Use of serial analysis of gene expression to reveal the specific regulation of gene expression profile in asthmatic rats treated by acupuncture
© Yin et al; licensee BioMed Central Ltd. 2009
Received: 25 November 2008
Accepted: 06 May 2009
Published: 06 May 2009
Asthma has become an important public health issue and approximately 300 million people have suffered from the disease worldwide. Nowadays, the use of acupuncture in asthma is increasing. This study intended to systematically analyze and compare the gene expression profiles between the asthmatic and acupuncture-treated asthmatic rat lung, and tried to gain insight into the molecular mechanism underlying the early airway response (EAR) phase of asthma treated by acupuncture.
Four tag libraries of serial analysis of gene expression (SAGE) were established from lung tissues of control rats (CK), asthmatic rats (AS), asthmatic rats treated by acupuncture (ASAC), and control rats treated by acupuncture (CKAC). Bioinformatic analyses were carried out by using the methods including unsupervised hierarchical clustering, functional annotation tool of the database for annotation, visualization, and integrated discovery (DAVID), gene ontology (GO) tree machine, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis.
There were totally 186 differentially expressed tags (P < 0.05, PCK/AS) between the libraries of CK and AS, 130 differentially expressed tags between libraries of AS/ASAC (P < 0.05, PAS/ASAC), and 144 differentially expressed tags between libraries of CK/CKAC (P < 0.05, PCK/CKAC). The gene expression profiles of AS and ASAC were more similar than other libraries via unsupervised SAGE clustering. By comparison of PCK/AS and PAS/ASAC, the DAVID genes functional classification was found to be changed from "immune response" to "response to steroid hormone stimulus", and the GO term "antigen processing and presentation of peptide antigen" disappeared in PAS/ASAC. Totally 3 same KEGG pathways were found among the three groups. Moreover, 21 specific tags of the acupuncture in treating asthma were detected using Venn diagrams.
Our SAGE research indicates that the gene expression profile of the EAR phase of asthma could be effectively and specifically regulated by acupuncture, which suggests that the gene expression of immune response and steroid hormone may play an important role in the treatment.
Asthma is a complex syndrome involving potentially permanent airway obstruction, airway hyperresponsiveness, and multicellular inflammation. It is estimated that approximately 300 million people have suffered from asthma worldwide and the burden of this disease in countries as well as families is increasing . Although inhaled steroids can significantly improve the symptoms, curative therapies are not yet available . Moreover, there are also significant concerns regarding the potential side effects from the long term use of conventional drugs, such as corticosteroids. Thus, an effective, low-risk, and non-drug strategy would provide a valuable and adjunctive treatment in asthma management . Complementary and alternative medicine (CAM), such as traditional Chinese herbal remedies, homeopathy as well as acupuncture, is widely applied in the asthma management. It is reported that CAM has been used among 59% of patients with asthma or rhinosinusitis in the United Kingdom, 41% in the United States, 26.5% in Germany, and 27.2% in Singapore .
Acupuncture literally means to puncture with a needle, which is an important therapy in traditional Chinese medicine (TCM) for at least 2,500 years . The theory of acupuncture holds that there are different types of energy flow (qi) in the human body, and the disruptions of qi are believed to be responsible for diseases. Acupuncture practitioners may use thin, solid, metallic needles to correct the imbalances of the flow via the stimulation of special points in the body, which are manipulated manually or by electrical stimulation. The therapy is developed as a relatively global system of medicine and is utilized to treat many diseases. The World Health Organization listed asthma and other 42 indications for acupuncture in 1979  and classified the diseases treated by acupuncture into four categories, 107 illnesses in 2002 . The National Institutes of Health (NIH) has accepted the validity of acupuncture treatment  and recommended it as an adjunctive treatment in comprehensive management programs of addiction, stroke rehabilitation, and asthma, etc [5, 9]. Acupuncture has traditionally been used to treat asthma in China and has been shown to be beneficial in acute asthma in short term [10, 11]. However, well-designed scientific researches in this field are needed and encouraged, which are not only important for elucidating the mechanism, but also useful for exploring new pathways in a systematic manner.
Acupuncture is a complex intervention on diseases and many studies have demonstrated that acupuncture can cause multiple biological responses and regulate many cellular and physiological processes, which could lead to changes of gene expression . These processes could occur at either the proximal or distal ends of acupuncture application, which may be mediated mainly by neural and humoral mechanisms [5, 12]. High-throughput technologies, such as microarray and serial analysis of gene expression (SAGE), may help to reveal and clarify the possible mechanism of acupuncture. In the nerve system, acupuncture was reported to regulate brain aging related genes , suppress several genes in the nociceptive pathways , and up-regulate genes in the spinal cord injury  by using microarray. In the immune and endocrine system, results of microarray have revealed that acupuncture could regulate genes to increase activities of natural killer cell  and superoxide dismutase , to keep the cytokines balance between Th1 and Th2 , and to induce hypocholesterolemic effects . However, these researches focused mainly on the nerve and immune systems, and the analyses of the generated data were limited. SAGE, as a powerful expression profiling method and much more accessible for dissecting the complex system, was applied to qualitatively and quantitatively evaluate the transcription of the genes via particular length without the prerequisite of a hybridization probe for each transcript . However, there has been no SAGE report concerning the mechanism of acupuncture in treating asthma so far.
In this study, four SAGE tag libraries were established respectively from lung tissues of control rats, asthmatic rats, asthmatic rats treated by acupuncture, and control rats treated by acupuncture. The study aimed to systematically analyze and compare the gene expression profiles in the lung of the four different groups, and attempted to examine the molecular mechanism of early airway response (EAR) phase of asthma treated by acupuncture.
Animal and experimental asthma model
Pathogen-free, male Sprague-Dawley (SD) rats (4 weeks old, 110–130 g, SLAC Laboratory Animal Co. Ltd., Shanghai, China), raised in a pathogen-free rodent facility and provided with food and water ad libitum, were randomly divided into four groups (each group contained 8 rats): control rats (CK), asthmatic rats (AS), asthmatic rats treated by acupuncture (ASAC), control rats treated by acupuncture (CKAC). The protocol of SD rat model of asthma was described as previously  and rats of CK and CKAC were sensitized and boosted to normal saline instead of OVA. Rats were kept in animal facilities approved by the Shanghai Committee for Accreditation of Laboratory Animal and the animal experiment conformed to the regulations of the State Science and Technology Commission.
Measurement of pulmonary function
The measurements of pulmonary resistance (RL), dynamic compliance (Cdyn), and respiratory rate (RR), were modified from Glaab T et al [24, 25]. Briefly, a rat was placed in supine position on a wood plate warmed by an incandescent lamp after anaesthesia. At the upper part of the trachea, a T-shape cutting was made and a T-shape cannula, which was directly attached to a heater controlled pneumotachograph (Series 3850A, Hans Rudolph, USA), was gently inserted into the trachea. Tidal flow was determined by the pneumotachograph connected to a differential pressure transducer (600D-011, AutoTran, USA). To measure transpulmonary pressure, a water-filled PE-90 tubing was inserted into the esophagus to the level of the midthorax (lower one-third of the esophagus) and coupled to a pressure transducer (PT14MX, Jialong Teaching Equipment, Shanghai). The pneumotachograph tidal flow signal was integrated with time to obtain tidal volume. RL and Cdyn were calculated over a complete respiratory cycle using an integration method over flows, volumes and pressures, and were continuously recorded with software (Shanghai Medical College, Fudan University) for physiology experiments. Respiratory parameters were averaged in 60 s segments and maximum RL, minimum Cdyn and change of RR values were taken and calculated as differential values subtracted from the corresponding baseline values (Figure 1). Then the rats were sacrificed. The lungs were excised right away, rinsed in ice-cold normal saline, dissected free from surrounding tissues in an ice-bath and frozen immediately in liquid nitrogen.
Construction, annotation, and confirmation of the SAGE libraries
Construction and annotation of the SAGE libraries were described as previously . The confirmation of the four SAGE libraries was performed by Quantitative Real-Time PCR (qRT-PCR) on an Applied Biosystems 7300 Real-Time PCR System using TOYOBO Realtime PCR Master Mix (Toyobo, Osaka, Japan). The threshold cycle number was determined using SDS v1.4 Software and the reactions were performed in triplicate. Total RNA (5 μg) was reversely transcribed into cDNA by using the RevertAid First Strand cDNA synthesis kit (Cat. No. K1622; Fermentas, EU). For qRT-PCR of the cDNA, primer pairs were designed to generate intron-spanning products of 101–150 bp (Primer sequences were listed in additional file 2). The generation of specific PCR products was confirmed by the melting curve and gel analysis. The expressional ratio was calculated according to the formula 2(Rt-Et)/2(Rn-En) as described previously . Transcripts with a twofold increase in expression were considered to be up-regulated and those with a 0.5-fold decrease in expression were considered to be down-regulated.
Bioinformatic analysis of SAGE tags
To identify genes preferentially regulated in the four groups, the two-way unsupervised clustering method based on tag copies was applied. This unguided approach allowed pattern discovery for subsequent supervised functional analysis. To reduce the magnitude effects of the extreme data, the genes with total tag counts less than 20 were filtered. The dendrogram of differentially expressed tags was created by using the TIGR MultiExperiment Viewer 4.0 http://www.tm4.org/mev.html, mainly with the average clustering and Euclidean distance.
Differentially expressed genes (P < 0.05) between SAGE libraries were functionally annotated and classified by using the functional annotation tool of database for annotation, visualization, and integrated discovery (DAVID) http://david.abcc.ncifcrf.gov/, which provided integrated solutions for the annotation and analysis of genome-scale datasets derived from high-throughput technologies.
Key regulatory processes in asthma were analyzed by Gene Ontology (GO) Tree Machine http://bioinfo.vanderbilt.edu/gotm/. GO Tree Machine generated a directed acyclic graph (DAG) for input gene sets, which was made to identify the most important GO categories and to suggest their potential biological importance.
The Kyoto encyclopedia of genes and genomes (KEGG) pathway is a collection of manually drawn pathway maps of the molecular interaction and reaction networks. The KEGG pathways of the differentially expressed genes between SAGE libraries were matched by using the DAVID Functional Annotation Tool.
One-way ANOVA (analysis of variance) followed by the least significant difference (LSD) test for post hoc analysis was used to analyze the significance of RL, Cdyn and RR among the four groups. Statistical analysis for the significance of each of the four SAGE libraries was made using Monte Carlo analysis. The enrichments of GO Tree Machine were statistically significant as determined by the hypergeometric test .
Measurements of pulmonary functions after OVA challenge
The comparisons of pulmonary resistance (kPa/ml/s) of the four groups
0.0003 ± 0.0038
0.0010 ± 0.0059
0.0085 ± 0.0237
0.0064 ± 0.0119
0.0065 ± 0.0160
0.0039 ± 0.0127
0.0062 ± 0.0120
0.0037 ± 0.0074
0.0055 ± 0.0090
0.0066 ± 0.0105
0.0317 ± 0.0394*
0.1969 ± 0.1051*
0.2363 ± 0.1197*
0.1874 ± 0.0897*
0.1292 ± 0.0787*
0.0893 ± 0.0575*
0.0622 ± 0.0512*
0.0416 ± 0.0393
0.0194 ± 0.0272
0.0062 ± 0.0205
-0.0016 ± 0.0153#
0.0283 ± 0.0539#
0.0695 ± 0.0716#
0.0762 ± 0.0611#
0.0810 ± 0.0764
0.0567 ± 0.0543
0.0359 ± 0.0564
0.0187 ± 0.0425
0.0124 ± 0.0373
0.0025 ± 0.0258
-0.0025 ± 0.0049
-0.0025 ± 0.0071
0.0011 ± 0.0101
0.0013 ± 0.0081
0.0007 ± 0.0098
0.0008 ± 0.0077
0.0018 ± 0.0082
0.0036 ± 0.0088
0.0054 ± 0.0076
0.0027 ± 0.0103
General analysis of SAGE libraries
Summary of serial analysis of gene expression data for the four libraries
By Comparing with the SAGE data between the libraries of CK and AS, there were totally 186 differentially expressed tags (P < 0.05, PCK/AS). Similarly, there were 130 differentially expressed tags between libraries of AS and ASAC (P < 0.05, PAS/ASAC), and 144 differentially expressed tags between libraries of CK and CKAC (P < 0.05, PCK/CKAC). See additional file 4 for the lists of differentially expressed tags of PCK/AS, PAS/ASAC, PCK/CKAC.
Confirmation of SAGE Results by qRT-PCR
Unsupervised SAGE clustering based on tag expression
The comparisons of the DAVID gene functional classification of PCK/AS, PAS/ASAC and PCK/CKAC
DAG analysis of gene categories of PCK/AS, PAS/ASAC and PCK/CKAC
Finding KEGG pathways of PCK/AS, PAS/ASAC and PCK/CKAC
Kyoto encyclopedia of genes and genomes (KEGG) pathways of differentially expressed tags
MAPK signaling pathway
T cell receptor signaling pathway
B cell receptor signaling pathway
Toll like receptor signaling pathway
ECM receptor interaction
WNT signaling pathway
Leukocyte transendothelial migration
Regulation of actin cytoskeleton
FCεRI signaling pathway
Antigen processing and presentation
Insulin signaling pathway
GnRH signaling pathway
ErbB signaling pathway
SNARE interactions in vesicular transport
The Venn diagram among PCK/AS, PAS/ASAC, and PCK/CKAC
The gene expression profiles of acupuncture in treating asthma were not yet well studied before. Our SAGE study, in which acupuncture served as a kind of biological perturbation of systems biology, has provided a molecular base of EAR phase of asthma treated by acupuncture and may provide clues for the further research.
The cluster analysis of gene expression profiles of the four libraries and genes with marked changes
Based on the global expression matrix, the result of the hierarchical cluster analysis indicated that the gene expression profiles of different libraries changed a lot when receiving different perturbation, such as OVA sensitization and acupuncture. It suggested that acupuncture could perturb the biological condition by regulating a number of genes. Moreover, the gene expression profiles of ASAC and CKAC were different, and the profiles of AS and ASAC were more similar than those in other groups, which indicated the specific effect of acupuncture in the EAR phase of asthma.
The genes with marked changes in expression levels probably have potential importance in treating asthma. CD24, referred to as heat-stable antigen, is an important co-stimulatory molecule in immunity . Previous studies have suggested that CD24 was involved in cell adhesion and signal transduction via phosphorylation of intracellular proteins, intracellular calcium mobilization, and activation of transcription factors . In our libraries, the expression of CD24 was found to be down-regulated 12 times in asthma while up-regulated 7 times by acupuncture. The result suggested that the gene may correlate with the immune-modulating effects of acupuncture in EAR phase of asthma.
The gene expressions of immune response and steroid hormone were regulated by acupuncture in the EAR phase of asthma
Several immune-related GO terms, such as antigen processing and presentation, antigen processing and presentation of peptide antigen, antigen processing and presentation of exogenous peptide antigen, disappeared in PAS/ASAC via GO Tree Machine. Moreover, the DAVID genes functional classification was found to shift from "immune response" in PCK/AS to "response to steroid hormone stimulus" in PAS/ASAC. The changes suggested that the immune response of EAR phase of asthma was attenuated by acupuncture, which may be realized through the release of the endogenous steroid hormone.
There were 14 genes in the DAVID genes functional classification "response to steroid hormone stimulus" in PAS/ASAC, in which the genes of MGP (Rn.2379) and Srd5a2 (Rn.9938) were interesting. MGP is a vitamin K-dependent protein that serves as a substrate for the enzyme γ-carboxylase . It plays a role in lung growth and development  and its expression was incited by dexamethasone . In our libraries, the expression of MGP was up-regulated by acupuncture, which may result from the release of endogenous steroid hormones in consideration of the DAVID gene classification. Srd5a2, an isozyme of the 5-alpha-reductase family, is present in a large number of cells. It plays a key role in the conversion of testosterone to dihydrotestosterone and in the removal of excess of potentially neurotoxic steroids . In our libraries, the expression of Srd5a2 was up-regulated in asthma but down-regulated by acupuncture. The changed expression suggested that Srd5a2 served as an adjustable gene target of acupuncture, which may relate with the genesis of steroid by acupuncture in the EAR phase of asthma.
Pathways regulated by acupuncture in the EAR phase of asthma
Pathway-level visualization of omics data provides an essential means for systems biology to capture the systematic properties of the inner activities of cells. Eight pathways were involved in the PAS/ASAC, in which tight junction pathway was commonly shared among PCK/AS, PAS/ASAC, and PCK/CKAC. Three pathways were the same between PCK/AS and PAS/ASAC, and 4 pathways were unique in PAS/ASAC.
The tight junction pathway was considered to be the background of OVA sensitization and acupuncture regulation. MAPK cascade is one of the three same pathways between PCK/AS and PAS/ASAC. It contributes to the amplification and specificity of the transmitted signals and plays discrete yet complementary roles in accentuating allergic airway inflammation . It is also reported that the MAPK pathway could regulate steroidogenesis . GnRH signaling pathway is one of the four unique pathways in PAS/ASAC, which is the central regulator of the reproductive hormonal cascade  and could enhance the basal steroidogenesis . Several signaling pathways are activated by GnRH, including MAPK, and protein kinase C , which could lead to the increase of corticotropin-releasing hormone-binding protein at mRNA level . In our study, the data suggested that GnRH signaling pathway may interact with other pathways and participate in the genesis of steroid by acupuncture in the EAR phase of asthma.
The specific and non-specific tags of acupuncture
From the Venn diagrams, the 21 tags were found to represent the specific genes of acupuncture in treating asthma. By using the DAVID classification analysis tool, 18 of the 21 tags were divided into 7 groups. The classification with highest score was "response to steroid hormone stimulus" (enrichment score = 1.71), which was in accordance with the classification of the PAS/ASAC. The genes involved in the classification were: MGP, Abca2, and Sult1a1, which demonstrated that the transportation and production of steroid hormone may be regulated by acupuncture in the EAR phase of asthma at the level of transcription.
Abca2 expressed in a broad range of tissues and previous studies indicated that the gene participated in the transport of steroids . Besides, the elevated expression of Abca2 was considered to be a conserved mechanism of cell survival . In our study, Abca2 was up-regulated in asthma, which may correlate with the cell survival in the EAR phase of asthma. Sult1a1 is a member of sulfotransferase families, primarily localized in the trans-Golgi apparatus and associated with the sulfation of steroids and various hormones [44, 45]. Our SAGE data demonstrated that Sult1a1 was up-regulated in asthma, which indicated the gene may catalyze the sulfation process of steroidgensis. However, the two genes both belong to the DAVID classification "homeostatic process" and the gene expressions of Abca2 and Sult1a1 after acupuncture were almost equivalent to those of the controls. It suggested that acupuncture could alleviate the OVA challenge by maintaining the internal equilibrium.
Seven common tags were found among the different groups when receiving the chemical and acupuncture stimulation both in normal and asthmatic conditions. Twenty-five tags were found to be the same when receiving the OVA sensitization or acupuncture treatment. Furthermore, 6 tags were identified as the non-specific genes of acupuncture in PAS/ASAC and PCK/CKAC. The above-mentioned genes belong mainly to cellular process, and cell communication, which suggested that they may serve as the background genes in EAR phase of asthma and acupuncture. For example, Syntaxin 5 is a Golgi-localized SNARE protein required for endoplasmic reticulum-Golgi traffic in yeast and Golgi reassembly following cell division in mammalian cells .
We have presented evidences of gene expression profiles in the lung of asthmatic rats and those treated by acupuncture via SAGE. The study indicates that the gene expression profile of the EAR phase of asthma could be effectively and specifically regulated by acupuncture at the transcriptional level, which suggests that the gene expression of immune response and steroid hormone may play an important role in the treatment.
This work was supported by the National Natural Science Foundation of China (No. 30873299, 90409014, 30701123), and the Shanghai Leading Academic Discipline Project (S30304). We are grateful to Ping Wan, Lei-Jun Zhuang for their excellent technical assistance and professor Zhi-Yong Ming, Nian-Qing Ding and Ka Bian for helpful advice in the preparation of this manuscript.
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