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.

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 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.

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.

[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.

[Studies of gene networks and singal transduction of kidney deficiency syndrome by syndrome differentiation through drug effects].

Three kinds of networks were summarily described in this review including the small intracellular molecular networks, the middle-scale networks of hypothalamus-pituitary-adrenal-thymus (HPAT) axis and the large network, neuroendocrine-immune (NEI) network, covering the whole organism and linking multiple systems together. The hypothesis was expressed that the "disease" or "syndrome" formed in the human body by the intervention from outside world is based on the changes of multi-molecular network. In this paper, the pattern and ability of signal transduction channel and the methods of studying changes in it were also described, and raised, herefrom, "to determine syndrome by drug effects (DSDE)" is the intervention means for studying syndrome in the light of systemic biological methods. We found Kidney-yang deficiency syndrome covered the NEI network and the regulating center located in hypothamus with Compound Bushen Recipe (CBR, Kidney-tonifying recipe). By intervention with EF, an effective component of CBR, it was found that EF can activate the immune system and the three networks, including growth axis, sex hormone axis and lymphocyte apoptosis network in HPAT axis through the downward pathway of NEI network to play its efficiency of molecular network. There are many regulation patterns of EF on networks. For example, in the network mechanism of lymphocyte apoptosis and proliferation, EF can reconstruct the balance of the opposite apoptosis related genes and proliferation related genes; EF can assemble and integrate co-stimulating molecules, transform growth factors (TGF), and several oncogenes to form an upstream factor network for initiating the proliferation and anti-apoptosis promotion; EF can simultaneously up-regulate the two opposite genes expression of IkappaB and NFkappaB in NIK/IKK/IkappaB/Rel/NFkappaB signal transduction channel, which could not only control the rising of NFkappaB in a moderate range, but also guarantee its predominant status to exert its hinge role in molecule regulating network, by which gene network regulation atlas in HPAT axis of Kidney-deficiency syndrome was observed.

[Study on relationship between estrogen receptor gene polymorphism and syndrome differentiation typing of female postmenopausal osteoporosis in Traditional Chinese medicine].

OBJECTIVE: To study the relationship between estrogen gene polymorphism and TCM Syndrome Differentiation of female postmenopausal osteoporosis in China. METHODS: Two hundred and forty-six Chinese postmenopausal women, age 44-80 years, mean 65.8 years, using molecular biological method to analyze the endonuclease Pvu II, Xba I restriction fragment length polymorphisms (RFLPs), with dual X-ray bone mineral density absorption meter to determine the bone mineral densities of lumbar vertebra (L1-4) and femur (intertrochanter, femur neck, Ward's region) separately. The subjects were divided into Kidney Yin deficiency type, Kidney Yang deficiency type and both Kidney Yin-Yang deficiency type, to observe the relationship between TCM and bone density as well as estrogen receptor gene polymorphism, Pp(Pvu II) and Xx(Xba I) were used to express RFLPs, the capital P and X to express the deficit of restricting sites. RESULTS: Bone mineral density of PPxx gene type (n = 21) was obviously lower than that of other gene types (n = 225), lumbar (-0.71 +/- 0.46) g/cm2, intertrochanter (-0.31 +/- 0.58) g/cm2, femur neck (-0.84 +/- 0.66) g/cm2, Ward's region (-0.96 +/- 0.85) g/cm2, the TCM Syndrome Differentiation typing of this gene type belonged to both Kidney Yin-Yang deficiency type. CONCLUSION: Estrogen receptor gene RFLPs is related to TCM Syndrome Differentiation typing.