Studies in animal models form the basis for our current understanding of the pathophysiology of asthma, and are central to the preclinical development of drug therapies[28]. Guinea pigs have been the most commonly used small animal species in preclinical studies related to asthma and COPD[29]. β 2 -adenoceptor agonists and antimuscarinic drugs prevent antigen-induced bronchoconstriction in actively sensitized guinea pigs in a dose-dependent manner. Histamine is the major mediator in guinea pigs but not in humans[30]. Asthma is a complex disease defined by reversible airway narrowing, acute and chronic airway inflammation, airway hyperresponsiveness (AHR) and airway tissue remodelling, in which accumulation of airway smooth muscle (ASM) is a prominent and widely reported feature[31]. In the pharmacodynamics study, the prolonged asthma time[12] and HE sections showed that QFXY had significant effects on asthma, reducing edema in airway mucosa and inflammatory cell infiltration in airway and vascular vessels. They were also beneficial to reducing airway remodelling.

Among up-regulated genes, the fold change of RHO almost ranked top (Additional file2). Among down-regulated genes, CLU and ENO1 had greater changes. Among 2D results, fold changes of Hsp90 and Serpin were of greater change (Table 1). Besides, references and literatures about every diff molecules were retrieved, of which some were related to the process of inflammation or asthma or lung diseases, such as GNB1[32, 33], MAPK3[34–37]. Altogether, with the combined consideration of fold changes and references, these genes (RHO, CLU, ENO1, GNB1, MAPK3) and proteins (Hsp90, Serpin) were selected for validation test.

The GO annotation suggested that QFXY might influence the inflammation, signal transduction, stress response, the apoptosis of endothelial and bronchial cells. Pathway analysis revealed that different genes were involved in the signaling pathways, including focal adhesion pathway, cell-extracellular matrix interactions pathway, TGF-beta signaling pathways, NK cell-mediated cytotoxic pathway and so on, which are all related with cell signaling, inflammation, mast cells and NK cells. Many asthma drugs also participated in those pathways in variety of mechanisms, targeting kinases, receptors or related proteins, affecting inflammation response, mitosis, angiogenesis, apoptosis, and anti-oxidation, to play a role in asthma. The qPCR change profile was basically in line with the microarray results, proving the reliability of microarray data. The commonly shared signal pathways of diff genes and diff proteins combined the genomics and proteomics together, to manifest the underlying mechanism of QFXY effects.

The Mapk3/Erk signaling cascade is a central Mapk pathway that plays a role in the regulation of various cellular processes such as proliferation, differentiation, development, and inflammation reactions and etc.[34–37]. Inhibition of this kinase strongly decreased the expression of pro-inflammatory genes encoding growth-regulated proteins (Gro-a, Gro-b and Gro-g) and interleukins (IL-1β, IL-6 and IL-8)[36]. Mapk can participate in the regulation of NF-κB transcriptional activity[38, 39]. Our previous study also presented decreasing erk expression and NF-κB inhibition[13].

Hsp90, as a molecular chaperone, has interactions with proteins, such as Akt and Raf-1[40]. Akt is a downstream effecter molecule of phosphoinositide 3-kinase and is thought to mediate many immune and inflammatory responses. It is also involved in the activation of NF-κB[41]. Amino acid residues 229–309 of Akt were involved in the binding to Hsp90 and amino acid residues 327–340 of Hsp90 β were involved in the binding to Akt[42]. Hsp90 plays an important role in maintaining Akt kinase activity. In our study, 2D and Western blot showed decreased Hsp90 after QFXY treatment, as well as less NF-κB activity[13], indicating QFXY may affect the binding of Hsp90 and Akt, which needs further confirmation.

GTP-binding protein beta1 subunit gene (GNB1), its up-regulation appears to be one of the candidate processes of sensitization. It also has NF-κB recognition sites[32]. The Ectodysplasin is involved in binding to its ligand EDA-A1 and activates the NF-κB intracellular signaling pathway by interaction through its death domain with the adaptor protein EDARADD[33]. Down-regulated GNB1 and EDARADD gene expression decreased NF-κB activity for anti-inflammation.

Serpins form an enormous superfamily of 40-60-kDa proteins found in almost all types of organisms. Most have evolved to finely regulate complex proteolytic pathways, such as blood coagulation, fibrinolysis, and inflammation[43, 44]. α1-antitrypsin (AAT) is an archetype member of the serpin supergene family. The reduced serum levels of AAT contribute to the development of chronic obstructive pulmonary disease (COPD). In addition to protease inhibition, AAT shows anti-inflammatory, immunomodulatory and antimicrobial properties[45, 46]. SerpinA1 is an endogenous anti-inflammatory factor, and its anti-inflammatory effects may be mediated through antioxidant activity[47]. Compared with the Model group, the HE sections of the QFXY group showed less inflammation and mucosa hyperplasia, and the 2D and qPCR proved higher SerpinA1 expression, which indicating specific ingredients in QFXY can activate SerpinA1.

Asthma is a disease characterized by persistent inflammation and structural changes in the airways referred to as airway remodelling., including smooth muscle hypertrophy, goblet cell hyperplasia, subepithelial fibrosis, and angiogenesis. Vascular remodelling in asthmatic lungs results from increased angiogenesis, mediated by vascular endothelial growth factor (VEGF). In addition, VEGF induces allergic inflammation, enhances allergic sensitization, and has a role in Th2 type inflammatory responses[48]. Matrix GLA protein (MGP) has a role in endothelial cell (EC) function[49]. MGP modulates the activity of transforming growth factor (TGF)-β superfamily, which is critical for morphogenesis and development[50]. MGP can stimulate VEGF expression through increased TGF-β activity in endothelial cells[51]. Comparing with the Model group, HE sections in the QFXY group showed less pulmonary consolidation, which means QFXY help alleviate lung tissue remodelling.

Asthma is featured by reversible airway obstruction. The lack of full reversibility in some asthmatic patients may be due to chronic airway remodelling[52]. It appears that inflammation and remodelling are interdependent processes that clearly influence the clinical long term evolution of asthma[53]. The ECM can act as a reservoir for an increasing number of growth factors. These growth factors can be rapidly released from the ECM to allow extracellular signaling regulated by the growth factors to proceed without the need for new protein synthesis[54].

In QFXY-asthma target network, Hsp90α, Mapk3, VIM were hub proteins suggesting that they may be some targets of QFXY pills. The complicated interaction network suggested that QFXY pills affected a complex system regulating inflammation and immune reactions. Seen from the above complex network, QFXY interacts with asthma related genes in both direct and indirect way, affecting several signal pathways. In the previous study, 55 ingredients have been identified, including 27 absorbable constituents in QFXY, among which there are 19 ingredients affect inflammatory pathways, typically they are sulfur containing alkynes, such as arctic acid; lignans, such as arctigenin; phenolic acids, such as sinapic acid; steroids, such as cholic acid[13]. In the following study, other effects of these ingredients, such as alleviating airway hyperresponsiveness (AHR) and airway tissue remodelling will be further explored.