Sanzi Yangqin Decoction Alleviates Allergic Asthma by Modulating Th1/Th2 Balance: Coupling Network Pharmacology with Biochemical Pharmacology

This study aimed to verify that Sanzi Yangqin Decoction (SYD) can relieve asthma in mice and explore the effect on TH1/Th2 balance. The targets of SYD and asthma were explored from the public database using various methods. The potential targets and signaling pathways were identified by KEGG enrichment analysis from DAVID database. Mice asthma models were established using OVA and aluminum hydroxide. Lung tissues of mice were stained with HE and Masson. The contents of IFN-γ, IL-4, and TNF-α in BALF and IgE in mouse serum were detected using ELISA. In addition, the changes in Th1 and Th2 cells of the spleen were detected by flow cytometry. Fourteen core targets including IL4, IFNG, and MMP9 were identified for the treatment of asthma by SYD. The content of IL-4 in the lung tissue and BALF was gradually decreased with the increase in SYD concentration, while the IFN-γ was gradually increased. The drug significantly reduced IgE levels in serum and TNF-α in BALF. The number of Th1 cells in the spleen increased, while Th2 cells decreased in a concentration-dependent manner. SYD can alleviate pulmonary inflammation, restore Th1/Th2 balance, and relieve asthma.


Introduction
Bronchial asthma (hereinafter referred to as asthma) is a chronic inflammatory disease of the airway involving a variety of cells and cellular elements. It is characterized by obvious heterogeneity [1], with airway inflammation and hyperresponsiveness as the main pathological features [2][3][4]. It is generally believed that chronic inflammation plays an important role in the occurrence and development of asthma [5,6]. Asthma is also often accompanied by pulmonary eosinophilia and excessive IgE secretion [7,8]. At present, the number of patients with asthma has exceeded 334 million globally [9]. In developed countries, the prevalence of asthma is stable or decreasing but rising rapidly in developing countries [2]. Studies have found that asthma is closely related to 1/ 2 imbalance [10]. IFN-c and IL-4, the representative cytokines secreted by 1 and 2 [11][12][13], can modulate 1/ 2 balance in the body [14][15][16][17].
Sanzi Yangqin Decoction (SYD) has often been applied to treat asthma clinically due to its efficacy in resisting inflammation, suppressing coughing, and relieving asthma [18,19]. At present, there are few studies on the mechanism of SYD in the treatment of asthma. In this study, possible mechanisms of action of SYD were screened based on network pharmacology, followed by experimental evaluation ( Figure 1). It points out the direction for the follow-up study on the molecular mechanism of SYD regulating 1/ 2 balance, provides a good reference for the clinical application and pharmacological mechanism research of SYD in the treatment of asthma, and enriches the relevant research reports.

Databases.
Twelve databases involved in the network pharmacological analysis are shown in Table 1.

Acquisition of Common Targets.
Active ingredients and potential targets of SYD were retrieved from TCMSP, SEA, and SwissTargetPrediction. Disease-related targets were harvested from GeneCards, CTD, and TTD with "asthma" as the index term. Venn diagram of R language was used to map potential targets of SYD to disease-related targets to yield common ones.

Screening of Core Targets.
Common targets were imported into STRING online analysis platform to plot a PPI diagram with the minimum confidence set to 0.9. e generated file "string interactions.tsv" was imported into Cytoscape for topological analysis. Common targets with a degree and betweenness centrality greater than mean values were screened as core targets.

Network Construction.
e common targets, diseaserelated targets, and compounds contained in SYD were imported into Cytoscape to construct a drug-target-disease network to inform topological analysis.

Enrichment Analysis.
To describe and annotate the functions of core targets and explore their biological processes and signaling pathways, we carried out an enrichment analysis on core targets screened out. is was based on the DAVID database, with the species limited to "Homo sapiens". GO terms can be subdivided into three nonoverlapping ontologies: molecular function (MF), biological process   analysis was conducted to screen out potential signaling pathways of SYD to treat asthma. e results of the enrichment analysis were determined under the screening condition of P < 0.05.
2.6. SYD Preparation. SYD, a Chinese medicine compound, composed of Fructus Perillae (10 g), Semen Sinapis Albae (10 g), and Semen Raphani (10 g). ey are dry and mature seeds of Raphanus sativus L. and Sinapis alba L. and the fruit of Perilla frutescens (L.) Britt., a labiate plant. Ten times the amount of water was added and decocted twice. e extracted liquid was filtered and concentrated to 0.3 g/mL. e qualified liquid was stored at −20°C for reserve. Mice in the blank group were treated with intraperitoneal injections of normal saline and aerosol inhalation. One h before stimulation, mice were given dexamethasone (2 mg/ kg) and SYD (15 g/kg, 30 g/kg, and 45 g/kg) by gavage. Mice in the blank and model groups were given normal saline by gavage ( Figure 2). Blood was collected via an orbital bleed to prepare serum. At the end of the experiment, mice were sacrificed with cervical disconnection, the spleen was harvested to prepare the single-cell suspension, and the obtained lung tissue was stored at −80°C for future use. All operations complied with the regulations on the administration of laboratory animals.

Q-PCR Detection of Core Targets Screened Based on
Network Pharmacology. Total RNA was extracted from the lung tissue using TRIzol reagent and reversely transcribed to cDNA. Total RNA with an OD260/OD280 between 1.8 and 2.0 was used for Q-PCR. Following the manufacturer's instructions, the pure RNA was reverse transcribed to cDNA using Prime-Script RT reagent kit with gDNA Eraser (Takara, Japan; 42°C 40 min, 85°C 5 min, 4°C store). e primers were designed by the software Primer Premier, as shown in Table 2. SYBR Green I nucleic acid gel stain was employed for Q-PCR. With GAPDH as the internal reference, 2 −ΔΔCt values were calculated as the relative expression levels of core target genes.

Histopathological Examination.
e fixed lung tissue was dehydrated with ethanol, routinely embedded in paraffin, and sliced into sections, which were stained with HE to observe the pathological changes under a microscope. e degree of inflammatory infiltration in the lung tissue was scored as follows: 0 point: no inflammatory cells; 1 point: focal infiltration of small amounts of inflammatory cells around the trachea or blood vessels and on the alveolar walls; 2 points: patchy or focal inflammatory cell infiltration around the trachea or blood vessels and on the alveolar walls, with the involving area less than 1/3 of the cross-sectional area of the lung; and 3 points: diffuse inflammatory cell infiltration around the trachea or blood vessels and in the lung interstitium, with the involving area reaching 1/3-2/3 of the cross-sectional area of the lung.

Detection of Related Indicators by ELISA.
Bronchoalveolar lavage fluid (BALF) was collected by inserting a catheter into the right main bronchus and flushing 800 μl of PBS in and out of the lungs three times (∼600 μl recovered) [20]. e contents of IL-4, IFN-c, and TNF-α in BALF and of IgE in serum were detected in strict accordance with the ELISA kit instructions.

Determination of IL-4 and IFN-c in the Lung Tissue by
Western Blot Assay. e total protein extracted from the lung tissue was quantified by the BCA method. From this, 25 μg of protein was loaded into each gel sample hole, separated by 10% SDS-PAGE, and then transferred to the PVDF membrane, which was incubated with primary and secondary antibodies. After being visualized using ECL reagent in the dark for 30 s, the bands were exposed using the gel imaging system. e average grey value was calculated using Image J software, and the relative protein expression levels of IL-4 and IFN-c were determined with β-actin as the internal reference.

Detection of 1 and 2 in the Spleen by Flow Cytometry.
To determine the 1/ 2 ratio in the spleen, we first prepared splenic single-cell suspension. With Brefeldin as the blocking agent, cells were incubated for 4 h with PMA and ionomycin to evaluate the 1 and 2 cells. After being permeabilized in Perm/Wash buffer solution in the dark for 20 min, the cells were incubated at 4°C in the dark with CD4-FITC, IFN-c-PE, and IL4-PE antibodies for 30 min, and then detected by flow cytometry (BD, Accuri C6 Plus, events: 10000). 2 cells were expressed by CD4 + and IL4 + and 1 cells by CD4 + and IFN-c + .

Active Ingredients of SYD.
Twenty active ingredients were screened out under the conditions of bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18% (Table 3). OB is the fraction (%) of an administered drug that reaches the systemic circulation, and DL is the resemblance of a compound to the existing drugs.

Enrichment Analysis.
e 60 common targets were imported into STRING online analysis platform, and 14 core targets were screened out based on degree and betweenness centrality (Figure 4(a)). ese were then subjected to enrichment analysis using the DAVID platform. GO enrichment analysis showed that SYD affected such BPs as cell proliferation and apoptosis, nucleic acid metabolism, and protein transcription and translation (Figure 4(b)). KEGG enrichment analysis revealed that SYD mainly acted on asthma-related signaling pathways (Figure 4(c)), with IL-4 and TNF-α being the main action targets in treating asthma (Figure 4(d)).

Alleviation of OVA-Induced Asthma by SYD. HE and
Masson staining results showed that compared to the model group, SYD effectively alleviated inflammation and collagen deposition in the lung tissue of mice and was increasingly effective with an increase in drug concentration ( Figure 5(a)). ere was a significant difference between the inflammation score and positive area in Masson (Figures 5(b) and 5(c)). In addition, SYD significantly reduced the content of TNF-α in BALF of model group mice and alleviated the inflammatory response ( Figure 5(d)).

Effects of SYD on the Key Proteins Expression.
Transcription and protein synthesis of core target genes, screened by network pharmacology, was detected by Q-PCR and the western blot assay. Results from Q-PCR showed that multiple genes in the core targets were differentially expressed, including IL4, IFNG, and MMP9 ( Figure 6(a)).
e results of western blot display that the content of IL-4 in the lung tissue was gradually decreased with the increase in SYD concentration, while the content of IFN-c was gradually increased. Quantitative analysis of the proteins showed that the differences in relative protein expression levels were statistically significant (Figures 6(b) and 6(c)). e contents of IL-4 and IFN-c in BALF were detected using ELISA kits. We found that the contents of IL-4 gradually decreased with an increase in drug concentration, while the contents of IFNc gradually increased (Figures 6(d) and 6(e)). Detection of IgE content in serum revealed that the IgE content gradually decreased with an increase in drug concentration (Figure 6(f )).

1/ 2 in the Spleen.
e percent of CD4 + IL-4 + T cells ( 2) in the model group was higher than those in the normal group, while percentages of CD4 + IFN-c + T cells ( 1) decreased significantly (Figures 7(b) and 7(d)). Flow cytometry showed that SYD increased the content of 1 cells in the spleen but reduced the content of 2 cells. Compared with the blank group, the 1/ 2 ratio in the model group was significantly lower. Compared with the model group, the 1/ 2 ratios in the medication groups were significantly higher (Figures 7(a) and 7(c)).

Discussion
In this study, 14 core targets of SYD for the treatment of asthma were identified based on network pharmacology. Enrichment analysis of core targets showed that SYD exerted therapeutic effects on asthma mainly by regulating IL-4mediated differentiation of 0 cells. e protective effect of SYD on OVA-induced asthma in mice was explored with dexamethasone as the control. In the initial stage of provocation, sneezing, and scratching were observed, while choking, shortness of breath, nodding breathing, and other asthma-specific manifestations were observed in the later stage of provocation, accompanied by reduced activity and dull hair. HE staining results showed that SYD effectively ameliorated the inflammatory cell infiltration around the bronchi and blood vessels of mice in the model group. Q-PCR showed that IL4, IFNG, and MMP9 genes were differentially expressed in the mouse lung tissue. Western blot assay indicated that SYD reduced the content of IL-4 and increased the content of IFN-c in the mouse lung tissue. In addition, the detection of IFN-c and IL-4 in BALF and IgE in mouse serum in each group showed that SYD significantly increased the content of IFN-c and reduced the contents of IL-4 and IgE. IFN-c and IL-4, as the representative cytokines secreted by 1 and 2, reflected the 1/ 2 balance affected by SYD. In general, SYD can increase the 1/ 2 ratio in asthmatic patients and restore it to normal balance.
1 and 2 cells are differentiated by the unstimulated 0 cells [31]. Under the stimulation of IL-12, 0 cells can be differentiated into 1 and secrete cytokines IFN-c and IL-2 [32][33][34]. IFN-c, as a specific cytokine secreted by 1, can activate macrophages, engulf the damaged tissue, and initiate bodily selfrepair to fight against asthma [35,36]. It can inhibit the differentiation of 2 cells induced by IL-4 to maintain 1/ 2 balance [37] and inhibit the generation of IgE. It promotes the synthesis of IgG and reduces the release of cellular inflammatory mediators to prevent asthma [38,39]. Under stimulation by IL-4, 0 can be differentiated into 2 and secrete cytokines such as IL-4 and IL-13 [40,41]. e representative IL-4 can bind to IL-4R on B cells to activate the JAK/STAT signal transduction system and promote B cells to produce specific IgE [42]. rough binding to mast cells, IgE can induce mast cell degranulation Evidence-Based Complementary and Alternative Medicine and histamine and leukotriene release [43]. e level of serum IgE, a core antibody mediating type I allergic reaction, is closely related to the severity of asthma [38]. Furthermore, IL-4 can also promote the expression of vascular cell adhesion molecules and allow eosinophilic granulocytes to accumulate and infiltrate the inflammatory site [41]. e commonly used drug dupilumab is a human-derived anti-IL-4Ra monoclonal antibody that can inhibit the binding of IL-4Ra to IL-4 and IL-13 and block the signaling pathways mediated by IL-4 and IL-13 [44]. After treating asthmatic mice with SYD, IFN-c levels in the lung tissue and BALF were increased significantly with  Figure 3: Network relationship of SYD in the treatment of asthma. (a) e intersection of drug and disease targets, the yellow area represents the range of SYD, and the blue area represents the range of asthma. (b) e 60 common targets of SYD and asthma were imported into STRING to generate the network diagram. e node size represents the degree value. e larger the diameter, the more critical the node is in the network. 6 Evidence-Based Complementary and Alternative Medicine     With the development of molecular biology, research on the clinical prevention and treatment of asthma is gradually deepening. Current studies have shown that the imbalance of 1/ 2 is closely related to the pathogenesis of asthma. e advantages of TCM in the regulation of 1/ 2 balance are mainly reflected in the aspects of multilink, multitarget, and multiapproach. rough molecular biological methods, lung tissues were detected by western blot. β-actin served as a reference gene. All experiments were repeated in triplicate to average. (d and e) ELISA was used to measure the total IL-4 and IFN-c content (pg/mL) in BALF of mice. (f ) ELISA was used to measure the total IgE content (ng/mL) in serum of mice (blank, n � 10; model, n � 7; SYD 15 g/kg, n � 8; SYD 30 g/kg, n � 8; SYD 45 g/kg, n � 9; dexamethasone, n � 9).
in-depth exploration of TCM is expected to bring a breakthrough for the clinical prevention of asthma. At present, the specific mechanism of TCM regulating the imbalance of 1/ 2 response is not very clear, and the author's followup research will focus on it.

Conclusion
It was confirmed that SYD alleviated the inflammation of mice with asthma, increased 1 cells and decreased 2 cells in the spleen, and restored the 1/ 2 balance to relieve asthma symptoms.     Data Availability e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest in this work.