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 21 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.

Manual acupuncture was performed for two weeks from the first day after the sensitization, once every other day (Figure 1). The acupuncture points, Dazhui (GV 14, between C7 and T1 vertebrae), bilateral Fengmen (BL12, foveola laterally between T2 and T3 vertebrae) and bilateral Feishu (BL13, foveola laterally between T3 and T4 vertebrae), were selected based on the theory of TCM in treating asthma 22. In consideration of the specific and non-specific effects of acupuncture 23, the same points in normal rats were selected and acted as control of ASAC, which could distinguish the specificity of acupuncture in treating asthma. The stainless needles (0.30 × 13 mm, Suzhou Medical Appliance Factory, Suzhou, China) were inserted about 5 mm deep into the skin. The needles were twisted approximately 360° evenly at the rate of 60 times/min for 20 s, manipulated every 5 min and withdrawn after 20 min. For the convenient manipulation of the acupuncture points on the back, rat was placed on the suspended shelf (50 × 45 mm, about 50 cm high from the ground, see additional file 1), which could easily make it calm and stand still without anesthesia. Acupuncture was performed by the same experienced practitioner and the animals were handled while awake, with special care to minimize stress. Both rats in ASAC and CKAC received the same acupuncture treatment.

The measurements of pulmonary resistance (RL), dynamic compliance (Cdyn), and respiratory rate (RR), were modified from Glaab T et al 2425. 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 and annotation of the SAGE libraries were described as previously 21. 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 26. 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.

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.html27, 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/28, 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/29. 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 29.

The allergen-specific early airway response to ovalbumin (OVA) in sensitized rats showed significant increases in RL and significant decreases in simultaneously measuring Cdyn and RR compared with those of the controls, thus indicating an allergen-specific EAR to OVA. By One-way ANOVA among the four groups, the significant differences (P < 0.05) were at 1–7 min for RL, 2 and 5 min for Cdyn, 2–4 min for RR after challenge. The LSD test for post hoc analysis indicated that RL in ASAC group was significantly decreased at 1–4 min after challenge in comparison with AS group (P < 0.05, Table 1), and Cdyn and RR in ASAC group were significantly increased at 2, 5 min and 2, 3 min respectively after challenge when compared with those of AS group (P < 0.05, see additional file 3). The results indicated that the immediate effects (1–3 min) of acupuncture were the most significant in the EAR phase of asthma.

The four SAGE libraries of rat lung were deposited in the SAGEmap database at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/geo, Accession numbers are GSM45195, GSM119459, GSM279944, and GSM279945). The information of the matched genes and ESTs of the 4 libraries was listed in the Table 2.

By Comparing with the SAGE data between the libraries of CK and AS, there were totally 186 differentially expressed tags (P < 0.05, P_CK/AS). Similarly, there were 130 differentially expressed tags between libraries of AS and ASAC (P < 0.05, P_AS/ASAC), and 144 differentially expressed tags between libraries of CK and CKAC (P < 0.05, P_CK/CKAC). See additional file 4 for the lists of differentially expressed tags of P_CK/AS, P_AS/ASAC, P_CK/CKAC.

To confirm the expression profiles among the four SAGE libraries, three differentially expressed genes of interest were chosen and their expression levels were evaluated by qRT-PCR. Dusp1 encodes a protein that catalyzes the dephosphorylation and inactivation of MAP kinase. S100A9 is a calcium binding protein that may be associated with acute inflammatory processes. MT-2 has a high content of cysteine residues that bind various heavy metals and could be transcriptionally regulated by both heavy metals and glucocorticoids. The results indicate that the expression profiles of representative genes in qRT-PCR analysis correspond to the SAGE data (Figure 2), which supports the validation of our quantitative data for further bioinformatic analysis.

Based on the global expression matrix, the dendrogram clearly identified CK from the other 3 libraries, while AS and ASAC displayed more similar patterns than CKAC on the gene expression profiles (Figure 3). The genes with over fivefold change or less than 0.2-fold between AS and ASAC were listed at the right side of the hierarchical clustering dendrogram. Seven genes were ranked with an expression over 5-fold increase: Fxyd3 (Rn.3896), Col6a2 (Rn.11889), Uba52 (Rn.4300), Tm9sf2 (Rn.11839), CD24 (Rn.6007), Mgp (Rn.2379), and ribosomal protein L31 (Rpl31, Rn.1101). Four genes were listed with an expression over 5-fold decrease: Ap1m1 (Rn.139185), Mgst2 (Rn.7854), Atp1b1 (Rn.8925), and Ywhaq (Rn.2502).

Six DAVID gene functional classifications of P_AS/ASAC with the enrichment score equal to or higher than 1.0 were found. They were cellular biosynthetic process, homeostatic process, response to steroid hormone stimulus, cell migration, cellular process, and cellular lipid metabolic process. The related gene functional classifications among P_CK/AS, P_AS/ASAC and P_CK/CKAC were compared (Figure 4). The classification of homeostasis process was only found in P_AS/ASAC. The DAVID gene functional classification was found to be changed from "immune response" in P_CK/AS to "response to steroid hormone stimulus" in P_AS/ASAC. (See additional file 5 for the gene lists of DAVID gene functional classification of P_CK/AS, P_AS/ASAC, P_CK/CKAC)

There were 25, 5 and 10 enriched GO categories of biological process in P_CK/AS, P_AS/ASAC and P_CK/CKAC respectively (Figure 5, 6, 7). Biosynthesis was only one overlapping GO term among them. The GO terms, such as antigen processing and presentation, cell migration, acetylcholine receptor signaling, muscarinic pathway disappeared in P_AS/ASAC, and new GO terms, like regulation of biosynthesis, regulation of liquid surface tension, epidermal growth factor receptor signaling pathway appeared in P_AS/ASAC when compared with P_CK/AS. Regulation of liquid surface tension was the only shared GO term between P_AS/ASAC and P_CK/CKAC.

For understanding functional roles of the differentially expressed genes, KEGG pathway analysis was assigned by applying the DAVID annotation tool. There were 15, 8, and 2 matched KEGG pathways (counts ≥ 3) in P_CK/AS, P_AS/ASAC and P_CK/CKAC respectively (see Table 3), in which tight junction pathway kept the same. Three pathways, MAPK signaling pathway, T cell receptor signaling pathway, focal adhesion, were identical between P_CK/AS and P_AS/ASAC. Although there are the same matching pathways among three groups, the involved genes were different. (See additional file 6)

By using the Venn diagram, 21 tags were attributed to the same group between P_CK/AS and P_AS/ASAC, 7 tags were found involved in the three groups, 6 tags were identified between P_AS/ASAC and P_CK/CKAC, and 25 tags were commonly clustered between P_CK/AS and P_CK/CKAC(Figure 8). (See additional file 7 for the gene lists of each group)

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 30. 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 31. 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.

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 P_AS/ASAC via GO Tree Machine. Moreover, the DAVID genes functional classification was found to shift from "immune response" in P_CK/AS to "response to steroid hormone stimulus" in P_AS/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 P_AS/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 32. It plays a role in lung growth and development 33 and its expression was incited by dexamethasone 34. 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 35. 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.

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 P_AS/ASAC, in which tight junction pathway was commonly shared among P_CK/AS, P_AS/ASAC, and P_CK/CKAC. Three pathways were the same between P_CK/AS and P_AS/ASAC, and 4 pathways were unique in P_AS/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 P_CK/AS and P_AS/ASAC. It contributes to the amplification and specificity of the transmitted signals and plays discrete yet complementary roles in accentuating allergic airway inflammation 36. It is also reported that the MAPK pathway could regulate steroidogenesis 37. GnRH signaling pathway is one of the four unique pathways in P_AS/ASAC, which is the central regulator of the reproductive hormonal cascade 38 and could enhance the basal steroidogenesis 39. Several signaling pathways are activated by GnRH, including MAPK, and protein kinase C 40, which could lead to the increase of corticotropin-releasing hormone-binding protein at mRNA level 41. 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.

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 P_AS/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 42. Besides, the elevated expression of Abca2 was considered to be a conserved mechanism of cell survival 43. 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 4445. 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 P_AS/ASAC and P_CK/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 46.