Medicina Sistémica, sistemica, Systemics

Effects of the phytotheraputic preparation Circulat on gene expression levels in cultured human fibroblasts were determined using whole-genome microarray analysis. Circulat is a systemic herbal formulation that was developed under the postulates of the Systemic Theory at Centro Médico Docente Adaptógeno de Venezuela, where it is practically used, in cooperation with Nulab Inc., USA .

Lyophilized Circulat water extract was added to the culture media to a final concentration of 2 mcg per ml and human fibroblast cells MRC-5 were cultured at 37 °C for 16 hours. Control fibroblasts were cultured in parallel under same conditions without the extract. Upon treatment, total RNA was isolated from the cells and used for microarray analysis. Both Circulat-treated and control experiments were carried out in triplicates yielding six samples that were processed independently.

Microarray analysis was performed in accordance with standard procedures recommended by Affymetrix, Inc using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. U133 Plus 2.0 arrays provide full genomic coverage and contain probes for more than 47,000 unique transcripts corresponding to more than 38,500 human genes. Following hybridization and scanning, raw data in the form of image files were converted to gene expression values using Affymetrix GeneChip Operating Software (GCOS). GCOS utilizes MAS 5.0 algorithm for data normalization, background subtraction, estimation of nonspecific binding, calculation of detection p-values, and generation of "presence" calls. Two-tailed Student's t-test assuming unequal sample variance was used to identify genes that displayed significant changes in the mean expression levels between control and treated samples with the t-test p-value less then 0.05. Only probes that were called "present" in at least two samples were considered in this analysis. In order to identify strongly regulated genes, we imposed an additional requirement for the mean fold change between the samples to be no less then 2. 52 genes satisfied these stringent criteria demonstrating that Circulate treatment resulted in significant changes in gene expression levels.

38 genes were upregulated, i.e. their expression levels were increased, and 14 genes were downregulated, i.e. their expression levels were decreased upon Circulat treatment by at least two fold. Downregulation does not mean a negative effect since positive effects of some drugs or stimuli are produced by the reduction of expression levels and activities of certain genes. The complete list of Circulat-responsive genes with mean expression values, fold change, t-test statistics, proteins encoded by them, brief descriptions of biological processes they participate in, molecular functions attributed to them, their likely subcellular localization as well as some other characteristics can be found in the attached supplement.

Clinical observations have firmly established that Circulat prevents development of severe manifestations of Type 2 diabetes. It is quite likely therefore, that at list some of the genes identified by our analysis as being regulated by Circulat are involved in pathogenesis or susceptibility to Type 2 diabetes. The obtained results provide an excellent opportunity to discover genes involved in the onset of Type 2 diabetes, which is the necessary prerequisite for our in-depth understanding of the disease as well as for development of new drugs and treatments. They also reveal the previously unknown molecular mechanisms of Circulat action and demonstrate the great potential of this preparation for normalization of gene expression altered in Type 2 diabetic patients.

Our analysis indicates that a large proportion of the identified genes are involved in energy metabolism and signaling pathways. This is consistent with what is currently known about molecular changes underlying Type 2 diabetes. Further analysis of these genes may reveal novel pathways involved in the disease development and progression and may yield new molecular targets for drug development. Several of likely candidates are listed below:

RTP1 receptor transporting protein 1

CYP3A4 cytochrome P450, family 3, subfamily A, polypeptide 4

RABGAP1L RAB GTPase activating protein 1-like

NMNAT2 nicotinamide nucleotide adenylyltransferase 2

STK38 serine/threonine kinase 38

ATF71P2 activating transcription factor 7 interacting protein 2

CH25H cholesterol 25-hydroxylase

GRK6 G protein-coupled receptor kinase 6

TMEM26 transmembrane protein 26

TRPM2 transient receptor potential cation channel, subfamily M, member 2.

It is important to point out that TRPM2 gene has been previously shown to be involved in the process of development of atherosclerosis, which is known to contribute to the pathogenesis of Type 2 diabetes. The observed reduction of expression levels of this gene by Circulat, therefore, is likely to be one of the main determinants of the positive effect of the preparation.

Microarray analysis is a new and extremely powerful technique that has been made possible by the latest achievements of molecular genetics and nanotechnology. It allows one to precisely monitor changes that take place inside the organism upon drug treatment on the molecular level and offers an objective way of estimating drug efficacy. This is the first study in which microarray analysis was used to reveal genome-wide expression changes in cultured human cells upon their treatment with a herbal preparation and it demonstrated the potential of such an approach for future development of herbal medicine.

Tissue culture work and microarray analyses were performed in DNA Sequencing Facility and Microarray Core Facility of the University of Pennsylvania School of Medicine, respectively.

Gene Symbols and Expression Data

Gene Symbol	Affymetrix Probe Id	Mean Experiment	Mean Control	Fold Change	t-test p-value
RTP1	1553823_a_at	15.66666667	3.166666667	4.947368421	0.006920496
CYP3A4	205998_x_at	90.63333333	20.8	4.357371795	0.010950177
CA12	233388_at	29.03333333	6.666666667	4.355	0.012863121
RABGAP1L	237650_at	36.46666667	8.6	4.240310078	0.008981609
NMNAT2	1556029_s_at	20.86666667	5.133333333	4.064935065	0.042171054
RLTPR	227216_at	32.6	9	3.622222222	0.024903156
CCL5	204655_at	33.86666667	9.6	3.527777778	0.033727623
GLYAT	206930_at	24.23333333	7.766666667	3.120171674	0.041978624
ZC3H12C	1559763_at	19.93333333	6.4	3.114583333	0.030189001
CEACAM6	211657_at	26.66666667	8.766666667	3.041825095	0.03325915
AHI1	220842_at	23	8.133333333	2.827868852	0.025509932
CYLC1	216809_at	25.83333333	9.3	2.777777778	0.004057367
FLJ20211	229864_at	64.7	24.93333333	2.594919786	0.046694254
AOF2	1555897_at	42.83333333	16.53333333	2.590725806	0.01310035
MGC40368	1569206_at	50.83333333	20.23333333	2.512355848	0.023463147
KIAA0690	1559127_x_at	56.13333333	22.53333333	2.49112426	0.048737734
SIRT2	1558331_at	33.96666667	13.76666667	2.467312349	0.028619776
NUP210	212316_at	42.06666667	17.06666667	2.46484375	0.043021154
OLFM2	223601_at	36.8	15.26666667	2.410480349	0.043482934
KIAA0802	240448_at	28.36666667	11.83333333	2.397183099	0.019624743
STXBP3	1560486_at	22.4	9.366666667	2.391459075	0.028410646
ULK1	209333_at	43.16666667	18.06666667	2.389298893	0.014691051
MGC39558	1562391_at	35.56666667	14.93333333	2.381696429	0.031269169
SETBP1	236317_at	33.4	14.03333333	2.380047506	0.046771831
KIAA1609	233423_at	29.03333333	12.26666667	2.366847826	0.049702477
ZBTB7A	219186_at	58.3	26.03333333	2.23943662	0.003884894
MAPT	206401_s_at	33.03333333	15.03333333	2.197339246	0.019562624
ANXA2P3	211241_at	162	73.96666667	2.190175755	0.035526956
ZBTB20	240216_at	35.76666667	16.53333333	2.163306452	0.040516242
FLJ40722	1564028_s_at	48.8	23.23333333	2.100430416	0.003223983
DYM	220774_at	34.5	16.43333333	2.099391481	0.007879413
NEURL	204888_s_at	33.3	15.86666667	2.098739496	0.049228493
OR2A9P /// OR2A20P	223971_at	40.76666667	19.46666667	2.094178082	0.043374012
CDYL	240594_at	38.33333333	18.5	2.072072072	0.040712976
STK38	244679_at	32.36666667	15.63333333	2.070362473	0.029048526
ATF7IP2	219870_at	38.2	18.76666667	2.035523979	0.01364187
MAF	209348_s_at	22.36666667	11.06666667	2.021084337	0.040223685
RP1-32F7.2	239481_at	19.8	9.833333333	2.013559322	0.029458491
DKFZp434D1428	223409_at	17.53333333	35.36666667	0.495758718	0.012493312
TIFA	238858_at	13.16666667	27.33333333	0.481707317	0.013270711
EVI2B	211742_s_at	19	39.5	0.481012658	0.044037999
CH25H	206932_at	14	29.56666667	0.473506201	0.021032921
GRK6	210981_s_at	44.86666667	95.96666667	0.467523446	0.00649051
DKK2	224199_at	7.033333333	16.03333333	0.438669439	0.049259205
TMEM26	1555025_at	8.266666667	19.26666667	0.429065744	0.044468546
WDR68	236134_at	11.63333333	28	0.41547619	0.013415944
ZFHX1B	1557797_a_at	13.63333333	33.4	0.408183633	0.028878705
MGC10986	218600_at	18.26666667	45.1	0.405025868	0.003551854
C21orf37	240801_at	7.333333333	18.83333333	0.389380531	0.026918272
TRPM2	205708_s_at	18.36666667	48.33333333	0.38	0.025004711
NIPSNAP3B	223764_x_at	5.666666667	15.9	0.35639413	0.040051049
FHL5	1555191_a_at	4.566666667	14.8	0.308558559	0.022784303

Gene Descriptions

Gene Symbol	Gene Description
RTP1	receptor transporting protein 1
CYP3A4	cytochrome P450, family 3, subfamily A, polypeptide 4
CA12	Carbonic anhydrase XII
RABGAP1L	RAB GTPase activating protein 1-like
NMNAT2	nicotinamide nucleotide adenylyltransferase 2
RLTPR	RGD, leucine-rich repeat, tropomodulin and proline-rich containing protein
CCL5	chemokine (C-C motif) ligand 5 /// chemokine (C-C motif) ligand 5
GLYAT	glycine-N-acyltransferase
ZC3H12C	zinc finger CCCH-type containing 12C
CEACAM6	carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen)
AHI1	Abelson helper integration site
CYLC1	cylicin, basic protein of sperm head cytoskeleton 1
FLJ20211	Hypothetical protein FLJ20211
AOF2	amine oxidase (flavin containing) domain 2
MGC40368	T-complex 11 (mouse) like 2
KIAA0690	KIAA0690
SIRT2	Sirtuin (silent mating type information regulation 2 homolog) 2 (S. cerevisiae)
NUP210	nucleoporin 210kDa
OLFM2	olfactomedin 2
KIAA0802	KIAA0802
STXBP3	syntaxin binding protein 3
ULK1	unc-51-like kinase 1 (C. elegans)
MGC39558	UDP-GalNAc:betaGlcNAc beta 1,3-galactosaminyltransferase, polypeptide 2
SETBP1	SET binding protein 1
KIAA1609	KIAA1609 protein
ZBTB7A	zinc finger and BTB domain containing 7A
MAPT	microtubule-associated protein tau
ANXA2P3	annexin A2 pseudogene 3
ZBTB20	Zinc finger and BTB domain containing 20
FLJ40722	hypothetical protein FLJ40722
DYM	Dymeclin
NEURL	neuralized-like (Drosophila)
OR2A9P /// OR2A20P	olfactory receptor, family 2, subfamily A, member 20 pseudogene
CDYL	Chromodomain protein, Y-like
STK38	Serine/threonine kinase 38
ATF7IP2	activating transcription factor 7 interacting protein 2
MAF	v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)
RP1-32F7.2	hypothetical protein FLJ37659
DKFZp434D1428	hypothetical protein DKFZp434D1428
TIFA	TRAF-interacting protein with a forkhead-associated domain
EVI2B	ecotropic viral integration site 2B
CH25H	cholesterol 25-hydroxylase
GRK6	G protein-coupled receptor kinase 6
DKK2	dickkopf homolog 2 (Xenopus laevis)
TMEM26	transmembrane protein 26
WDR68	WD repeat domain 68
ZFHX1B	Zinc finger homeobox 1b
MGC10986	hypothetical protein MGC10986
C21orf37	chromosome 21 open reading frame 37
TRPM2	transient receptor potential cation channel, subfamily M, member 2
NIPSNAP3B	nipsnap homolog 3B (C. elegans)
FHL5	four and a half LIM domains 5, activator of CREM in testis

Biological Process Summary

Gene Symbol	Gene Ontology Biological Process Description
RTP1	---
CYP3A4	electron transport /// lipid metabolism /// xenobiotic metabolism /// xenobiotic metabolism
CA12	one-carbon compound metabolism
RABGAP1L	---
NMNAT2	biosynthesis /// NAD biosynthesis /// pyridine nucleotide biosynthesis
RLTPR	---
CCL5	calcium ion homeostasis /// exocytosis /// cell motility /// chemotaxis /// inflammatory response /// cellular defense response /// response to oxidative stress /// cell adhesion /// signal transduction /// cell-cell signaling /// sensory perception /// response to virus /// chemotaxis /// inflammatory response /// immune response /// immune response
GLYAT	acyl-CoA metabolism /// response to toxin
ZC3H12C	---
CEACAM6	signal transduction /// cell-cell signaling
AHI1	---
CYLC1	spermatogenesis /// cell differentiation /// development /// spermatogenesis
FLJ20211	---
AOF2	electron transport /// transcription /// regulation of transcription, DNA-dependent /// chromatin modification
MGC40368	---
KIAA0690	---
SIRT2	chromatin silencing at rDNA /// chromatin silencing at telomere /// regulation of transcription, DNA-dependent /// protein amino acid ADP-ribosylation /// redox signal response /// cell cycle /// mitosis /// regulation of exit from mitosis /// negative regulation of transcription /// histone deacetylation /// regulation of phosphorylation /// negative regulation of striated muscle development /// cell division /// redox signal response /// regulation of exit from mitosis /// negative regulation of transcription /// histone deacetylation /// regulation of phosphorylation /// negative regulation of striated muscle development /// chromatin silencing /// chromatin silencing /// gene silencing
NUP210	---
OLFM2	---
KIAA0802	---
STXBP3	vesicle docking during exocytosis /// protein transport /// vesicle-mediated transport /// transport
ULK1	protein amino acid phosphorylation /// signal transduction /// protein amino acid phosphorylation
MGC39558	protein amino acid glycosylation
SETBP1	---
KIAA1609	---
ZBTB7A	transcription /// negative regulation of transcription, DNA-dependent /// cartilage development /// regulation of transcription, DNA-dependent
MAPT	microtubule cytoskeleton organization and biogenesis /// apoptosis /// negative regulation of microtubule depolymerization /// negative regulation of microtubule depolymerization
ANXA2P3	---
ZBTB20	transcription /// regulation of transcription, DNA-dependent
FLJ40722	---
DYM	---
NEURL	nervous system development /// protein ubiquitination
OR2A9P /// OR2A20P	G-protein coupled receptor protein signaling pathway
CDYL	chromatin assembly or disassembly /// spermatogenesis /// metabolism
STK38	protein amino acid phosphorylation /// protein kinase cascade /// protein modification /// protein amino acid phosphorylation
ATF7IP2	---
MAF	transcription /// regulation of transcription, DNA-dependent /// transcription from RNA polymerase II promoter
RP1-32F7.2	---
DKFZp434D1428	---
TIFA	---
EVI2B	---
CH25H	metabolism /// lipid metabolism
GRK6	protein amino acid phosphorylation /// signal transduction /// regulation of G-protein coupled receptor protein signaling pathway
DKK2	development /// Wnt receptor signaling pathway /// negative regulation of Wnt receptor signaling pathway
TMEM26	---
WDR68	---
ZFHX1B	regulation of transcription, DNA-dependent /// nervous system development /// negative regulation of transcription /// transcription /// regulation of transcription
MGC10986	---
C21orf37	---
TRPM2	cation transport /// sodium ion transport /// calcium ion transport /// transport /// ion transport /// calcium ion transport
NIPSNAP3B	---
FHL5	---

Molecular Function Summary

Gene Symbol	Gene Ontology Molecular Function Description
RTP1	---
CYP3A4	iron ion binding /// oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen /// oxygen binding /// heme binding /// metal ion binding /// unspecific monooxygenase activity /// quinine 3-monooxygenase activity /// monooxygenase activity /// monooxygenase activity /// oxidoreductase activity /// monooxygenase activity /// testosterone 6-beta-hydroxylase activity /// monooxygenase activity
CA12	carbonate dehydratase activity /// zinc ion binding /// lyase activity /// metal ion binding /// carbonate dehydratase activity /// zinc ion binding
RABGAP1L	---
NMNAT2	nicotinamide-nucleotide adenylyltransferase activity /// transferase activity /// nucleotidyltransferase activity
RLTPR	---
CCL5	chemokine activity /// cytokine activity /// chemokine activity /// signal transducer activity /// receptor binding
GLYAT	acyltransferase activity /// transferase activity /// glycine N-acyltransferase activity /// acyltransferase activity
ZC3H12C	---
CEACAM6	receptor activity
AHI1	---
CYLC1	structural molecule activity /// structural molecule activity
FLJ20211	---
AOF2	oxidoreductase activity
MGC40368	---
KIAA0690	Binding
SIRT2	DNA binding /// transcription factor binding /// zinc ion binding /// hydrolase activity /// NAD-dependent histone deacetylase activity /// histone acetyltransferase binding /// histone deacetylase binding /// tubulin deacetylase activity /// ubiquitin binding /// metal ion binding /// protein binding /// transcription factor binding /// NAD-dependent histone deacetylase activity /// histone acetyltransferase binding /// histone deacetylase binding /// tubulin deacetylase activity /// ubiquitin binding /// zinc ion binding
NUP210	porin activity /// protein binding
OLFM2	latrotoxin receptor activity
KIAA0802	---
STXBP3	---
ULK1	nucleotide binding /// protein serine/threonine kinase activity /// protein-tyrosine kinase activity /// protein binding /// ATP binding /// transferase activity /// protein kinase activity /// protein serine/threonine kinase activity /// kinase activity
MGC39558	galactosyltransferase activity /// transferase activity
SETBP1	DNA binding
KIAA1609	---
ZBTB7A	DNA binding /// protein binding /// zinc ion binding /// transcriptional repressor activity /// histone acetyltransferase binding /// metal ion binding /// nucleic acid binding /// DNA binding
MAPT	structural constituent of cytoskeleton
ANXA2P3	---
ZBTB20	DNA binding /// protein binding /// zinc ion binding /// metal ion binding /// nucleic acid binding
FLJ40722	---
DYM	---
NEURL	ubiquitin-protein ligase activity /// zinc ion binding /// metal ion binding
OR2A9P /// OR2A20P	rhodopsin-like receptor activity /// olfactory receptor activity
CDYL	chromatin binding /// catalytic activity
STK38	nucleotide binding /// magnesium ion binding /// protein serine/threonine kinase activity /// protein-tyrosine kinase activity /// ATP binding /// transferase activity /// magnesium ion binding /// protein kinase activity /// protein serine/threonine kinase activity /// ATP binding /// kinase activity /// metal ion binding
ATF7IP2	---
MAF	DNA binding /// RNA polymerase II transcription factor activity
RP1-32F7.2	---
DKFZp434D1428	---
TIFA	---
EVI2B	---
CH25H	catalytic activity /// steroid hydroxylase activity
GRK6	nucleotide binding /// G-protein coupled receptor kinase activity /// protein-tyrosine kinase activity /// signal transducer activity /// ATP binding /// transferase activity /// protein kinase activity /// protein serine/threonine kinase activity /// kinase activity /// receptor activity
DKK2	---
TMEM26	---
WDR68	protein binding
ZFHX1B	transcription factor activity /// zinc ion binding /// transcriptional repressor activity /// phosphatase regulator activity /// SMAD binding /// metal ion binding /// nucleic acid binding /// DNA binding
MGC10986	zinc ion binding /// metal ion binding
C21orf37	---
TRPM2	calcium channel activity /// calcium ion binding /// hydrolase activity /// sodium ion binding /// ADP-ribose diphosphatase activity /// ion channel activity /// cation channel activity /// calcium channel activity /// receptor activity
NIPSNAP3B	---
FHL5	zinc ion binding /// metal ion binding

Cellular Localization Summary

Gene Symbol	Gene Oontology Cellular Component Description
RTP1	integral to membrane
CYP3A4	membrane fraction /// endoplasmic reticulum /// microsome /// microsome /// cell surface /// membrane /// membrane fraction
CA12	integral to membrane /// integral to membrane
RABGAP1L	---
NMNAT2	---
RLTPR	---
CCL5	extracellular space /// extracellular region
GLYAT	Mitochondrion
ZC3H12C	---
CEACAM6	integral to plasma membrane /// membrane
AHI1	---
CYLC1	cytoskeleton /// acrosomal matrix /// cytoskeleton
FLJ20211	---
AOF2	Nucleus
MGC40368	---
KIAA0690	nucleus /// integral to membrane
SIRT2	chromatin silencing complex /// cytoplasm /// cytoskeleton /// microtubule /// cytoplasm /// microtubule /// chromatin silencing complex /// microtubule
NUP210	integral to membrane /// outer membrane
OLFM2	Membrane
KIAA0802	---
STXBP3	---
ULK1	---
MGC39558	Membrane
SETBP1	Nucleus
KIAA1609	---
ZBTB7A	nucleus /// nucleus
MAPT	cytosol /// cytoskeleton /// microtubule /// microtubule associated complex /// plasma membrane /// axon
ANXA2P3	---
ZBTB20	Nucleus
FLJ40722	---
DYM	integral to membrane
NEURL	ubiquitin ligase complex
OR2A9P /// OR2A20P	integral to membrane
CDYL	chromatin /// nucleus
STK38	nucleus /// nucleus
ATF7IP2	integral to membrane
MAF	chromatin /// nucleus
RP1-32F7.2	---
DKFZp434D1428	---
TIFA	---
EVI2B	integral to plasma membrane
CH25H	membrane fraction
GRK6	---
DKK2	extracellular space /// extracellular region
TMEM26	---
WDR68	---
ZFHX1B	nucleus /// nucleus
MGC10986	---
C21orf37	---
TRPM2	integral to plasma membrane /// membrane /// integral to membrane
NIPSNAP3B	---
FHL5	Nucleus

Gene Family Summary

Gene Symbol	Ensembl Family Description
RTP1	RECEPTOR TRANSPORTING
CYP3A4	CYTOCHROME P450
CA12
RABGAP1L
NMNAT2