that metabolism abnormality existed in only splenasthenic syndrome or all syndrome of deficiency of QI.③The similar comparison of genes of substance and energy metabolism from patients of splenasthenic syndrome and that of rat models of splenasthenic syndromeDifferentially expressed genes from rat models of splenasthenic syndrome were collected from publishing paper, and were analyzed according to above mentioned method③to approach characteristic metabolic genes and pathways of splenasthenic syndrome.(3)Fluorescent quantitation PCR verification of differentially expressed genes from splenasthenic-splenogastric hygropyrexia syndrome1)Gastric mucosa collectionFour patients who meet criteria of chronic gastritis, splenasthenic syndrome and Hp-uninfection, and4patients who meet criteria of chronic gastritis, splenogastric hygropyrexia syndrome and Hp-infection were collected in Guangdong province hospital of Chinese medicine.Their gastric mucosas were taken200mg in gastroscope, and were preserved in liquid nitrogen.(3)Fluorescent quantitation PCR of differentially expressed genesHLA-DRB1,SCGN, COX7B and SULT1A4, which were from differentially expressed genes of Hp-uninfected splenasthenic syndrome-and Hp-infected splenogastric hygropyrexia syndrome and the latter three genes were also differentially expressed gene of splenasthenic-splenogastric hygropyrexia syndrome, were used to quantitation PCR experiment by the method of SYBR Green I.The primers were designed using primer express2.0software.Total RNA extraction and fluorescent quantitation PCR were performed according to direction of kits.Relative amount of experiment and control group was calculated by the method of△△Ct.Results1.Differentially expressed genes related to substrance and energy metabolism from the patients of splenasthenic syndrome(1)Differentially expressed genes from patients of splenasthenic syndrome-healthy volunteers①287gene sets including49cellular components,60molecular functions and178biological processes were obtained by GO analysis and11pathways were obtained by KEGG analysis.GO biological process and KEGG pathway showed genes related to metabolism of lipid, protein, nucleic acid, carbohydrate and trace element were differentially expressed in the patients of splenasthenic syndrome;②Fifteen differentially expressed genes related to metabolism were obtained by class comparison between groups of arrays, they occupied75%(15/20) of total differentially expressed genes,14genes were down-regulated and11gene productions have enzyme activities,they participated in the metabolism process related to lipid, protein, nucleic acid and carbohydrate.ACAA2↓and CYP20A1↓were participated in lipid metabolism, and showed fatty acid degradation and cholesterol metabolic transformation decreased;RPS28↓,UBXN1↓,UBE2D2↓,ASL↓, ASS1↓,PCYOX1L↓,ALDH9A1↓,B3GNT1↓,GCNT1↓and PPP1R3C↓were participated in protein metabolism, and showed that protein biosynthesis and modification of glycosylation and phosphorylation decreased,protein ubiquitination disturbed, amino acid metabolism related in urea cycle and autonomic nerve decreased, protein; RMI1↓, SMARCD3↓and PARP1↑were participated in nucleic acid metabolism, and showed that DNA duplication and transcription decreased, DNA damage repair increased; B3GNT11↓, GCNT1↓and PPP1R3C↓were participated in carbohydrate metabolism, and showed that biosynthesis of glycan and glycogen decreased.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and healthy volunteers could not be separated.(2) Differentially expressed genes from patients of splenasthenic-splenogastric hygropyrexia syndrome①373gene sets including58cellular components,59molecular functions and256biological processes were obtained by GO analysis.These GO biological process included metabolism of lipid, protein, nucleic acid, carbohydrate, trace element and energy. Two pathways obtained by KEGG analysis were ribosome and lactoflavin metabolism.② Fifty-six differentially expressed genes related to metabolism were obtained by class comparison between groups of arrays, they occupied71%(56/79) of total differentially expressed genes,45genes were down-regulated and30gene productions have enzyme activities, they participated in the metabolism process related to lipid, protein, nucleic acid, carbohydrate, trace element and energy metabolism.ACADVL↓, LRP11↑,SULT1A4↓, CRLS1↑,GPCPD1↓, PIGL↓, B3GNT1↓, ST8SIA4↓and FUT9↑were participated in lipid metabplism, and showed that (3-oxidation of fatty acid cut down, cholesterol intake increased and its metabolic transformation decreased, phospholipid and glycolipid metabolism were abnormal;ASRGL1↓, AARSD1↓,EBNA1BP2↓, PUM2↑, MRPL52↓, C120RF65↓, PSMB8↓, PSME2↓, UBA7↓, RNF11↑,FBXO44↓,ZFYVE26↓,CHMP2A↓, SSR4↓,SNX4↑,RAB3B↓,RABL2A↓,GOLGA2↓, KDELR1↓,PHPT1↓,ACPP↓,PTPRF↓, CRKL↓,HDAC7↓,ADPRHL2↓,B3GNT1↓, ST8SIA4↓,DDOST↓and FUT9↑were participated in protein metabolism, and showed that protein biosynthesis, ubiquitination and targeted transport decreased, posttranslation modification of phosphorylation and glycosylation decreased;TOP2A↓, SF3A3↓,CREB3↓,CRTC2↓,NR1D2↑, MED6↓, GTF2IRD1↓, C1ORF83↓, ZNF773↓,ZMYND11↑,DFFB↓,FLJ35220↓and ADPRHL2↓, were participated in nucleic acid metabolism, and showed that DNA duplication and transcription decreased, DNA damage repair increased; AGL↑, PTPRF↓, B3GNT1↓, FUT9↑,ST8SIA4↓,SULT1A4↓,DDOST↓and PIGL↓were participated in carbohydrate metabolism, and showed that glycogen degradation increased and glycoconjugate biosynthesis decreased;COMMD1↓,FTL↓,SLC39A6↑,CHRFAM7A↓,SCGN↑and S100A6↓were participated in trace metabolism, and showed copper and ferri ion metabolism decreased, zinc ion metabolism increased, calcium ion metabolism were abnormal;AK3↓and COX7B↓were participated in energy metabolism, and showed energy metabolism blocked.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and those of splenogastric hygropyrexia syndrome could be separated.(3)Differentially expressed genes from patients of Hp-uninfected splenasthenic-Hp-infected splenogastric hygropyrexia syndrome①703gene sets including114cellular components,114molecular functions and475biological processes were obtained by GO analysis and29pathways obtained by KEGG analysis.The GO biological process and KEGG pathway showed that Hp infection caused host differential expression of genes related to protein metabolism, signal pathway, immunity and inflammation reaction, cystoskeleton etc.②Thirty-four annonated differentially expressed genes which obtained by class comparison between groups of arrays, were participated in protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on.The82%of these genes (28/34) were categorized in three molecular interaction networks involved in gene expression, cancer progress, antigen presentation and inflammatory response.Taken together, these data indicated that Hp infection could alter cellular gene expression processes, escape host defense mechanism, increase inflammatory and immune responses, activate NF-κB and Wnt/β-catenin signaling pathway, disturb metal ion homeostasis,and induce carcinogenesis.③Clustering analysis of differentially expressed genes showed patients of Hp-uninfected splenasthenic syndrome and those of Hp-infected splenogastric hygropyrexia syndrome could be separated.2.The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome, peoples of syndrome or constitution of deficiency of Ql and rat models of splenasthenic syndrome(1)The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic-splenogastric hygropyrexia syndrome and patients of splenasthenic syndrome-healthy volunteer①The GO biological process from the two microarray data showed genes related to metabolism of lipid, protein, nucleic acid, carbohydrate and trace element were differentially expressed in the patients of splenasthenic syndrome;KEGG pathway from the two microarray data showed that genes related to protein metabolism were differentially expressed.②The results of class comparison between groups of arrays showed, differentially expressed genes related to substrance and energy metabolism from patients of splenasthenic-splenogastric hygropyrexia syndrome and patients of splenasthenic syndrome-healthy volunteer showed obviously difference.The dissimilarity was the numbers and metabolic process of differentially expressed genes.The former was more than the latter, metabolic processes included lipid, protein, nucleic acid, carbohydrate, trace element and energy. The latter was participated in lipid, protein, nucleic acid and carbohydrate metabolism. Moreover, differentially expressed gene name was also difference.The similarity was followed as (1) Many genes related to substrance and energy metabolism were differentially expressed in the two microarray data, and they occupated over70%of total differentially expressed genes, and most of these genes were enzyme genes or enzyme activity gene, and their expressions were down-regulated in patients of splenasthenic syndrome;(2)B3GNT1expression was down-regulated in the two microarray data;(3) In the metabolic pathway, the results of two microarray data from patients of splenasthenic syndrome showed cholesterol metabolic transformation and β-oxidation of fatty acid decreased, ribosome assembly blocked, protein modification of phosphorylation and glycosylation decreased, protein ubiquitation decreased, DNA duplication and transcription decreased,DNA damage repair increased, tissue glycogen biosyntheis decreased and degradation increased, and glycan biosynthesis decreased.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and those of splenogastric hygropyrexia syndrome could be separated, while patients of splenasthenic syndrome and healthy volunteers could not be separated.(2) The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome and syndrome or constitution of deficiency of QIDifferentially expressed genes related to substrance and energy metabolism from patients of splenasthenic syndrome and syndrome or constitution of deficiency of QI showed obviously difference.(1)Differentially expressed genes related to metabolism from patients of splenasthenic syndrome were similar to that of constitution of deficiency of QI in expression tendency and metabolic pathway, and both existed metabolic block, but patients of splenasthenic syndrome more obvious.For these results, patients of other syndrome or constitution of deficiency of QI had completely different results;(2) ST, RAB and S100might be mark genes of splenasthenic syndrome, while RP, PTP and UBE2might be mark genes of syndrome of deficiency of QI;(3)In metabolic pathway, differentially expressed genes related to lipid, carbohydrate, protein and nucleic acid metabolism from patients of splenasthenic syndrome were down-regulated, while that of syndrome or constitution of deficiency of QI were up-regulated and participated in less metabolic process;(4)Protein ubiqutination abnormality was commonness of syndrome of deficiency of QI,but splenasthenic syndrome and constitution of deficiency of QI decreased, other syndrome or constitution of deficiency of QI increased;(5) DNA damage might be common pathological phenomenon of syndrome and constitution of deficiency of QI.3.The similar comparison of differentially expressed genes related to metabolism from patients and animal models of splenasthenic syndromeDifferentially expressed genes related to metabolism from patients and animal models of splenasthenic syndrome had obvious differentence in numbers and expression tendency. But thirteen differentially expressed genes were repeatedly appeared in microarray results of patients and animal models of splenasthenic syndrome, they were B3GNT1,CYP, SLC39A, SNX, FUT, SLC2A, GCNT, ACAD, LDH, RP, PPP1, S100and PTPR. That RP and PTPR were down-regulated also appeared in syndrome of deficiency of QI;(1)Except for RP, all these genes production were enzyme and transporter;(2) These genes were participated in lipid, carbohydrate, protein and tace element metabolism, and showed that glucose transport and degradation, glycogen synthesis and degradation, and glycan synthesis, were disordered;Fatty acid and cholesterol metabolism were abnormal; Ribosome assembly blocked, protein modification of phosphorylation decreased and that of glycosylation was abnormal, and targeted transport increased; Zinc ion metabolism increased and calcium ion metabolism increased.3.Fluorescent quantitation PCR verification of differentially expressed genes from splenasthenic-splenogastric hygropyrexia syndromeThe results of fluorescent quantitation PCR for HLA-DRB1,SCGN,COX7B and SULT1A4, which were from differentially expressed genes of Hp-uninfected splenasthenic syndrome-and Hp-infected splenogastric hygropyrexia syndrome and the latter three genes were also differentially expressed gene of splenasthenic-splenogastric hygropyrexia syndrome,and that of microarray experiment were similar, but the ratio of the former was lower than the latter. ACAA2of differentially expressed genes from splenasthenic syndrome-healthy volunteers was also verified by our research groups previously.Conclusions1.It was different to other syndrome of deficiency of QI that the patients of splenasthenic syndrome showed nutrition dysmetabolism by down-regulation of metabolic genes, especially enzyme genes.Dysmetabolism showed that cholesterol metabolic transformation and β-oxidation of fatty acid decreased, ribosome assembly was blocked, protein modification of phosphorylation and glycosylation decreased, protein ubiquitation decreased, DNA duplication and transcription decreased, DNA damage repair increased, tissue glycogen biosyntheis decreased and degradation increased, and glycan biosynthesis decreased. All of these results might be the one of important reason causing nutrition dysmetabolism of splenasthenic syndrome.All these results acted out macro-metabolism sketch of patients of splenasthenic syndrome, and supported discussion related to metabolism for pathophysiology of spleen in Neijing, which provided some clues for modern research and annotation of scientic essence of spleen picture.At the same time, the macro-metabolism sketch of patients of splenasthenic syndrome well revealed molecular pathophysiology background of spleen, which provided theoretical basis and academic guide for metabolic disease research based on the treatment from spleen.2.The differentially expressed genes related to metabolism from analysis of gene microarray data and the determination results of biochemical indicator related to metabolism from literature report were confirmed each other and revealed pathophysiology of spleen mainly included digest and absorb,and metabolism of substance.3.The B3GNT1,ST, RAB,CYP, SLC39A, SNX, FUT, SLC2A, GCNT, ACAD, LDH, RP, PPP1, S100and PTPR, might be closely related to the occurrence of splenasthenic syndrome, while RP, PTP and UBE2might be closely related to the occurrence of syndrome of deficiency of QI.All of these genes provided some clues for research of splenasthenic syndrome.4.The analysis of gene expression profiling showed the clinical manifestation between splenasthenic and splenogastric hygropyrexia syndrome was more obvious, while between splenasthenic syndrome and healthy volunteers was not obvious,which provided theoretical basis for classification of cases of disease in Chinese medicine.5.Hp infection could affect host on protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on, which provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases. |
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