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Transcriptome analysis of blood stasis syndrome in subjects with hypertension
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J Tradit Chin Med 2016 April 15; 36(2): 173-180 ISSN 0255-2922 © 2016 JTCM. All rights reserved.
CLINICAL STUDY TOPIC
Transcriptome analysis of blood stasis syndrome in subjects with hypertension
He Ling, Fang Meixia, Chen Liguo, Zhou Jianhua, Yuan Jing, Xu Jing, Shan Yan, Xu Qingyun, Xiong Tingting aa He Ling, Fang Meixia, Chen Liguo, Zhou Jianhua, Yuan Jing, Xu Jing, Shan Yan, Xu Qingyun, Xiong Tingting, Medical School of Jinan University, Guangzhou 510632, China Supported by National Scientific Fund (Assessing MicroRNA-mediated Endothelial Cell Injury in Blood Stasis, No. 81173157) and Guangdong Scientific Fund (Assessing MicroRNA-mediated Endothelial Cell Injury in Blood Stasis, No. 10151063201000045) Correspondence to: Prof. Chen Liguo, Medical School of Jinan University, Guangzhou 510632, China. [email protected]. cn Telephone: +86-20-85226476 Accepted: August 19, 2015
alyzed. Genes enriched significantly and their pathways were determined, as were network interactions, and encoded proteins. Gene identities were confirmed by real-time polymerase chain reactions. RESULTS: Compared with cells cultured in sera of the blood stasis groups, those culture in sera of healthy individuals and of the non-blood stasis group showed 11 and 301 differences, respectively in stasis-related genes. Genes identified as differing between the blood stasis and healthy groups included activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-β, and Jun proto-oncogene (JUN). Pathway and protein interaction network analyses showed that these genes were associated with endoplasmic reticulum stress. Cells cultured in sera of patients with blood stasis and Qi deficiency, Qi stagnation, heat retention, and cold retention were compared with cells cultured in sera of patients with the other types blood stasis syndrome. The comparison showed differences in expression of 28, 28, 34, and 32 specific genes, respectively.
Abstract OBJECTIVE: To screen for mRNAs associated with blood stasis syndrome and to explore the genetic mechanisms of blood stasis syndrome in hypertension. METHODS: This study involved groups of patients with hypertension and blood stasis, including those with Qi deficiency, Qi stagnation, cold retention and heat retention; as well as hypertensive patients without blood stasis and healthy individuals. Human umbilical vein endothelial cells were co-cultured with the sera of these healthy individuals and patients with blood stasis syndrome. Total RNA was extracted from these cells and assessed by a high-throughput sequencing method (Solexa) and digital gene expression. Differentially expressed genes among these six groups were compared using whole genome sequences, and mRNAs associated with blood stasis syndrome identified. Differences in gene use and gene ontology function were anJTCM | www. journaltcm. com
CONCLUSION: The pathogenesis of blood stasis syndrome in hypertension is related to endoplasmic reticulum stress and involves the differential expression of the activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-β, and JUN genes. © 2016 JTCM. All rights reserved. Key words: Hypertension; Blood stasis; RNA, messenger; Endoplasmic reticulum stress 173
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INTRODUCTION
tively. These patients were recruited from the cardiovascular department, the outpatient TCM department, and patients hospitalized at the First Affiliated Hospital of Jinan University. Patients in this group did not differ significantly from the group of hypertensive patients with blood stasis syndrome in blood pressure, sex distribution, mean arterial pressure, or average age (each P > 0.05). The third group consisted of 30 healthy volunteers (17 males, 13 females), recruited from the Department of Traditional Chinese Medicine, School of Medicine, Jinan University. Their blood pressures were within normal ranges and differed significantly (P < 0.01) from blood pressures in hypertensive patients, both with and without blood stasis syndrome. High blood pressure was defined according to World Health Organization / International Hypertension Alliance guidelines (1999),11 and blood stasis syndrome was diagnosed according to the 2011 revised criteria.12 Patients with target organ damage, diabetes, or severe cardiovascular, cerebrovascular, or renal disease were excluded.
Hypertension is a highly prevalent clinical syndrome characterized by high arterial pressure that can seriously damage the heart, brain, and kidneys. In traditional Chinese medicine (TCM), blood stasis is believed to play an important role in the occurrence and development of hypertension. Current studies on the mechanism of blood stasis syndrome have been limited to observing changes in blood, focusing on the relationship between vascular changes and the initiation of blood stasis syndrome. Moreover, blood stasis biology has been associated with changes in vascular endothelial cell (VEC) function,1 with VEC damage closely associated with the initiation and development of blood stasis syndrome.2,3 Endoplasmic reticulum stress (ERS) has been shown to damage VECs,4 indicating that ERS may induce blood stasis by causing endothelial cell injury. Our research group has focused on a blood stasis syndrome model, including the effects of VEC injury and mechanisms of repair, as well as on the effects of drugs and the molecular / genetic mechanisms underlying blood stasis. We and others have found that different types of blood stasis have different biochemical foundations, but also have common pathological and physiological characteristics.5-10 Because of the complex pathogenesis of blood stasis syndrome, including the effects of various factors, screening for and identifying biomarkers associated with this syndrome may help in its diagnosis and treatment. An established model of endothelial cell injury-blood stasis syndrome was used to screen for genes associated with the initiation of hypertension-related blood stasis syndrome among groups of hypertensive patients, with and without blood stasis syndrome, and healthy individuals. The genetic pathways of identified genes were investigated to determine the molecular mechanism underlying the development of blood stasis syndrome in hypertension.
Serum collection All included individuals were instructed to fast and not take any drugs overnight prior to a blood draw. Forearm venous blood was aseptically collected into sterile, non-anticoagulation vacuum tubes, allowed to coagulate, and centrifuged at 2784´g at 4 ℃ for 15 min. Serum was removed, placed in an aseptic Eppendorf tube (Gibco, Guangzhou, China), and incubated in a water bath at 56 ℃ for 30 min to inactivate serum complement. Finally, these samples were stored at －20 ℃. Establishing a cell model The cell model of hypertension and blood stasis,13 involved the use of human umbilical vascular endothelial cells (HUVECs; CRL-1730), obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in Dulbecco's Modified Eagle's medium (DMEM); (Gibco) containing 10% fetal bovine serum at 37 ℃ in a 5% CO2 incubator. To each culture flask was added serum, to a concentration of 10%, plus 90% F12K medium (Gibco), and the cells were cultured at 37 ℃ in a 5% CO2 incubator for 24 h. The cells were collected, and total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed by agarose gel electrophoresis.
MATERIALS AND METHODS Three groups of individuals were analyzed. The first group included 40 patients (21 males, 19 females), of mean age (66 ± 7) years, diagnosed with blood stasis syndrome with hypertension from December 2011 to June 2012 at the Second Hospital affiliated with Guangzhou Medical College. Their mean systolic and diastolic blood pressures were (157 ± 13) and (104 ± 7) mm Hg, respectively. TCM syndrome differentiation rules have identified four types of patients with blood stasis syndrome plus hypertension: blood stasis syndrome with Qi deficiency, Qi stagnation, cold retention, and heat retention. Ten patients were included in each subgroup. Another group of 12 patients [6 males, 6 females, of mean age (65 ± 9) years] had hypertension but without blood stasis. Their mean systolic and diastolic b; ppd pressures were (166 ± 8) and (104 ± 8) mm Hg, respecJTCM | www. journaltcm. com
mRNA sequence analysis and gene screening mRNA transcriptome sequencing was performed using second-generation IIlimina Solexa sequencing technology by the Shanghai Megiddo Biotechnology Co., Ltd. (Shanghai, China), The original statistical data were assessed while retaining high-quality sequences. Statistical analyses of each fragment per kilobase of exons per million fragments mapped (FPKM), as well as the transcript value in the expressed samples, were compared. 174
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Finally, differences in group transcripts and relative gene expression were analyzed among the six groups of samples.
residue at the dye front. The ratio of A260 to A280 was 1.8-2, showing that the quality of these total RNA samples was in full compliance with requirements for chip analysis.
Gene function analysis Differentially expressed genes were analyzed using Goatools (https: //github.com/tanghaibao/GOatools) in the GO database, along with a PATHWAY enrichment analysis of differential gene use with the software KO-Based Annotation System (KOBAS) (http: //kobas. cbi.pku.edu.cn/home.do). False positive rates were compared by Fisher's exact tests. A P value ≤ 0.05 was considered significant. Differences in the expression of significantly enriched genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify cellular pathways. The Human Protein Reference Database (http: // www.hprd.org/Release 9), was used to assess interactions among these genes and interaction data were determined using Cytoscape software (http://www.cytoscape.org/) for network visualization.
Differential gene expression of blood stasis syndrome Gene expression by each cell sample was assessed by differential gene expression (DGE) analysis, with a P value of < 0.01, an False Discovery Rate (FDR) of < 0.001, and a |log2Ratio| of ≥ 1 considered significant (Figure 1). The four groups with hypertension and blood stasis groups showed significant differences in mRNA expression compared with the control group. Specifically, the groups with Qi deficiency, Qi stagnation, cold retention, and heat retention showed significant differences in 76 (42 downregulated and 34 upregulated), 51 (34 downregulated and 17 upregulated), 61 (39 downregulated and 22 upregulated) and 62 (39 downregulated and 23 upregulated) mRNAs, respectively. The four groups with hypertension and blood stasis also showed significant differences compared with the group with hypertension but without blood stasis. Specifically, the groups with Qi deficiency, Qi stagnation, cold retention, and heat retention showed significant differences in 579 (117 downregulated and 459 upregulated), 475 (98 downregulated and 337 upregulated), 621 (135 downregulated and 486 upregulated) and 469 (103 downregulated and 366 upregulated) mRNAs, respectively. In addition, the group with hypertension but without blood stasis differed from the healthy group in 529 (423 downregulated and 106 upregulated) mRNAs.
Quantitative real-time polymerase chain reaction (qRT-PCR) PCRs were performed according to the manufacturer's instructions, with primers designed based on the sequences in the gene database. The2-ΔΔCT method was used to assess differential gene expression. Log2 ratios were determined by comparing gene expression in the five hypertensive groups with that in healthy controls.
RESULTS RNA extraction Total RNA was extracted from HUVECs incubated with serum from each of the groups of subjects. The 28S and 18S rRNA bands were clear, with no observed
Differential mRNA expression analysis Integrated analyses of differential mRNA expression in the six groups are shown in Table 1.
600 Gene number
500 400 300 200 100 0
QBS vs NC QSBS vs NC CBS vs NC
HBS vs NC NBS vs NC QDBS vs NBS QSBS vs NBS CBS vs NBS HBS vs NBS Groups
Upregulated gene number Downregulated gene number Figure 1 Numbers of mRNAs differentially expressed between pairs of groups QDBS: Qi deficiency and blood stasis; QSBS: Qi stagnation and blood stasis; CBS: cold retention and blood stasis; HBS: heat retention and blood stasis; NBS: hypertension without blood stasis; NC: healthy controls. Compare with NC group, the groups with QDBS, QSBS, CBS, and HBS showed significant differences in 76 (42 downregulated and 34 upregulated), 51 (34 downregulated and 17 upregulated), 61 (39 downregulated and 22 upregulated) and 62 (39 downregulated and 23 upregulated) mRNAs, respectively. Compared with NBS group, the groups with QDBS, QSBS, CBS, and HBS showed significant differences in 579 (117 downregulated and 459 upregulated), 475 (98 downregulated and 337 upregulated), 621 (135 downregulated and 486 upregulated) and 469 (103 downregulated and 366 upregulated) mRNAs, respectively. In addition, the group with NBS differed from NC group in 529 (423 downregulated and 106 upregulated) mRNAs. JTCM | www. journaltcm. com
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He L et al. / Clinical Study Table 1 Specific gene statistics in blood stasis syndrome Total
Group
mRNA Difference Upregulated
Downregulated
Qi deficiency and blood stasis syndrome vs normal and vs hypertension alone
22
10
12
Qi stagnation and blood stasis syndrome vs normal and vs hypertension alone
19
11
8
cold retention and blood stasis syndrome vs normal and vs hypertension alone
14
7
7
heat retention and blood stasis syndrome vs normal and vs hypertension alone
14
7
7
All four groups with blood stasis syndrome vs hypertension alone
301
239
62
All four groups with blood stasis syndrome vs normal
11
2
9
Qi deficiency and blood stasis syndrome in endemic hypertension
28
14
14
Qi stagnation and blood stasis syndrome in endemic hypertension
28
8
20
cold retention and blood stasis syndrome in endemic hypertension
32
10
22
Heat retention and blood stasis syndrome in endemic hypertension
34
9
25
Qi deficiency and blood stasis syndrome
124
67
57
Qi stagnation and blood stasis syndrome
81
34
47
cold retention and blood stasis syndrome
161
83
78
heat retention and blood stasis syndrome
69
38
31
GO enrichment analysis of differentially expressed genes in different blood stasis syndromes Patients with blood stasis and heat retention, cold retention, Qi deficiency, and Qi stagnation were compared with the group with hypertension but without blood stasis. GO enrichment analysis demonstrated that differentially expressed genes are related to multiple stress responses, cell differentiation, apoptosis, and autophagy in VECs. These genes are also immune related (P < 0.05) (Figure 2). Five of the 20 pathways identified were directly associated with endoplasmic reticulum stress (ERS), including PERK-regulated gene expression, chaperone activation of IRE1alpha, chaperone activation by activating transcriptional factor 6 (ATF6)-alpha, and the release of eIF4E and the ATF-2 transcription factor network. Other pathways included the interleukin (IL)-6 and IL-2 inflammatory pathways, the p53 and p38 apoptosis pathways, the mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) pathways, the mitogen-activated protein kinase (MAPK) signaling pathway, and signal transduction and amino acid metabolism pathways (Table 2).
Confirmation of differential mRNA expression by qRT-PCR qRT-PCR analysis comparing mRNA expression in the four groups with hypertension and blood stasis syndrome and the group with hypertension alone and the control group confirmed findings on the differential expression of ATF4, ATF3, DDIT3, TRIB3, CEBPB, and JUN mRNAs (Figure 4).
DISCUSSION Hypertension is a polygenic disease resulting from different genetic and environmental factors14 and can enhance the risk of cardiac and cerebrovascular diseases. Hypertensive patients with blood stasis syndrome show increases in hemorheology, hemodynamics, and microcirculation.15 ERS can damage VECs by promoting the expression of p38 mitogen activated protein kinase (MAPK) and its downstream molecules, along with oxidative stress. These findings suggest that ERS may be caused by endothelial cell injury resulting from blood stasis, consistent with results showing that ERS inhibition is effective in treating vascular-related diseases.16 The ER is important for protein synthesis and folding, Ca2 + storage, and lipid synthesis.17 Modifications of secreted, transmembrane, and ER resident proteins can help them fold into their native conformations, eventually forming functional proteins.18 Changes in the inner cellular environment, such as hypoxia, Ca2+ homeostasis, oxidative stress, or abnormal glycosylation, can result in the accumulation of unfolded or misfolded proteins in the ER, resulting in ER dysfunction or ERS. Protein accumulation leads to a series of follow-up reactions, called an unfolded protein response (UPR). Reducing the amount of protein in the ER re-
Protein expression and blood stasis syndrome Comparisons of gene expression by cells incubated with sera from patients with hypertension alone and those with hypertension and blood stasis revealed evidence of differential expression of specific genes in protein networks. Genes associated with ERS included activating transcription factor 4 (ATF4), activating transcription factor 3 (ATF3), DNA-damage inducible transcription factor 3 (DDIT3), Tribbles homolog 3 (TRIB3), CCAAT / enhancer binding protein-β (CEBPB), JUN, Jun dimerization protein 2 and Early growth response 1 (Figure 3). JTCM | www. journaltcm. com
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Positive regulation of cell death Cellular response to external stimulus Negative regulation of cellular process Primary metabolic process Programmed cell death Biological regulation Response to oxygen-containing compound Single-organism cellular process Cellular response to stimulus Regulation of apoptotic process Single-organism process Regulation of programmed cell death Response to external stimulus Regulation of protein metabolic process Regulation of cell death Negative regulation of biological process Positive regulation of cellular process Cellular response to organic substance Postive regulation of biol Response to unfolded protein Cellular process Response to unfolded protein Biological_process Response to undoplasmic reticulum stress Cellular response to chemical stimulus Response to chemical Response to stimulus Cellular response to stress Response to organic substance Response to stress
A
Anion binding Cytokine activity Prephenate dehydratase activity Interleukin-8 receptor binding Fibronectin binding Growth factor activity Amino acid transmembrane transporter activity Receptor binding Utral amino acid transmembrane transporter activity Molecular_function Binding Protein binding Organelle lumen Membrane-enclosed lumen Intracellular organelle Organelle Nuclear part Pigment granule Melanosome Nucleus Extracellular region part Intracellula membrane-bounded organelle membrane-bounded organelle Extracellular space Cytoplasm Cytosol Cytoplasmic part Intracellular Cell Cell part Intracellular part Cellular_component
B
C
Figure 2 GO gene protein analysis related to blood stasis syndrome A: Biological processes; B: molecular functions, C: cellular components. The ordinate indicates significant enrichment using the GO term; the abscissa represents the enrichment factor; GO: gene ontology.
sults in the repair of misfolded proteins and the degradation of unfolded and misfolded proteins in the ER, restoring ER function and ensuring the release of mature proteins. Overexposure to the altered cellular involvement may overwhelm UPR capacity, leading to apoptosis.19-24 Stress and UPR can activate three transcription factors, inositol enzyme (IRE-1), pancreatic endoplasmic protein kinase (PERK), and activating transcription factor 6 (ATF6), resulting in three steps to induce cell apoptosis, involving PERK, ATF4, and the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α).25 PERK pathway activation occurs by inhibiting protein during early ERS, resulting in the phosphorylation of elF2α and ATF4 activation; this in turn promotes the activation and expression of CCAAT-enhancer-binding protein homologous protein (CHOP) and induces cell apoptosis.26 ATF4 is a key molecule increased in ERS-induced apoptosis.27 The transcription factor CEBPB is involved in many biological processes, including cell differentiation, proliferation, metabolism, tumors, inflammation, and apoptosis. The C/EBP homologous protein CHOP is an important signaling molecule in the ERS-associated apoptosis pathway.28 DDIT3, also known as GADD153, CHOP, and C / EBP, is a memJTCM | www. journaltcm. com
ber of the C / EBP family of transcription factors. Its high expression may affect protein folding in the ER and trigger an unfolded protein response and apoptosis induced by a specific ER cell pathway network, causing cell death. This protein plays an important role in induced cell cycle arrest, promoting cell differentiation and apoptosis.29 ATF3 is a member of the ATF/CERB transcription factor family that inhibits transcription by binding to the gadd153 / CHOP promoter and corresponding sites. ATF3 plays an important role in the transcriptional regulation of cell apoptosis, with any factor that upregulates ATF3 expression inducing cell apoptosis.30 The v-Jun sarcoma virus 17 gene homologue JUN binds to the promoter regions of many genes, regulating the expression of many genes in various malignant tumors. TRIB3 is a serine/threonine protein kinase and a member of a newly discovered protein gene family that can inhibit mitosis. Overexpression of TRIB3 can inhibit the MAPK pathway, adjust the NF-κB pathway, and induce apoptosis.31 Thus, all of these genes and ERS are closely related to cell apoptosis. Our results, using gene chip technology, showed differences in mRNA expression among patients with hypertension plus blood stasis, hypertension alone, and nor177
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He L et al. / Clinical Study Table 2 Identification of gene pathways and involved genes differentiating patients with hypertension plus blood stasis syndrome and those with hypertension alone No.
Pathway
1
Number of genes
Background gene number
PERK regulated gene expression
5
45
2
Amino acid and oligopeptide SLC transporters
8
341
3
Activation of Chaperones by IRE1alpha
4
51
4
Amino acid transport across the plasma membrane
6
190
5
Serine biosynthesis
3
23
6
Signaling mediated by p38-alpha and p38-beta
5
343
7
IL6-mediated signaling
7
349
8
Axonal growth stimulation
2
18
9
Activation of Chaperones by ATF6-alpha
2
26
10
p53 signaling pathway
7
691
11
Vitamin B6 metabolism
2
42
12
Release of eIF4E
2
57
13
Disease
16
4993
14
mTOR signaling pathway
5
553
15
ATF-2 transcription factor network
6
494
16
IL12 and STAT4 dependent signaling pathways in Th1 development
3
103
17
Neurophilin interactions with VEGF and VEGFR
2
28
18
Circadian clock
3
210
19
VEGF signaling pathway
4
693
20
MAPK signaling pathway
9
2468
Notes: PERK: pancreatic endoplasmic reticulum kinase; SLC: solute-carrier; IRE1: inositol-requiring protein 1; IL-6: interleukin-6; ATF6: activating transcriptional factor 6; eIF4E: eukaryotic initiation factor 4E; mTOR: mammalian target of rapamycin; ATF2: activating transcription factor-2; IL-12: interleukin-12; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor; MAPK: mitogenactivated protein kinase. A CEBPB
TSC22D3
ATF3
TSC22D3 ATF3
CEBPB
JDP2
JUN
DDIT3
B
JDP2 JUN
DDIT3 ATF4
TRIB3
ATF4 TRIB3
CEBPG
C
TSC22D3
JUND JDP2
ATF4
EGR1 CEBPB
CEBPG
DDIT3 TRIB3
ATF3
TRIB3
JUN
ATF3
MYC
D
CEBPG
ATF4
CEBPB
DDIT3
CEBPG
RASSF1
Figure 3 Correlations between blood stasis and gene expression network interactions A: Qi stagnation and blood stasis (QSBS) vs hypertension without blood stasis (NBS); B: Qi deficiency and blood stasis (QDBS) vs NBS; C: cold retention and blood stasis (CBS) vs NBS; D: heat retention and blood stasis (HBS) vs NBS. ATF4: activating transcription factor 4; ATF3: activating transcription factor 3; JUN: Jun proto-oncogene; CEBPB: CCAAT/enhancer binding protein-β; DDIT3: DNA-damage inducible transcription factor 3; TRIB3: Tribbles homolog 3; CEBPG: CCAAT/enhancer binding protein gamma; TSC22D3: TSC22 domain family member 3; JDP2: Jun dimerization protein 2; MYC: v-myc avian myelocytomatosis viral oncogene homolog ; RASSF1: Ras association (Ral GDS/AF-6) domily member 1; EGR1: Early growth response 1.
mal controls. ATF4, ATF3, DDIT3, TRIB3, CEBPB, and JUN mRNAs were more highly expressed in the JTCM | www. journaltcm. com
blood stasis groups. GO and PATHWAY analysis showed that multiple genes and a signal pathway were 178
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Log2 ratio
He L et al. / Clinical Study 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 ATF4 -1.0 0.6
Log2 ratio
0.4
6 A 5
B
4 3 2 1 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 0 ATF4 QSBS vs NC 6 C 5
0.2 0.0
-0.2
ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 QSBS vs NBS D
4 3 2 1 0
Log2 ratio
Log2 ratio
-0.4 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 -0.6 QDBS vs NC QDBS vs NBS 6 1.8 F E 1.6 5 1.4 4 1.2 1.0 3 0.8 2 0.6 1 0.4 0.2 0 0.0 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 CBS vs NBS CBS vs NC G 5.0 0.6 H 4.5 0.4 4.0 0.2 3.5 3.0 0.0 2.5 -0.2 2.0 -0.4 1.5 1.0 -0.6 0.5 -0.8 0.0 -1.0 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 HBS vs NBS HBS vs NC Sequencec Qpcr Figure 4 DGE sequencing and quantitative detection of PCR gene expression ratio A: Qi deficiency and blood stasis (QDBS) vs healthy controls (NC); B: Qi deficiency and blood stasis vs hypertension without blood stasis (NBS); C: Qi stagnation and blood stasis (QSBS) vs NC; D: QSBS vs NBS; E: cold retention and blood stasis (CBS) vs NC; F: CBS vs NBS; G: heat retention and blood stasis (HBS) vs NC; H: HBS vs NBS stasis. The longitudinal coordinates for each pair of groups represents the log2 ratio of relative expression. ATF4: activating transcription factor 4; ATF3: activating transcription factor 3; TRIB3: Tribbles homolog 3; DDIT3: DNA-damage inducible transcription factor 3; CEBPB: CCAAT/enhancer binding protein-β; JUN: Jun proto-oncogene; TSC22D3: TSC22 domain family member 3.
related to ERS. Determining the relationships of ATF4, ATF3, DDIT3, TRIB3, CEBPB, and JUN, as well as of ERS, with the pathogenesis of blood stasis in hypertension requires additional in vitro and in vivo studies.
4
5
REFERENCES 1 2
3
Li L, Tian J, Cai Y, et al. Basic research on blood stasis biology. Shandong Zhong Yi Za Zhi 2010; 29(12): 863-865. Lin J, Zhou H, Zheng L, et al. The effect of Xuefuzhuyu decoction on VEGF and PDGF in human umbilical vein endothelial cells. Zhong Xi Yi Jie He Xin Nao Xue Guan Za Zhi 2009, 7(10): 1182-1184. Gao D, Wu L, Jiao Y, et al. Experimental study on effects of Xuefuzhuyu decoction on endothelial progenitor cells
JTCM | www. journaltcm. com
6
7
179
differentiation. Zhong Guo Zhong Yi Ji Chu Yi Xue Za Zhi 2009; 15(12): 917-919. Zeng W, Guo YH, Qi W, et al. 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulum stress of endothelial cells stimulated by uremic serum. Life Sci 2014; 103(1): 15-24. Chen L, Qu Y, Ge H, et al. Effect of Buyang Huanwu Decoction on expression of adhesion molecules in vascular endothelial cells of blood stasis rats. Zhong Cao Yao 2005; 36 (5): 224-227. Chen L, Qu Y, Hu X, et al. Effects of xijiao dihuang decoction on the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats. Zhong Guo Bing Li Sheng Li Za Zhi 2006; 22(3): 547-550. Hu X, Chen L, Qu Y. The effect of Shenghua tea on VEAM of rat. Zhong Cheng Yao 2006; 28(9): 1330-1333. April 15, 2016 | Volume 36 | Issue 2 |
He L et al. / Clinical Study 8
9
10
11
12
13
14 15
16
17
18
19
Chen L, Chen C, Qu Y, et al. Effects of Xue fu Zhu yu decoction on the expression of adhesion molecules in vascular endothelial cells of blood stasis rat. Shandong Zhong Yi Yao Da Xue Xue Bao 2006; 30(5): 395-398. Liu H, Wu X, Li D, et al. Chinese experimental prescription Lixuehuoxue decoction on the expression of leukocyte series of adhesion molecules in rats with sepsis. Zhong Guo Shi Yan Fang Ji Xue Za Zhi 2009; 15 (1): 44-47. Wei Z, Cheng X. Influence of Chinese herbal remedies with actions of activating blood circulation and rem oving stasis combined with HBO on platelet membrane glycoprotein and leukocyte adhesion molecules of cerebral ischemia and reperfusion. Beijing Zhong Yi Yao Da Xue Xue Bao 2009; 15(1): 44-47. Lin J, Wu K. The World Health Organization/International Hypertension Alliance guidelines (1999). Gao Xue Ya Za Zhi 1999; 7(2): 97-100. Du J, Shi Z. Diagnosis and treatment guidance for blood stasis syndrome by integrative medicine. Zhong Guo Zhong Xi Yi Jie He Za Zhi 2011; 31(6): 839-842. Hu X. The research on vascular endothelial cell (VEC) injury model of blood stasis syndrome (BSS) associated with hypertension disease. Guangzhou: Jinan University, 2007: 31-33. Tang M. Primary study of gene mechanism in hypertension development. Int Med J 2006; 27(1): 61-64. Jia H. Study on the treatment of blood stasis syndrome in hypertension. Zhong Guo Shi Yong Yi Yao Za Zhi 2013; 8 (5): 49-51. Liu J, HE Y. Effect of endoplasmic reticulum stress in the progression of renal diseases. Sheng Li Ke Xue Jin Zhan 2010, 41(6): 439-442. Zhang XQ, Xu CF, Yu CH, Chen WX, Li YM. Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol 2014; 20 (7): 1768-1776. Zhang H, Li X. Mechanism of the endoplasmic reticulum stress in diabetic nephropathy. Chin J Nephrol 2011; 27 (2): 135-138.
JTCM | www. journaltcm. com
20
21 22
23
24
25
26 27
28
29 30
31
180
Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007; 8(7): 519-529. Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 2008; 7(12): 1013-1030. Minamino T, Kitakaze M. ER stress in cardiovascular disease. J Mol Cell Cardiol 2010; 48(6): 1105-1110. Eizirik DL, Cardozo AK, Cnop M. The role for endoplasmic reticulum stress in diabetes mellitus. Endocr Rev 2008; 29(1): 42-61. Lindenmeyer MT, Rastaldi MP, Ikehata M, et al. Proteinuria and hyperglycemia induce endoplasmic reticulum stress. J Am Soc Nephrol 2008; 19(11): 2225-2236. Hu G, He K, Fan L. Endoplasmic reticulum stress-induced apoptosis and myocardial ischemia reperfusion injury of the Chinese medical. Zhong Hua Bai Jian Yi Xue Za Zhi 2010, 12(1): 72-75. Fels DR, Koumenis C. The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biol Ther 2006; 5(7): 723-728. Pillai S. Birth pangs: the stressful origins of lymphocytes. J Clin Invest 2005; 115(2): 224-227. Li Z, Zhoua I, Tang C. Molecular Mechanism on Endoplasmic Reticulum Stress Responses. Zhong Guo Sheng Wu Hua Xue Fen Zi Sheng Wu Xue Bao 2004; 20(3): 283-288. Yu L. Identification of the target genes of transcription factor CEBPB in acute promyelocytic Leukemia cells induced by all-trans retinoic acid. Hunan: Central South University, 2013: 11-12. Ma L. The Member of Transcription factor ATF3/CERB: ATF3. J Mol Biol 2007; 4(2): 147-149. Wang Y. Study on the methylation status of DDIT3 gene promoter in hematologic malignancies. Jiangsu: Jiangsu university, 2009: 7-8. Shang Y. The role and the signal transduction mechanism of Tribble3 on macrophage apoptosis in atherosclerosis. Shandong: Shandong University, 2010: 75-78.
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J Tradit Chin Med 2016 April 15; 36(2): 173-180 ISSN 0255-2922 © 2016 JTCM. All rights reserved.
CLINICAL STUDY TOPIC
Transcriptome analysis of blood stasis syndrome in subjects with hypertension
He Ling, Fang Meixia, Chen Liguo, Zhou Jianhua, Yuan Jing, Xu Jing, Shan Yan, Xu Qingyun, Xiong Tingting aa He Ling, Fang Meixia, Chen Liguo, Zhou Jianhua, Yuan Jing, Xu Jing, Shan Yan, Xu Qingyun, Xiong Tingting, Medical School of Jinan University, Guangzhou 510632, China Supported by National Scientific Fund (Assessing MicroRNA-mediated Endothelial Cell Injury in Blood Stasis, No. 81173157) and Guangdong Scientific Fund (Assessing MicroRNA-mediated Endothelial Cell Injury in Blood Stasis, No. 10151063201000045) Correspondence to: Prof. Chen Liguo, Medical School of Jinan University, Guangzhou 510632, China. [email protected]. cn Telephone: +86-20-85226476 Accepted: August 19, 2015
alyzed. Genes enriched significantly and their pathways were determined, as were network interactions, and encoded proteins. Gene identities were confirmed by real-time polymerase chain reactions. RESULTS: Compared with cells cultured in sera of the blood stasis groups, those culture in sera of healthy individuals and of the non-blood stasis group showed 11 and 301 differences, respectively in stasis-related genes. Genes identified as differing between the blood stasis and healthy groups included activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-β, and Jun proto-oncogene (JUN). Pathway and protein interaction network analyses showed that these genes were associated with endoplasmic reticulum stress. Cells cultured in sera of patients with blood stasis and Qi deficiency, Qi stagnation, heat retention, and cold retention were compared with cells cultured in sera of patients with the other types blood stasis syndrome. The comparison showed differences in expression of 28, 28, 34, and 32 specific genes, respectively.
Abstract OBJECTIVE: To screen for mRNAs associated with blood stasis syndrome and to explore the genetic mechanisms of blood stasis syndrome in hypertension. METHODS: This study involved groups of patients with hypertension and blood stasis, including those with Qi deficiency, Qi stagnation, cold retention and heat retention; as well as hypertensive patients without blood stasis and healthy individuals. Human umbilical vein endothelial cells were co-cultured with the sera of these healthy individuals and patients with blood stasis syndrome. Total RNA was extracted from these cells and assessed by a high-throughput sequencing method (Solexa) and digital gene expression. Differentially expressed genes among these six groups were compared using whole genome sequences, and mRNAs associated with blood stasis syndrome identified. Differences in gene use and gene ontology function were anJTCM | www. journaltcm. com
CONCLUSION: The pathogenesis of blood stasis syndrome in hypertension is related to endoplasmic reticulum stress and involves the differential expression of the activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-β, and JUN genes. © 2016 JTCM. All rights reserved. Key words: Hypertension; Blood stasis; RNA, messenger; Endoplasmic reticulum stress 173
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INTRODUCTION
tively. These patients were recruited from the cardiovascular department, the outpatient TCM department, and patients hospitalized at the First Affiliated Hospital of Jinan University. Patients in this group did not differ significantly from the group of hypertensive patients with blood stasis syndrome in blood pressure, sex distribution, mean arterial pressure, or average age (each P > 0.05). The third group consisted of 30 healthy volunteers (17 males, 13 females), recruited from the Department of Traditional Chinese Medicine, School of Medicine, Jinan University. Their blood pressures were within normal ranges and differed significantly (P < 0.01) from blood pressures in hypertensive patients, both with and without blood stasis syndrome. High blood pressure was defined according to World Health Organization / International Hypertension Alliance guidelines (1999),11 and blood stasis syndrome was diagnosed according to the 2011 revised criteria.12 Patients with target organ damage, diabetes, or severe cardiovascular, cerebrovascular, or renal disease were excluded.
Hypertension is a highly prevalent clinical syndrome characterized by high arterial pressure that can seriously damage the heart, brain, and kidneys. In traditional Chinese medicine (TCM), blood stasis is believed to play an important role in the occurrence and development of hypertension. Current studies on the mechanism of blood stasis syndrome have been limited to observing changes in blood, focusing on the relationship between vascular changes and the initiation of blood stasis syndrome. Moreover, blood stasis biology has been associated with changes in vascular endothelial cell (VEC) function,1 with VEC damage closely associated with the initiation and development of blood stasis syndrome.2,3 Endoplasmic reticulum stress (ERS) has been shown to damage VECs,4 indicating that ERS may induce blood stasis by causing endothelial cell injury. Our research group has focused on a blood stasis syndrome model, including the effects of VEC injury and mechanisms of repair, as well as on the effects of drugs and the molecular / genetic mechanisms underlying blood stasis. We and others have found that different types of blood stasis have different biochemical foundations, but also have common pathological and physiological characteristics.5-10 Because of the complex pathogenesis of blood stasis syndrome, including the effects of various factors, screening for and identifying biomarkers associated with this syndrome may help in its diagnosis and treatment. An established model of endothelial cell injury-blood stasis syndrome was used to screen for genes associated with the initiation of hypertension-related blood stasis syndrome among groups of hypertensive patients, with and without blood stasis syndrome, and healthy individuals. The genetic pathways of identified genes were investigated to determine the molecular mechanism underlying the development of blood stasis syndrome in hypertension.
Serum collection All included individuals were instructed to fast and not take any drugs overnight prior to a blood draw. Forearm venous blood was aseptically collected into sterile, non-anticoagulation vacuum tubes, allowed to coagulate, and centrifuged at 2784´g at 4 ℃ for 15 min. Serum was removed, placed in an aseptic Eppendorf tube (Gibco, Guangzhou, China), and incubated in a water bath at 56 ℃ for 30 min to inactivate serum complement. Finally, these samples were stored at －20 ℃. Establishing a cell model The cell model of hypertension and blood stasis,13 involved the use of human umbilical vascular endothelial cells (HUVECs; CRL-1730), obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in Dulbecco's Modified Eagle's medium (DMEM); (Gibco) containing 10% fetal bovine serum at 37 ℃ in a 5% CO2 incubator. To each culture flask was added serum, to a concentration of 10%, plus 90% F12K medium (Gibco), and the cells were cultured at 37 ℃ in a 5% CO2 incubator for 24 h. The cells were collected, and total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed by agarose gel electrophoresis.
MATERIALS AND METHODS Three groups of individuals were analyzed. The first group included 40 patients (21 males, 19 females), of mean age (66 ± 7) years, diagnosed with blood stasis syndrome with hypertension from December 2011 to June 2012 at the Second Hospital affiliated with Guangzhou Medical College. Their mean systolic and diastolic blood pressures were (157 ± 13) and (104 ± 7) mm Hg, respectively. TCM syndrome differentiation rules have identified four types of patients with blood stasis syndrome plus hypertension: blood stasis syndrome with Qi deficiency, Qi stagnation, cold retention, and heat retention. Ten patients were included in each subgroup. Another group of 12 patients [6 males, 6 females, of mean age (65 ± 9) years] had hypertension but without blood stasis. Their mean systolic and diastolic b; ppd pressures were (166 ± 8) and (104 ± 8) mm Hg, respecJTCM | www. journaltcm. com
mRNA sequence analysis and gene screening mRNA transcriptome sequencing was performed using second-generation IIlimina Solexa sequencing technology by the Shanghai Megiddo Biotechnology Co., Ltd. (Shanghai, China), The original statistical data were assessed while retaining high-quality sequences. Statistical analyses of each fragment per kilobase of exons per million fragments mapped (FPKM), as well as the transcript value in the expressed samples, were compared. 174
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Finally, differences in group transcripts and relative gene expression were analyzed among the six groups of samples.
residue at the dye front. The ratio of A260 to A280 was 1.8-2, showing that the quality of these total RNA samples was in full compliance with requirements for chip analysis.
Gene function analysis Differentially expressed genes were analyzed using Goatools (https: //github.com/tanghaibao/GOatools) in the GO database, along with a PATHWAY enrichment analysis of differential gene use with the software KO-Based Annotation System (KOBAS) (http: //kobas. cbi.pku.edu.cn/home.do). False positive rates were compared by Fisher's exact tests. A P value ≤ 0.05 was considered significant. Differences in the expression of significantly enriched genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify cellular pathways. The Human Protein Reference Database (http: // www.hprd.org/Release 9), was used to assess interactions among these genes and interaction data were determined using Cytoscape software (http://www.cytoscape.org/) for network visualization.
Differential gene expression of blood stasis syndrome Gene expression by each cell sample was assessed by differential gene expression (DGE) analysis, with a P value of < 0.01, an False Discovery Rate (FDR) of < 0.001, and a |log2Ratio| of ≥ 1 considered significant (Figure 1). The four groups with hypertension and blood stasis groups showed significant differences in mRNA expression compared with the control group. Specifically, the groups with Qi deficiency, Qi stagnation, cold retention, and heat retention showed significant differences in 76 (42 downregulated and 34 upregulated), 51 (34 downregulated and 17 upregulated), 61 (39 downregulated and 22 upregulated) and 62 (39 downregulated and 23 upregulated) mRNAs, respectively. The four groups with hypertension and blood stasis also showed significant differences compared with the group with hypertension but without blood stasis. Specifically, the groups with Qi deficiency, Qi stagnation, cold retention, and heat retention showed significant differences in 579 (117 downregulated and 459 upregulated), 475 (98 downregulated and 337 upregulated), 621 (135 downregulated and 486 upregulated) and 469 (103 downregulated and 366 upregulated) mRNAs, respectively. In addition, the group with hypertension but without blood stasis differed from the healthy group in 529 (423 downregulated and 106 upregulated) mRNAs.
Quantitative real-time polymerase chain reaction (qRT-PCR) PCRs were performed according to the manufacturer's instructions, with primers designed based on the sequences in the gene database. The2-ΔΔCT method was used to assess differential gene expression. Log2 ratios were determined by comparing gene expression in the five hypertensive groups with that in healthy controls.
RESULTS RNA extraction Total RNA was extracted from HUVECs incubated with serum from each of the groups of subjects. The 28S and 18S rRNA bands were clear, with no observed
Differential mRNA expression analysis Integrated analyses of differential mRNA expression in the six groups are shown in Table 1.
600 Gene number
500 400 300 200 100 0
QBS vs NC QSBS vs NC CBS vs NC
HBS vs NC NBS vs NC QDBS vs NBS QSBS vs NBS CBS vs NBS HBS vs NBS Groups
Upregulated gene number Downregulated gene number Figure 1 Numbers of mRNAs differentially expressed between pairs of groups QDBS: Qi deficiency and blood stasis; QSBS: Qi stagnation and blood stasis; CBS: cold retention and blood stasis; HBS: heat retention and blood stasis; NBS: hypertension without blood stasis; NC: healthy controls. Compare with NC group, the groups with QDBS, QSBS, CBS, and HBS showed significant differences in 76 (42 downregulated and 34 upregulated), 51 (34 downregulated and 17 upregulated), 61 (39 downregulated and 22 upregulated) and 62 (39 downregulated and 23 upregulated) mRNAs, respectively. Compared with NBS group, the groups with QDBS, QSBS, CBS, and HBS showed significant differences in 579 (117 downregulated and 459 upregulated), 475 (98 downregulated and 337 upregulated), 621 (135 downregulated and 486 upregulated) and 469 (103 downregulated and 366 upregulated) mRNAs, respectively. In addition, the group with NBS differed from NC group in 529 (423 downregulated and 106 upregulated) mRNAs. JTCM | www. journaltcm. com
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He L et al. / Clinical Study Table 1 Specific gene statistics in blood stasis syndrome Total
Group
mRNA Difference Upregulated
Downregulated
Qi deficiency and blood stasis syndrome vs normal and vs hypertension alone
22
10
12
Qi stagnation and blood stasis syndrome vs normal and vs hypertension alone
19
11
8
cold retention and blood stasis syndrome vs normal and vs hypertension alone
14
7
7
heat retention and blood stasis syndrome vs normal and vs hypertension alone
14
7
7
All four groups with blood stasis syndrome vs hypertension alone
301
239
62
All four groups with blood stasis syndrome vs normal
11
2
9
Qi deficiency and blood stasis syndrome in endemic hypertension
28
14
14
Qi stagnation and blood stasis syndrome in endemic hypertension
28
8
20
cold retention and blood stasis syndrome in endemic hypertension
32
10
22
Heat retention and blood stasis syndrome in endemic hypertension
34
9
25
Qi deficiency and blood stasis syndrome
124
67
57
Qi stagnation and blood stasis syndrome
81
34
47
cold retention and blood stasis syndrome
161
83
78
heat retention and blood stasis syndrome
69
38
31
GO enrichment analysis of differentially expressed genes in different blood stasis syndromes Patients with blood stasis and heat retention, cold retention, Qi deficiency, and Qi stagnation were compared with the group with hypertension but without blood stasis. GO enrichment analysis demonstrated that differentially expressed genes are related to multiple stress responses, cell differentiation, apoptosis, and autophagy in VECs. These genes are also immune related (P < 0.05) (Figure 2). Five of the 20 pathways identified were directly associated with endoplasmic reticulum stress (ERS), including PERK-regulated gene expression, chaperone activation of IRE1alpha, chaperone activation by activating transcriptional factor 6 (ATF6)-alpha, and the release of eIF4E and the ATF-2 transcription factor network. Other pathways included the interleukin (IL)-6 and IL-2 inflammatory pathways, the p53 and p38 apoptosis pathways, the mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) pathways, the mitogen-activated protein kinase (MAPK) signaling pathway, and signal transduction and amino acid metabolism pathways (Table 2).
Confirmation of differential mRNA expression by qRT-PCR qRT-PCR analysis comparing mRNA expression in the four groups with hypertension and blood stasis syndrome and the group with hypertension alone and the control group confirmed findings on the differential expression of ATF4, ATF3, DDIT3, TRIB3, CEBPB, and JUN mRNAs (Figure 4).
DISCUSSION Hypertension is a polygenic disease resulting from different genetic and environmental factors14 and can enhance the risk of cardiac and cerebrovascular diseases. Hypertensive patients with blood stasis syndrome show increases in hemorheology, hemodynamics, and microcirculation.15 ERS can damage VECs by promoting the expression of p38 mitogen activated protein kinase (MAPK) and its downstream molecules, along with oxidative stress. These findings suggest that ERS may be caused by endothelial cell injury resulting from blood stasis, consistent with results showing that ERS inhibition is effective in treating vascular-related diseases.16 The ER is important for protein synthesis and folding, Ca2 + storage, and lipid synthesis.17 Modifications of secreted, transmembrane, and ER resident proteins can help them fold into their native conformations, eventually forming functional proteins.18 Changes in the inner cellular environment, such as hypoxia, Ca2+ homeostasis, oxidative stress, or abnormal glycosylation, can result in the accumulation of unfolded or misfolded proteins in the ER, resulting in ER dysfunction or ERS. Protein accumulation leads to a series of follow-up reactions, called an unfolded protein response (UPR). Reducing the amount of protein in the ER re-
Protein expression and blood stasis syndrome Comparisons of gene expression by cells incubated with sera from patients with hypertension alone and those with hypertension and blood stasis revealed evidence of differential expression of specific genes in protein networks. Genes associated with ERS included activating transcription factor 4 (ATF4), activating transcription factor 3 (ATF3), DNA-damage inducible transcription factor 3 (DDIT3), Tribbles homolog 3 (TRIB3), CCAAT / enhancer binding protein-β (CEBPB), JUN, Jun dimerization protein 2 and Early growth response 1 (Figure 3). JTCM | www. journaltcm. com
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Positive regulation of cell death Cellular response to external stimulus Negative regulation of cellular process Primary metabolic process Programmed cell death Biological regulation Response to oxygen-containing compound Single-organism cellular process Cellular response to stimulus Regulation of apoptotic process Single-organism process Regulation of programmed cell death Response to external stimulus Regulation of protein metabolic process Regulation of cell death Negative regulation of biological process Positive regulation of cellular process Cellular response to organic substance Postive regulation of biol Response to unfolded protein Cellular process Response to unfolded protein Biological_process Response to undoplasmic reticulum stress Cellular response to chemical stimulus Response to chemical Response to stimulus Cellular response to stress Response to organic substance Response to stress
A
Anion binding Cytokine activity Prephenate dehydratase activity Interleukin-8 receptor binding Fibronectin binding Growth factor activity Amino acid transmembrane transporter activity Receptor binding Utral amino acid transmembrane transporter activity Molecular_function Binding Protein binding Organelle lumen Membrane-enclosed lumen Intracellular organelle Organelle Nuclear part Pigment granule Melanosome Nucleus Extracellular region part Intracellula membrane-bounded organelle membrane-bounded organelle Extracellular space Cytoplasm Cytosol Cytoplasmic part Intracellular Cell Cell part Intracellular part Cellular_component
B
C
Figure 2 GO gene protein analysis related to blood stasis syndrome A: Biological processes; B: molecular functions, C: cellular components. The ordinate indicates significant enrichment using the GO term; the abscissa represents the enrichment factor; GO: gene ontology.
sults in the repair of misfolded proteins and the degradation of unfolded and misfolded proteins in the ER, restoring ER function and ensuring the release of mature proteins. Overexposure to the altered cellular involvement may overwhelm UPR capacity, leading to apoptosis.19-24 Stress and UPR can activate three transcription factors, inositol enzyme (IRE-1), pancreatic endoplasmic protein kinase (PERK), and activating transcription factor 6 (ATF6), resulting in three steps to induce cell apoptosis, involving PERK, ATF4, and the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α).25 PERK pathway activation occurs by inhibiting protein during early ERS, resulting in the phosphorylation of elF2α and ATF4 activation; this in turn promotes the activation and expression of CCAAT-enhancer-binding protein homologous protein (CHOP) and induces cell apoptosis.26 ATF4 is a key molecule increased in ERS-induced apoptosis.27 The transcription factor CEBPB is involved in many biological processes, including cell differentiation, proliferation, metabolism, tumors, inflammation, and apoptosis. The C/EBP homologous protein CHOP is an important signaling molecule in the ERS-associated apoptosis pathway.28 DDIT3, also known as GADD153, CHOP, and C / EBP, is a memJTCM | www. journaltcm. com
ber of the C / EBP family of transcription factors. Its high expression may affect protein folding in the ER and trigger an unfolded protein response and apoptosis induced by a specific ER cell pathway network, causing cell death. This protein plays an important role in induced cell cycle arrest, promoting cell differentiation and apoptosis.29 ATF3 is a member of the ATF/CERB transcription factor family that inhibits transcription by binding to the gadd153 / CHOP promoter and corresponding sites. ATF3 plays an important role in the transcriptional regulation of cell apoptosis, with any factor that upregulates ATF3 expression inducing cell apoptosis.30 The v-Jun sarcoma virus 17 gene homologue JUN binds to the promoter regions of many genes, regulating the expression of many genes in various malignant tumors. TRIB3 is a serine/threonine protein kinase and a member of a newly discovered protein gene family that can inhibit mitosis. Overexpression of TRIB3 can inhibit the MAPK pathway, adjust the NF-κB pathway, and induce apoptosis.31 Thus, all of these genes and ERS are closely related to cell apoptosis. Our results, using gene chip technology, showed differences in mRNA expression among patients with hypertension plus blood stasis, hypertension alone, and nor177
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He L et al. / Clinical Study Table 2 Identification of gene pathways and involved genes differentiating patients with hypertension plus blood stasis syndrome and those with hypertension alone No.
Pathway
1
Number of genes
Background gene number
PERK regulated gene expression
5
45
2
Amino acid and oligopeptide SLC transporters
8
341
3
Activation of Chaperones by IRE1alpha
4
51
4
Amino acid transport across the plasma membrane
6
190
5
Serine biosynthesis
3
23
6
Signaling mediated by p38-alpha and p38-beta
5
343
7
IL6-mediated signaling
7
349
8
Axonal growth stimulation
2
18
9
Activation of Chaperones by ATF6-alpha
2
26
10
p53 signaling pathway
7
691
11
Vitamin B6 metabolism
2
42
12
Release of eIF4E
2
57
13
Disease
16
4993
14
mTOR signaling pathway
5
553
15
ATF-2 transcription factor network
6
494
16
IL12 and STAT4 dependent signaling pathways in Th1 development
3
103
17
Neurophilin interactions with VEGF and VEGFR
2
28
18
Circadian clock
3
210
19
VEGF signaling pathway
4
693
20
MAPK signaling pathway
9
2468
Notes: PERK: pancreatic endoplasmic reticulum kinase; SLC: solute-carrier; IRE1: inositol-requiring protein 1; IL-6: interleukin-6; ATF6: activating transcriptional factor 6; eIF4E: eukaryotic initiation factor 4E; mTOR: mammalian target of rapamycin; ATF2: activating transcription factor-2; IL-12: interleukin-12; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor; MAPK: mitogenactivated protein kinase. A CEBPB
TSC22D3
ATF3
TSC22D3 ATF3
CEBPB
JDP2
JUN
DDIT3
B
JDP2 JUN
DDIT3 ATF4
TRIB3
ATF4 TRIB3
CEBPG
C
TSC22D3
JUND JDP2
ATF4
EGR1 CEBPB
CEBPG
DDIT3 TRIB3
ATF3
TRIB3
JUN
ATF3
MYC
D
CEBPG
ATF4
CEBPB
DDIT3
CEBPG
RASSF1
Figure 3 Correlations between blood stasis and gene expression network interactions A: Qi stagnation and blood stasis (QSBS) vs hypertension without blood stasis (NBS); B: Qi deficiency and blood stasis (QDBS) vs NBS; C: cold retention and blood stasis (CBS) vs NBS; D: heat retention and blood stasis (HBS) vs NBS. ATF4: activating transcription factor 4; ATF3: activating transcription factor 3; JUN: Jun proto-oncogene; CEBPB: CCAAT/enhancer binding protein-β; DDIT3: DNA-damage inducible transcription factor 3; TRIB3: Tribbles homolog 3; CEBPG: CCAAT/enhancer binding protein gamma; TSC22D3: TSC22 domain family member 3; JDP2: Jun dimerization protein 2; MYC: v-myc avian myelocytomatosis viral oncogene homolog ; RASSF1: Ras association (Ral GDS/AF-6) domily member 1; EGR1: Early growth response 1.
mal controls. ATF4, ATF3, DDIT3, TRIB3, CEBPB, and JUN mRNAs were more highly expressed in the JTCM | www. journaltcm. com
blood stasis groups. GO and PATHWAY analysis showed that multiple genes and a signal pathway were 178
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Log2 ratio
He L et al. / Clinical Study 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 ATF4 -1.0 0.6
Log2 ratio
0.4
6 A 5
B
4 3 2 1 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 0 ATF4 QSBS vs NC 6 C 5
0.2 0.0
-0.2
ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 QSBS vs NBS D
4 3 2 1 0
Log2 ratio
Log2 ratio
-0.4 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 -0.6 QDBS vs NC QDBS vs NBS 6 1.8 F E 1.6 5 1.4 4 1.2 1.0 3 0.8 2 0.6 1 0.4 0.2 0 0.0 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 CBS vs NBS CBS vs NC G 5.0 0.6 H 4.5 0.4 4.0 0.2 3.5 3.0 0.0 2.5 -0.2 2.0 -0.4 1.5 1.0 -0.6 0.5 -0.8 0.0 -1.0 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 ATF4 ATF3 TRIB3 DDIT3 CEBPB JUN TSC2205 HBS vs NBS HBS vs NC Sequencec Qpcr Figure 4 DGE sequencing and quantitative detection of PCR gene expression ratio A: Qi deficiency and blood stasis (QDBS) vs healthy controls (NC); B: Qi deficiency and blood stasis vs hypertension without blood stasis (NBS); C: Qi stagnation and blood stasis (QSBS) vs NC; D: QSBS vs NBS; E: cold retention and blood stasis (CBS) vs NC; F: CBS vs NBS; G: heat retention and blood stasis (HBS) vs NC; H: HBS vs NBS stasis. The longitudinal coordinates for each pair of groups represents the log2 ratio of relative expression. ATF4: activating transcription factor 4; ATF3: activating transcription factor 3; TRIB3: Tribbles homolog 3; DDIT3: DNA-damage inducible transcription factor 3; CEBPB: CCAAT/enhancer binding protein-β; JUN: Jun proto-oncogene; TSC22D3: TSC22 domain family member 3.
related to ERS. Determining the relationships of ATF4, ATF3, DDIT3, TRIB3, CEBPB, and JUN, as well as of ERS, with the pathogenesis of blood stasis in hypertension requires additional in vitro and in vivo studies.
4
5
REFERENCES 1 2
3
Li L, Tian J, Cai Y, et al. Basic research on blood stasis biology. Shandong Zhong Yi Za Zhi 2010; 29(12): 863-865. Lin J, Zhou H, Zheng L, et al. The effect of Xuefuzhuyu decoction on VEGF and PDGF in human umbilical vein endothelial cells. Zhong Xi Yi Jie He Xin Nao Xue Guan Za Zhi 2009, 7(10): 1182-1184. Gao D, Wu L, Jiao Y, et al. Experimental study on effects of Xuefuzhuyu decoction on endothelial progenitor cells
JTCM | www. journaltcm. com
6
7
179
differentiation. Zhong Guo Zhong Yi Ji Chu Yi Xue Za Zhi 2009; 15(12): 917-919. Zeng W, Guo YH, Qi W, et al. 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulum stress of endothelial cells stimulated by uremic serum. Life Sci 2014; 103(1): 15-24. Chen L, Qu Y, Ge H, et al. Effect of Buyang Huanwu Decoction on expression of adhesion molecules in vascular endothelial cells of blood stasis rats. Zhong Cao Yao 2005; 36 (5): 224-227. Chen L, Qu Y, Hu X, et al. Effects of xijiao dihuang decoction on the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats. Zhong Guo Bing Li Sheng Li Za Zhi 2006; 22(3): 547-550. Hu X, Chen L, Qu Y. The effect of Shenghua tea on VEAM of rat. Zhong Cheng Yao 2006; 28(9): 1330-1333. April 15, 2016 | Volume 36 | Issue 2 |
He L et al. / Clinical Study 8
9
10
11
12
13
14 15
16
17
18
19
Chen L, Chen C, Qu Y, et al. Effects of Xue fu Zhu yu decoction on the expression of adhesion molecules in vascular endothelial cells of blood stasis rat. Shandong Zhong Yi Yao Da Xue Xue Bao 2006; 30(5): 395-398. Liu H, Wu X, Li D, et al. Chinese experimental prescription Lixuehuoxue decoction on the expression of leukocyte series of adhesion molecules in rats with sepsis. Zhong Guo Shi Yan Fang Ji Xue Za Zhi 2009; 15 (1): 44-47. Wei Z, Cheng X. Influence of Chinese herbal remedies with actions of activating blood circulation and rem oving stasis combined with HBO on platelet membrane glycoprotein and leukocyte adhesion molecules of cerebral ischemia and reperfusion. Beijing Zhong Yi Yao Da Xue Xue Bao 2009; 15(1): 44-47. Lin J, Wu K. The World Health Organization/International Hypertension Alliance guidelines (1999). Gao Xue Ya Za Zhi 1999; 7(2): 97-100. Du J, Shi Z. Diagnosis and treatment guidance for blood stasis syndrome by integrative medicine. Zhong Guo Zhong Xi Yi Jie He Za Zhi 2011; 31(6): 839-842. Hu X. The research on vascular endothelial cell (VEC) injury model of blood stasis syndrome (BSS) associated with hypertension disease. Guangzhou: Jinan University, 2007: 31-33. Tang M. Primary study of gene mechanism in hypertension development. Int Med J 2006; 27(1): 61-64. Jia H. Study on the treatment of blood stasis syndrome in hypertension. Zhong Guo Shi Yong Yi Yao Za Zhi 2013; 8 (5): 49-51. Liu J, HE Y. Effect of endoplasmic reticulum stress in the progression of renal diseases. Sheng Li Ke Xue Jin Zhan 2010, 41(6): 439-442. Zhang XQ, Xu CF, Yu CH, Chen WX, Li YM. Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol 2014; 20 (7): 1768-1776. Zhang H, Li X. Mechanism of the endoplasmic reticulum stress in diabetic nephropathy. Chin J Nephrol 2011; 27 (2): 135-138.
JTCM | www. journaltcm. com
20
21 22
23
24
25
26 27
28
29 30
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Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007; 8(7): 519-529. Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 2008; 7(12): 1013-1030. Minamino T, Kitakaze M. ER stress in cardiovascular disease. J Mol Cell Cardiol 2010; 48(6): 1105-1110. Eizirik DL, Cardozo AK, Cnop M. The role for endoplasmic reticulum stress in diabetes mellitus. Endocr Rev 2008; 29(1): 42-61. Lindenmeyer MT, Rastaldi MP, Ikehata M, et al. Proteinuria and hyperglycemia induce endoplasmic reticulum stress. J Am Soc Nephrol 2008; 19(11): 2225-2236. Hu G, He K, Fan L. Endoplasmic reticulum stress-induced apoptosis and myocardial ischemia reperfusion injury of the Chinese medical. Zhong Hua Bai Jian Yi Xue Za Zhi 2010, 12(1): 72-75. Fels DR, Koumenis C. The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia resistance and tumor growth. Cancer Biol Ther 2006; 5(7): 723-728. Pillai S. Birth pangs: the stressful origins of lymphocytes. J Clin Invest 2005; 115(2): 224-227. Li Z, Zhoua I, Tang C. Molecular Mechanism on Endoplasmic Reticulum Stress Responses. Zhong Guo Sheng Wu Hua Xue Fen Zi Sheng Wu Xue Bao 2004; 20(3): 283-288. Yu L. Identification of the target genes of transcription factor CEBPB in acute promyelocytic Leukemia cells induced by all-trans retinoic acid. Hunan: Central South University, 2013: 11-12. Ma L. The Member of Transcription factor ATF3/CERB: ATF3. J Mol Biol 2007; 4(2): 147-149. Wang Y. Study on the methylation status of DDIT3 gene promoter in hematologic malignancies. Jiangsu: Jiangsu university, 2009: 7-8. Shang Y. The role and the signal transduction mechanism of Tribble3 on macrophage apoptosis in atherosclerosis. Shandong: Shandong University, 2010: 75-78.
April 15, 2016 | Volume 36 | Issue 2 |