Combined 1H NMR fecal metabolomics and 16S rRNA gene sequencing to reveal the protective effects of Gushudan on kidney-yang-deficiency-syndrome rats via gut-kidney axis.

Based on traditional Chinese medicine (TCM) theory, kidney is regarded as governing the bones and dominating the storage of essence ('jing' in Chinese). Gushudan (GSD) is a traditional Chinese medicine prescription with the effects of strengthening bone and nourishing kidney, which has been used to treat osteoporosis for years. Several anti-osteoporosis effects of GSD have been investigated based on metabolomics in previous studies. However, the specific mechanism of GSD on kidney tonifying and its alterations in gut microbiota are still unclear. In this study, 1H NMR fecal metabolomics and 16 S rRNA gene sequencing technology were integrated to comprehensively explore the microbiota and metabolic changes in kidney-yang-deficiency-syndrome (KYDS) rats and to elucidate the protective mechanism of GSD through the gut-kidney axis. GSD significantly regulated the levels of 12 out of 31 potential metabolites and the abundance of 11 out of 16 potential microbial biomarkers related to KYDS, respectively. Fecal metabolomics showed that GSD could reserve the abnormal levels of gut microbial-mediated metabolites of KYDS rats, such as tryptophan, lysine, dimethylamine, creatinine, acetate and butyrate, which mainly involved in amino acid metabolism, methylamine metabolism, energy metabolism and short-chain fatty acid metabolism. Specifically, GSD could promote butyrate-producing bacteria g_Lachnospiraceae_NK4A136_group and lactate-producing bacteria g_Lactobacillus. Interestingly, there was a strong relationship between altered fecal metabolites and perturbed intestinal microflora in genus. For example,lysine was negatively correlated with g_Lactobacillus, while acetate was positively correlated with g_Barnesiella. In conclusion, the study showed that the gut-kidney axis had scientific implications, which not only offered new insights into the in-depth understanding of the pathogenesis of KYDS, but also provided further evidence for the efficacy evaluation of GSD.

The Effect of Shen Qi Wan Medicated Serum on NRK-52E Cells Proliferation and Migration by Targeting Aquaporin 1 (AQP1).

BACKGROUND Shen Qi Wan (SQW) as a well-known formula for the amelioration of kidney yang deficiency syndrome (KYDS), and it has been widely employed in traditional Chinese medicine (TCM). This study aimed to investigate the effect and underlying mechanism of SQW medicated serum on proliferation and migration in NRK-52E cells. MATERIAL AND METHODS We employed the real-time cell analysis (RTCA) system to investigate the effect of SQW medicated serum on proliferation and migration in NRK-52E cells. In addition, the migration was further investigated by using a wound-healing assay. The mRNA and protein expression level of aquaporin 1 (AQP1) of NRK-52E cells with SQW medicated serum-treated were quantified by real-time quantitative polymerase chain reaction (q-PCR) and western blot assay, respectively. Furthermore, NRK-52E cells were transfected with lentivirus AQP1-RNAi to assess migratory cell abilities in vitro. RESULTS The migratory abilities of NRK-52E cells were significantly increased after SQW medicated serum treatment (P<0.05), and no significant difference in cell proliferation. In addition, SQW medicated serum was significantly upregulated the mRNA and protein expression level of AQP1 in NRK-52E cells (P<0.05). Additionally, the in vitro metastasis test proved that knockdown of AQP1 suppressed migratory abilities according to RTCA and wound healing test while was reversed by SQW medicated serum (P<0.05). CONCLUSIONS Our study demonstrates that SQW medicated serum effectively promotes the migration of NRK-52E cells by increasing AQP1 expression, and AQP1 may be as a therapeutic target of SQW for renal injury treatment under KYDS.

A Novel Strategy Facilitates Reference Gene Selection by RT-qPCR Analysis in Kidney Yang Deficiency Syndrome Mice Infected with the Influenza A (H1N1) Virus.

In reverse transcription-quantitative polymerase chain reaction (RT-qPCR) studies, endogenous reference genes are routinely used to normalize the expression of target gene studies. In order to precisely evaluate the relative expression of genes in the cells of mice suffering from Kidney Yang Deficiency Syndrome (KYDS) in response to influenza A virus (IAV) H1N1 using RT-qPCR, it is crucial to identify reliable reference genes. In the present study, 15 candidate reference genes (Actb, ß2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhae, 18S rRNA, Rpl13, Ubc, Rpl32, and Ppia) were investigated in lung cells from KYDS mice infected with IAV H1N1. NormFinder, BestKeeper, and GeNorm were used to assess the stability of reference genes. The results were authenticated over extended experimental settings by a group of 10 samples. In the present study, we explored a novel method using dual-gene combinations; the difference in gene expression between the model and normal control groups was statistically analyzed by an independent-samples t-test, and the difference in the mean value between the two groups was compared. A P value > 0.05 and the lowest absolute value of the difference indicated the optimal reference two-gene combination. Four additional host innate immune system-related genes (TLR3, TLR4, TLR7, and RIG-I) were analyzed together with the two treatment datasets to confirm the selected reference genes. Our results indicated that none of these 15 candidate reference genes can be used as reference gene individually for relative quantitative fluorescence PCR analysis; however, the combination of Grcc10 and Ppia, based on the process of calculating the higher P value and lower difference values between groups, was the best choice as a reference gene for the lung tissue samples in KYDS mice infected with IAV. This technique may be applied to promote the selection process of the optimal reference gene in other experiments.

Expression profiling-based clustering of healthy subjects recapitulates classifications defined by clinical observation in Chinese medicine.

Differences between healthy subjects and associated disease risks are of substantial interest in clinical medicine. Based on clinical presentations, Traditional Chinese Medicine (TCM) classifies healthy people into nine constitutions: Balanced, Qi, Yang or Yin deficiency, Phlegm-dampness, Damp-heat, Blood stasis, Qi stagnation, and Inherited special constitutions. In particular, Yang and Yin deficiency constitutions exhibit cold and heat aversion, respectively. However, the intrinsic molecular characteristics of unbalanced phenotypes remain unclear. To determine whether gene expression-based clustering can recapitulate TCM-based classification, peripheral blood mononuclear cells (PBMCs) were collected from Chinese Han individuals with Yang/Yin deficiency (n = 12 each) and Balanced (n = 8) constitutions, and global gene expression profiles were determined using the Affymetrix HG-U133A Plus 2.0 array. Notably, we found that gene expression-based classifications reflected distinct TCM-based subtypes. Consistent with the clinical observation that subjects with Yang deficiency tend toward obesity, series-clustering analysis detected several key lipid metabolic genes (diacylglycerol acyltransferase (DGAT2), acyl-CoA synthetase (ACSL1), and ATP-binding cassette subfamily A member 1 (ABCA1)) to be down- and up-regulated in Yin and Yang deficiency constitutions, respectively. Our findings suggest that Yin/Yang deficiency and Balanced constitutions are unique entities in their mRNA expression profiles. Moreover, the distinct physical and clinical characteristics of each unbalanced constitution can be explained, in part, by specific gene expression signatures.

High-throughput chinmedomics-based prediction of effective components and targets from herbal medicine AS1350.

This work was designed to explore the effective components and targets of herbal medicine AS1350 and its effect on "Kidney-Yang Deficiency Syndrome" (KYDS) based on a chinmedomics strategy which is capable of directly discovering and predicting the effective components, and potential targets, of herbal medicine. Serum samples were analysed by UPLC-MS combined with pattern recognition analysis to identify the biomarkers related to the therapeutic effects. Interestingly, the effectiveness of AS1350 against KYDS was proved by the chinmedomics method and regulated the biomarkers and targeting of metabolic disorders. Some 48 marker metabolites associated with alpha-linolenic acid metabolism, fatty acid metabolism, sphingolipids metabolism, phospholipid metabolism, steroid hormone biosynthesis, and amino acid metabolism were identified. The correlation coefficient between the constituents in vivo and the changes of marker metabolites were calculated by PCMS software and the potential effective constituents of AS1350 were also confirmed. By using chinmedomics technology, the components in AS1350 protecting against KYDS by re-balancing metabolic disorders of fatty acid metabolism, lipid metabolism, steroid hormone biosynthesis, etc. were deduced. These data indicated that the phenotypic characterisations of AS1350 altering the metabolic signatures of KYDS were multi-component, multi-pathway, multi-target, and overall regulation in nature.

Genome-wide association study on susceptibility genes associated with yang-deficiency constitution: A small sample case-control study.

OBJECTIVE: To explore susceptibility genes associated with yang-deficiency constitution using single nucleotide polymorphism (SNP) genotyping. METHODS: Based on an epidemiological survey, 30 volunteers with yang-deficiency constitution and 30 volunteers with a balanced constitution were included according to the Classification and Determination Standards of Constitutions in Traditional Chinese Medicine. Peripheral blood was collected and DNA was extracted from white blood cells. A genome-wide association study (GWAS) was conducted by SNP 6.0 genotyping at the Beijing CapitalBio Corporation Ltd. A minimum association P-value (Fisher's exact value) of less than 10(-4) in the allele, genotype, dominant, and recessive models served as the standard for significant association of SNP with yang-deficiency constitution. RESULTS: Among the four genetic models, a total of 42 SNPs were significantly associated with yang-deficiency constitution (Fisher's exact P-values P<10(-4)). These SNPs were adjacent to more than 20 genes, including RGS6, mGluR5, GAPDHL19, and IKZF1. CONCLUSION: Yang-deficiency constitution exhibits the characteristics of polygenic inheritance. This pilot study suggests that the polymorphisms in RGS6, mGluR5, GAPDHL19, and IKZF1 are associated with changes in cyclic adenosine monophosphate and cyclic guanosine monophosphate levels, memory, metabolic energy status, and immune function, respectively in people with yang-deficiency constitution.

Effect of polypeptide 2B1 on condition of dampness pattern in rats in terms of traditional Chinese medicine.

OBJECTIVE: This study investigated how polypeptide 2B1 is involved in regulating and governing dampness in rat models with dampness pattern defined in terms of Traditional Chinese Medicine. METHODS: We randomly divided 48 SPF 10-week-old male Sprague-Dawley (SD) rats into a normal group, normal + Aristolochic acid I (AA-I) for 5 min group, normal + AA-I for 60 min group, dampness pattern group (DS-Group), dampness pattern + AA-I for 5 min group, and dampness pattern + AA-I for 60 min group. Groups were then treated accordingly. We took out the lung, stomach, liver, spleen, kidney, large intestine, and small intestine tissues to detect gene and protein expression of organic anion transporter polypeptide 2B1 (OATP2B1). RESULTS: Gene expression of OATP2B1 in spleen, kidney, and small intestine of rats with dampness pattern was lower than that in normal rats (P < 0.05). The gene expressions of OATP2B1 in liver, stomach, large intestine, and small intestine were lower than that in control rats at different time points after being stimulated by AA-I (P < 0.05). CONCLUSION: There is coordination among multiple viscera in handling the condition of dampness, and the mechanism underlying the action may rely on regulating the expression of OATP2B1.

[Effect of electroacupuncture on spleen lymphocyte apoptosis-related gene expression in aging rats with yang deficiency].

OBJECTIVE: To observe effects of electroacupuncture (EA) of "Guanyuan" (GV 4)-"Housanli" (ST 36)-"Baihui" (GV 20,"Shuanggu Yitong" acupoint prescription) on the expression of splenic lymphocyte apoptosis-related genes Fas, Bax, and Bcl-2 in aging rats with yang defiency. METHODS: Thirty Sprague-Dawley aged female rats were randomly and e-ic qually divided into normal control, yang deficiency model (model) and EA groups. The yang deficiency model was established by subcutaneous injection of D-galactose (125 mg/kg) and hydrocortisone(1.5 mg/100 g). EA (2 Hz, 1 mA) was applied to "Guanyuan"(GV 4), "Housanli" (ST 36) and "Baihui" (GV 20) for 15 min, once daily, 6 times per week for 4 weeks. The expression of pro-apoptotic genes Fas, Bax and anti-apoptosis gene Bcl-2 in the spleen tissues were detected by real-time quantitative RT-PCR. RESULTS: In comparison with the normal control group, splenic Fas mRNA and Bax mRNA expression levels were upregulated significantly, and Bcl-2 mRNA expression level was down-regulated considerably in the model group (P < 0. 01). After EA intervention, splenic Fas mRNA and Bax mRNA expression levels were down-regulated markedly, while Bcl-2 mRNA expression up-regulated remarkably in the EA group (P < 0.05, P < 0.01). CONCLUSION: EA of acupoint prescription ["Guanyuan" (GV 4), "Housanli" (ST 36) and "Baihui" (GV 20)] can down-regulate spleen Fas mRNA and Bax mRNA expression, and up-regulate Bcl-2 mRNA expression in aging rats with yang deficiency, which may contribute to its effect in improving yang deficiency in clinic.

Effect of kidney-reinforcing, blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells from chronic aplastic anemia patients.

OBJECTIVE: To explore the effect of kidney-reinforcing, blood-activating and stasis-removing recipes on adhesion molecule expression of bone marrow mesenchymal stem cells (MSCs) from patients with chronic aplastic anemia (CAA). METHODS: We used three Traditional Chinese Medicine recipes, namely a kidney-reinforcing recipe (KRR), blood-activating and stasis-removing recipe (BASRR), and kidney-reinforcing, blood-activating and stasis-removing recipe (KRBASRR), and a normal saline control to prepare herbal medicine serum in Sprague Dawley rats. Thirty CAA patients were enrolled in the experimental group, including 17 kidney-Yang deficient patients and 13 kidney-Yin deficient patients. Ten healthy individuals were included in the control group. MSCs were isolated from bone marrow samples, and the cell density was observed to measure their proliferation ability by microscopy on days 2, 7, and 14 after isolation. In addition, the expression of adhesion molecules of bone marrow MSCs (CD106, CD49d, CD31 and CD44) were detected by flow cytometry after 48 h of treatment with the four different herbal medicine serums. RESULTS: The proliferation of MSCs from kidney-Yang deficient and kidney-Yin deficient patients was weaker than that of MSCs from the control group. The expression of all adhesion molecules of bone marrow MSCs from CAA patients was obviously lower than that in the control group (P < 0.01). The expression of CD49d and CD31 in MSCs from patients with a kidney-Yin deficiency was lower than in those with a kidney-yang deficiency (P < 0.05 and P < 0.01, respectively). For kidney-Yang deficient patients, CD31 expression in the KRBASRR group was significantly higher than that in the BASRR group (P < 0.01), while CD44 in the KRBASRR group was significantly higher than that in both KRR and BASRR groups (P < 0.01). For kidney-Yin deficient patients, CD106 and CD49d expression in the KRBASRR group was obviously higher than that in the KRR group (P < 0.05), while CD31 and CD44 expression in the KRBASRR group was significantly higher than that in both KRR and BASRR groups (P < 0.05 and P < 0.01, respectively). CONCLUSION: The bone marrow microenvironment in CAA patients is abnormal. The effect of KRBASRR may be better than that of KRR and BASRR for kidney-Yang deficient and kidney-Yin deficient patients by improving the expression levels of MSC adhesion molecules.

Association of APOE polymorphisms and insulin resistance with TCM syndromes in type 2 diabetes patients with macroangiopathy.

To investigate the association between apolipo-protein E (APOE) polymorphisms and insulin resistance and Traditional Chinese Medicine (TCM) syndromes in type 2 diabetes mellitus (T2DM) with macroangiopathy, 60 patients with T2DM macroangiopathy were enrolled and divided into three groups: dryness-heat due to deficiency of yin, Qi-Yin deficiency, and Yin-Yang deficiency, according to the TCM syndromes, with a control group of 20 healthy individuals. APOE genotype analysis was performed with polymerase chain reaction amplification and restriction fragment length polymorphism, and the results showed that the proportion of the ε4/4 and ε3/4 genotypes and frequencies of the ε4 and ε3 alleles were higher in the Qi-Yin deficiency group (P<0.05). Among the T2DM macroangiopathy patients, the E4 group had the largest number of cases, as well as a significantly longer disease course compared to the E2 group (P<0.05). The insulin resistance index (IRI), insulin action index and body mass index (BMI) of patients in the Yin-Yang deficiency group were significantly different from those of patients with dryness-heat due to deficiency of yin and Qi-Yin deficiency. Furthermore, correlation analysis of the BMI and IRI of patients in the Yin-Yang deficiency group revealed a correlation coefficient r=0.696 (P<0.01) and a typical correlation between them. In conclusion, the Qi-Yin deficiency in T2DM patients with macroangiopathy is associated with the APOE E4 and E3 genotypes. Thus, the APOE gene polymorphism can, to some degree, reflect the TCM syndrome types of T2DM patients with macroangiopathy. Insulin resistance plays an important role in the occurrence of T2DM macroangiopathy and is closely associated with the Yin-Yang deficiency according to the TCM differentiating types.

[Differential gene expressions in kidney Yang deficiency in individuals with sub-health status].

OBJECTIVE: To study the gene expression profiles in kidney Yang deficiency in individuals with sub-health status. METHODS: The subjects identified to have Kidney-yang deficiency and sub-health status were enrolled in this study with healthy subjects as the normal control. Using gene chip technique and MAS software, the gene expression profiles in kidney Yang deficiency in the subjects with sub-health status were analyzed. RESULTS: Characteristic profiles of the differentially expressed genes were found between the kidney Yang deficiency group and normal control group. Among the total of 75 differentially expressed genes, 22 were up-regulated (with a ratio greater than 2) and 53 were down-regulated (with a ratio less than 0.5) in kidney Yang deficiency group. CONCLUSION: Gene chip technique allows rapid and high-throughput screening for gene expression in kidney Yang deficiency and sub-health status, and a initial evidence of kidney Yang deficiency-related genes has been obtained.

Identification of Linkage Disequilibrium SNPs from a Kidney-Yang Deficiency Syndrome Pedigree.

In order to probe the genetic traits of Kidney-yang Deficiency Syndrome (KDS), we employed a national standard of KDS diagnosis for the collection of KDS subjects. Each candidate KDS subject from a typical family was diagnosed by 5 independent physicians of Traditional Chinese Medicine (TCM), and repeated for 3 years, all on the first Saturday of December. Fifteen samples of genomic DNA were isolated and genotyped by Affymetrix 100 K arrays of single nucleotide polymorphism (SNP). Then appropriate tools were used for the analysis of linkage disequilibrium (LD) and bioinformatic mining of LD SNPs. The results indicated that our procedure of TCM diagnosis can effectively collect KDS subjects and therefore provide substantial basis for the linkage analysis of KDS. Five SNPs (i.e. rs514207, rs1054020, rs7685923, rs10515889 and rs10516202) were identified as LD SNPs from this KDS family, representing an unprecedented set of LD SNPs derived from TCM syndrome. These SNPs demonstrate midrange linkage disequilibrium within the KDS family. Two genes with established functions were identified within 100 bp of these SNPs. One is Homo sapiens double cortin domain containing 5, which interacts selectively with mono-, di- or tri-saccharide carbohydrate and involves certain signaling cascades. Another one, leucyl-tRNA synthetase, is also a pleiotropic gene response to cysteinyl-tRNA aminoacylation and protein biosynthesis. In conclusion, KDS is involved in special SNP linkage disequilibrium in the intragenic level, and genes within the flanks of these SNPs suggest some essential symptoms of KDS. However, definitive evidence to confirm or exclude these loci and to establish their biological activities will be required.

Insufficient activity of MAPK pathway is a key monitor of Kidney-Yang Deficiency Syndrome.

OBJECTIVE: To explore the genetic characteristics and molecular regulator of Kidney-Yang Deficiency Syndrome (KDS). DESIGN: A typical KDS family was collected using a questionnaire of cold feeling and a 40-item scoring table of KDS based on Traditional Chinese Medicine (TCM), by single-blind method repeated annually over three years. Their transcriptomes were assayed by microarray and validated by RT-PCR and ELISA. Simultaneously, 10 healthy volunteers were recruited as controls and the same protocols were performed. RESULTS: This typical KDS family has 35 members, of whom 11 were evaluated as having severe KDS and 6 as having common KDS. Results of the cDNA microarray revealed that there were 420 genes/expressed sequence tags differentially expressed in KDS transcriptomes, indicating a global functional impairment in the mass-energy-information carrying network of KDS patients, involving energy metabolism, signal transduction, development, cell cycle, and immunity. Pathway analysis by gene set enrichment assay (GSEA) and other tools demonstrated that mitogenic activated protein kinase (MAPK) is among the most insufficiently activated pathways, while the oxidative phosphorylation and glycolysis/gluconeogenesis pathways, the two main pathways relevant to ATP synthesis, were among the most excessively activated pathways in KDS patients. Results of RT-PCR and ELISA confirmed the status of insufficient activity of the MAPK pathway. CONCLUSION: KDS patients undergo overall attenuated functions in the mass-energy-information carrying network. The marked low level of energy output in KDS may be primarily attributed to the insufficient activity of the MAPK pathway, which may be a key monitor for the abnormal energy metabolism and other impaired activities in KDS.

[Establishment of a quantitative mathematical model of Shen-deficiency syndrome based on whole-genome transcriptional profiles].

OBJECTIVE: To establish a quantitative mathematical model of Shen-deficiency syndrome of TCM by utilizing whole-genome transcriptional profiles. METHODS: The 4, 10, 18, 24 months old SD rats were used, 24-months aged rats intervened by Epimedium Flavonoids (EF) were adopted in the experiment. Rats' hypothalamus, pituitary, adrenal, lymphocytes, bone, liver, and kidney, and spleen were taken for determining whole-genome mRNA expression with gene chip, and a quantitative nerve network model was established by utilizing the gene expression profile of different aged rats, then the model was used to evaluate the effects of EF on Shen-deficiency syndrome. RESULTS: Totally 199 genes showing age-dependent characteristics were screened out from the 7 kinds of tissue, most of them were neuro-endocrine immune related genes. Evaluation based on the mathematical model showed the age of hypothalamus, pituitary, adrenal, liver, kidney, bone, and spleen in the 24-months rats after EF intervention was 12.64, 10.87, 8.10, 12.70, 11.93, 13.14, and 10.13 months, respectively. CONCLUSION: A quantitative mathematical model can be established based on the gene expression profile, it is suitable for estimating the efficacy of Shen-tonifying drugs. EF can make the gene expression of elder close to the young state, suggesting that EF has action in improving Shen-deficiency syndrome and delaying senescence.

[Comparison of gene expression profiles between rats of Shen deficiency syndrome and Shen-yang deficiency syndrome differentiated according to therapeutic efficacy of drugs used].

OBJECTIVE: To explore similarity and difference of connotation between Shen deficiency syndrome (SDS) and Shen-yang deficiency syndrome (SYDS) on the molecular level. METHODS: The senescent SD rats and corticosterone-treated rats were adopted for models of SDS and SYDS respectively, their syndrome was differentiated according to the therapeutic efficacy of treatment with epimedium flavonoids (EF). The gene expression profiles of hypothalamas, pituitary, adrenal gland and lymphocytes (HPAT axis) were detected before and after EF treatment using gene chip provided by Affymetrix company. RESULTS: As compared with the young rats, the ageing rats and corticosterone-treated rats showed a significant down-regulation in highly consistent pattern, of various neurotransmitters of HPAT axis firstly, followed with that of growth and sex hormone related genes. EF could reverse the above genes expression in both models, and for SYDS model rats, it could also significantly up-regulate the gene expressions of heat shock protein, cytochrome P450 and thyroid stimulating hormone (TSH). CONCLUSION: Both SDS and SYDS model rats show connotation of Shen deficiency, and the substantial base of Shen-yang deficiency syndrome resides in the process of oxidative phosphorylation of energy metabolism accelerated by thyroid hormone.

[Research on gene expression of warm-hot drug treating cold syndrome].

OBJECTIVE: To study the therapeutic molecular mechanism of the warm-hot drugs treating cold syndrome. METHOD: A brother and his sister with deficiency-cold syndrome were chosen and treated with appropriate Chinese formula consisted chiefly of warm-hot drugs for 45 days. Then microarray technique was applied for comparing the gene expression difference of sister who had significant effect, the data was dialed with multiple analysis method and the results were mined though gene function and pathway. RESULT: 276 differential genes were obtained, which were related to metabolism and 18 pathways. CONCLUSION: Warm-hot drugs work on the gene expression of metabolism. It may be exerting the curative action by gene network and there is distinct difference between gene expression of curative effect and syndrome.

Establishment of a discriminant mathematical model for diagnosis of deficiency-cold syndrome using gene expression profiling.

OBJECTIVE: To screen diagnostic markers of Deficiency-Cold syndrome by gene expression profile and to establish a discriminant mathematical milliliters model for the clinical diagnosis of this syndrome based on a support vector machine (SVM). METHODS: A family suffering from Deficiency-Cold syndrome is chosen for this study. This family has 5 patients with Deficiency-Cold syndrome and 10 normal members. The peripheral blood samples for these 5 patients and 5 normal members are tested by using cDNA microarray with 18,816 clones to get their differential expression genes. These genes are further explored to understand their biological functions and pathways through existing databases. A SVM model for clinical diagnosis is then developed based on these differential expression genes. RESULTS: A total of 83 differential expression genes were identified between patients and normal members, in which 21 genes were recorded in the FATIGO database and 16 genes were related to metabolism. Eight (8) pathways were sorted out in the KEGG database, and half pathways were associated with human metabolism. A discriminant mathematical model based on a support vector machine successfully predicted a normal person and a patient with heavy Deficiency-Cold syndrome based on their gene differential expression profiles. Thus, this model may classify the Deficiency-Cold syndrome. CONCLUSION: This work demonstrates that the differential expression genes can be used to identify normal persons and patients with Deficiency-Cold syndrome. Deficiency-Cold syndrome is mainly associated with the metabolism-related gene regulations. In addition, the discriminant mathematical model based on a support vector machine is applicable to the clinical diagnosis for Deficiency-Cold syndrome.

Familial characteristics of kidney-yang deficiency and cold syndrome.

Family investigation is a reliable model to study the effects of both genetic and environmental factors on human health. This article studies kidney-yang deficiency syndrome and cold syndrome through family investigation and cDNA microarray technology, exploring the effects of both genetic and environmental factors on the health of family members. Particularly, these two syndromes were first assessed by the accumulated clinical scores measured by 40-item scoring tables among 15 family members. The family patterns were obtained and the correlation of these two syndromes was determined. Then the gene differential expression profiles among 12 family members were obtained using an 18,816 clones cDNA microarray. The profiles of the patients with typical kidney-yang deficiency syndrome and cold syndrome were compared to those of normal members and 89 differential expression genes were found. Further, only 22 genes were identified as known functions, and most (16 genes) were associated with the regulation of metabolism, temperature feeling, and growth. Therefore, the formation and development of these two syndromes have not only genetic but also environmental factors, including living conditions and lifestyle.

[Syndrome differentiation through drug effects in mapping the two regulatory pathways of gene networks in Shen deficiency syndrome].

OBJECTIVE: To study the regulatory pathways and rules of the gene networks in Shen deficiency syndrome. METHODS: Tissues of hypothalamus, pituitary, adrenal, lymphocyte, bone, liver and kidney were taken as samples from 4 months' and 24 months' old SD rats and rats after treatment with Epimedium flavonoids (EF), differences of gene expression profile in Shen deficiency syndrome were studied repeatedly with gene chip rat expression set U230 2.0 array from Affymetrix Co. RESULTS: Gene expressions in the aged rats all decreased including neurotransmitter of gamma-aminobutyric acid (gammaGABA), gonadotropin releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), thyroid stimulating hormone (TSH), growth hormone-releasing hormone receptor (GHRH), insulin-like growth factor (IGF) and binding proteins (IGFBP) in hypothalamus, pituitary and adrenal (HPA axis), cell growth-related gene, growth factor related protein, and immune regulatory genes such as interferon gamma (IFN-gamma), interleukin 4 (IL-4) and interleukin 6 (IL-6) in lymphocytes, parathyroid hormone (PTH), calcitonin, procollagen, collagen, connective tissue growth factor in bone, and oxidative phosphorylation genes such as cytochrome P450 and NADH dehydrogenase, glutamate dehydrogenase related with protein metabolism, and glucose-6-phosphatase related with glucose metabolism in liver, most of which were up-regulated after treatment with EF as well as genes related with ageing and cell cycle, such as cyclin B, metabolism related genes and proteins of sodium and chloride channel in kidney. CONCLUSION: Dysfunction of the two regulatory pathways of gene networks as nerve-endocrine-immunity and nerve-endocrine-bone metabolism exists in Shen deficiency syndrome differentiated by effects of drugs, which could be improved by strengthening Shen therapy.

[Expression of bcl-2 gene in spleen deficiency syndrome in colorectal carcinoma and the regulatory effect of Jianpikangfu decoction].

OBJECTIVE: To investigate the correlation between spleen deficiency syndrome in colorectal carcinoma and bcl-2 gene expression, and observe the regulatory effect of Jianpikangfu decoction. METHODS: Forty-five advanced colorectal carcinoma patients with spleen deficiency were randomized into Jianpikangfu decoction treatment group with also symptomatic treatment with western medicine and control group in which the patients were given expectant treatment with western medicine. The activity of salivary amylase and bcl-2 expression in the tumor tissues were detected before and after the treatment. RESULTS: Jianpikangfu decoction in combination with western medicine treatment produced more obvious inhibition of reduction in salivary amylase activity than exclusive western medicine treatment (t=7.822, P<0.01), and significantly lowered the positivity rate of bcl-2 expression (chi2=4.286, P<0.05) in the tumor tissues, which, however, displayed no obvious changes in response to exclusive western medicine treatment. CONCLUSION: Jianpikangfu decoction can inhibit the decrease in salivary amylase activity and regulate bcl-2 gene expression in colorectal carcinoma patients with spleen deficiency syndrome.