The Study on the Molecular Mechanism of Erchen Tang on Asthma Based on Network Pharmacology

Objective: To explore the active ingredients of ECT and their targets of asthma and investigate the potential mechanism of ECT on asthma. Methods: Firstly, the active ingredients and target of ECT were screened for BATMAN and TCMSP, functional analysis was nished via DAVID. Then, animal model was induced by ovalbumin (OVA) and aluminum hydroxide. Eosinophil (EOS) counts, EOS active substance Eosinophilic cationic protein (ECP) and eotaxin levels were detected followed the instruction. Pathological changes of lungs tissue were examined by H&E staining and transmission electron microscopy. Interleukin (IL-4, IL-10, IL-13, TNF-α), TIgE and IgE level in bronchoalveolar lavage uid (BALF) were measured by ELISA. Finally, the protein expression of TGF-β / STAT3 pathway to lung tissue was detected by Western Blot. Results: A total of 450 compounds and 526 target genes were retrieved in Erchen tang. Functional analysis indicated that its treatment of asthma was associated with inammatory factor and brosis. In the animal experiment, the results showed that ECT signicantly regulated inammatory cytokine (IL-4, IL-10, IL-13, TNF-α) levels in (P<0.05, P<0.01) , reduced EOS number (P<0.05) and also ECP and Eotaxin levels in blood (P<0.05) in BALF and / or plasma. Bronchial tissue injury was obviously improved on ECT treatment. Associated protein in TGF-β / STAT3 pathway were signicantly regulated by ECT (P<0.05). Conclusion: This study originally provided the evidence that the Erchen tang was effective against the treatment of asthma symptoms, and its underlying mechanism might be regulation of inammatory factor secretion and TGF-β/STAT3 signaling pathway.


Background
Asthma is a common disorder characterized by airway in ammation and by declining in lung function with airway remodeling in a subset of asthmatics [1]. Related studies have determined that the in ammation of asthma is exacerbated by Th2 activity [2]. Activated Th2 cells secrete more in ammatory cytokines such as IL-4 and IL-13, thus causing airway hyperresponsiveness (AHR), eosinophil in ltration, goblet cell hyperplasia, and excessive mucus secretion [3]. IL-13 and IL-4 partly share the same receptor and signaling pathways and both are deeply involved in IgE synthesis, eosinophil activation, mucus secretion and airways remodeling [4]. Furthermore, in airway remodeling, TGF-β / STAT3 pathway plays an important role, in which JAK activated by TGF-β and cause the up-regulated STAT3 level [5], which was already known that both TGF-β and STAT3 were tightly related to airway brosis [6,7]. Present treatment of Cough Variant Asthma (CVA) patients with bronchodilators, glucocorticoids and leukotriene receptor antagonists cannot fundamentally alleviate chronic in ammation of the airway [8,9]. Therefore, it is urgent to develop drugs with signi cant therapeutic effects on asthma by regulating the TGF-β / STAT3 pathway to suppress airway in ammation.
Recent studies indicated that extracts from Chinese medicine or other herbs have immune regulation function [10] and good effect on the treatment of asthma [11]. Erchen tang (ECT), as a famous prescription of traditional Chinese medicine rst collected in ancient Chinese medicine book 'Taiping Huimin Heji Ju Fang', was included in Chinese pharmacopoeia [12], has achieved good results such as less coughing and less sputum in clinical treatment of various types of bronchial asthma [13,14]. In laboratory studies, the therapeutic effects of drugs on asthma are usually studied on animal model induced by ovalbumin (OVA) and aluminum hydroxide [15], and rats are often used in traditional Chinese medicine pharmacodynamics study as asthma models [16], [17]. It was proved by animal experiments that ECT could regulate the cytokine levels (such as IL-1, IL-6 and TNF-α) in serum [18] and prevent the airway remodeling by inhibit the expression of MMP-9 in lung tissue on asthma rats induced by ovalbumin (OVA) and aluminum hydroxide [19], and its improvement in lung tissue and the pulmonary function of chronic bronchitis model rats was also proved by Gao Miaoran [20]. However, there are still lots of unclear points of ECT, such as its active ingredients and the speci c target of the active ingredient, to solve these problems, we further used network pharmacology to lucubrate the active ingredients and the mechanisms of ECT.
Network pharmacology (NP) is a useful discipline that emphasizes maximizing drug e cacy and minimizing adverse effect via the multiple regulation of the signaling pathway [21]. It has been well applied to the study of the mechanisms of traditional Chinese medicine (TCM) [22] for almost all TCM and worldwide ethnomedicine exert therapeutic effect by targeting multiple molecules of the human body. Such as prediction of potential targets and pathways of cordycepin on the proliferation of MCF-7 breast cancer cells [23] and screen of active compounds of Curcumae Rhizoma [24]. Since the active ingredients and mechanism of ECT in treating asthma is not clear yet, NP is an effective way to create the targets-pathways interaction, and the targets related to asthma was collected to create pathways-disease interactions, as well as further related bioinformatics analysis, was employed to explain the potential mechanism of treatment of ECT on asthma.
In this study, we used network pharmacology to explore the active ingredients of ECT and their targets of asthma. Thereby, the effect of Erchen Tang on asthma and the potential molecular mechanisms of TGFβ/STAT signaling pathway were studied by asthma animal experiments. After the experiment, the rats were euthanized by intravenous injection of 0.3% sodium pentobarbital. Elisa kit were all bought from Sigma (America). Horseradish peroxidase-conjugated goat anti-rabbit/antimouse/anti-goat antibody, Tissue protein extraction reagent, BCA Protein Quantitation Kit, SDS-loading Buffer, SDS-PAGE gel preparation kit, High sensitivity chemiluminescence detection kit and cDNA reverse transcription kit was all bought from Beijing Quanjin Biotechnology Co., Ltd. (Beijing, China).

Target gene screening and Functional enrichment analysis
Erchen tang was constituted by Pinellia ternata (Thunb.) Makino, Smilax glabra Roxb., Glycyrrhiza aspera Pall., and Citrus reticulata Blanco. Firstly, we separately queried the chemical composition of these four Chinese herbal medicines from the database of BATMAN and TCMSP. Then we combined the compounds of each medicinal material and imported them into TCMSP to get their target genes. We further normalized these genes and got human related genes in database of Metascape. Finally, we used DAVID to perform functional enrichment analysis of human related genes, the mechanism map was obtained from KEGG.
1.4 Animal experiment 1.4.1 Animal model of asthma One week after adaptive feeding, except for the normal control group, other groups were nihilated of ovalbumin (OVA) and aluminum hydroxide: on the 0th and 12th day of the experiment, each rat was intraperitoneally injected 1 mL of 10% OVA solution (containing sterile antigen solution of Class II OVA 1 mg and 100 mg of aluminum hydroxide gel) of sensitization, and then challenged with 5% OVA for 30 min every day for 5 days, then every other day, for 6 weeks.

Experimental material
24 h after last injection, 20% urethane solution was used for anesthetizing by intraperitoneal injection. Blood was taken from the abdominal aorta, partially placed in the EDTA anticoagulation tube, and the rest of the blood was placed in a non-anticoagulant tube, 3000 r/min, centrifuged for 15 min and collected the serum. The blood vessels of the neck were separated from the nerves, and the main bronchus was separated, the left bronchus was ligated, the right pulmonary alveoli through 1 cm below the main bronchus was rinsed by injecting 0.6 mL 37% isotonic sodium chloride solution each time for three times. Total 1.35 ~ 1.55 mL liquid (bronchoalveolar lavage uid, BALF) was collected, the supernatant wad used to detect indicators such as IL-4 and IL-13. One part of the right lung was xed with 4% paraformaldehyde, and the other part of the right lung was frozen in liquid nitrogen. Take a suitable amount of cryopreserved lung tissue, and 9 times of low-temperature refrigerated physiological saline was added to prepare 10% tissue homogenate.

Histopathology and tissue imaging
After dehydration, embedding and Sect. 4 ~ 5 µm of tissue, hematoxylin staining was carried out for 6 ~ 10 min. The mixture was separated by 1% hydrochloric acid solution, diluted ammonia water was used for 10 min, then eosin staining for 10 s. Tissue in ammation was observed after xation. Tissue imaging was observed after 3% uranyl acetate-lead citrate dyed. The computerized image analyzer (Pathological image analyzer, Lycra, Japan) was used to measure the bronchial lumen and outer circumference (Pi, Pe), the number of bronchial smooth muscle cells (Pi). Calculating the basement membrane area (WAi), airway smooth muscle layers area (WAm), and make WAi, WAm and the ratio of Pi was measured as a standardization, representing bronchial basement membrane thickness (WAi/Pi) and smooth muscle layer thickness (WAm/Pi), respectively.

Eosinophil count, EOS active substance ECP and Eotaxin levels
BALF was centrifuged at 2000 rpm for 5 min (Benchtop centrifuge, Shanghai Anting Scienti c Chemical Instrument Factory), the supernatant of it was discarded, and the cell sediment was diluted in 1 mL of pre-cooled PBS. The precipitate of BALF was resuspended for total cell counts recorded with on a hemocytometer. Slide were prepared by using a cytospin instrument. At least 500 in ammatory cells were counted in each sample. Total EOS number per microscope eld was determined by counting EOS under an optical microscope (Microplate scanning microplate reader American BioTek instrument). Then the absolute EOS number was determined. EOS active substance ECP and Eotaxin in blood were detected by Elisa Kit according to the manufacture instructions.
1.4.6 Enzyme-Linked Immunosorbent Assay (ELISA) Interleukin (IL-4, IL-10, IL-13, TNF-α), TIgE and IgE level in serum were measured by ELISA using monoclonal antibody-based mouse interleukin ELISA kit according to the manufacturer's instruction. All data represent the mean and standard error of mean from at least three separate experiments and were compare by analysis of variance (ANOVA).

Detection of the expression of TGF-β/STAT3 signaling pathway
The expression of TGF-β\STAT3 signaling pathway in BALF was detected by western blot (Bio-Rad electrophoresis system, Bole Life Medical Products Co., Ltd.). Western blot analysis was performed according to the instruction. Brie y, lung homogenate was centrifuged in 5000 r/min for 5 min, sedimentary tissue was collected and lysed on ice in a RIPA buffer with protease inhibitor 1% PMSF, and then clari ed by centrifugation. Total cell lysates were resuspended in SDS sample buffer and resolved by SDS-PAGE. Proteins were transferred to PVDF membrane and then blocked with 5% BSA / TBST buffer for 1 h at room temperature. The blocked PVDF membrane was directly placed in primary antibody solution and incubated overnight at 4 ℃ and then transferred to second antibody solution and incubated at room temperature for 2 h. After that was color development, the gel imaging system (Gel imaging system, American Bio-RAD company) was used to scan and lm. All experiments were performed in triplicate.
The collected BALF was centrifuged, and the supernatant was collected. The RPMI 1640 culture solution was resuspended,the cell sediment was diluted and cultured at 37 °C in a 5% CO 2 incubator for 2 h to extract macrophages and then lymphocytes were removed. Rinsing with PBS solution (containing 10% FBS), centrifuging, and further separating with precooling separation solution. Finally, interface cells of 1.105 to 1.090 were collected and rinsed with PBS (containing 10% FBS). Separated EOS was adjusted to a cell concentration of 2 × 10 5 with RPMI 1640 complete medium containing 10% FBS, and 10 U/ML IL-5 cytokine was added, and cultured in a culture plate at 5% CO 2 and 37 ° C. After 24 h, the total protein was extracted and quanti ed, and the proteins related to TGF-β\STAT3 signaling pathways were detected. The detection method was the same as the previous one. It should be noted that the protein extraction reagent was the mammalian protein extraction reagent (containing 1% PMSF). Morphological identi cation of separated EOS above was observed by Wright-Giems staining on room temperature with Giems dye liquor. After washing, rst rinse the slide with PBS solution, then rinse with small stream of water, seal and observe.

Statistical analysis
Data are presented as the mean ± standard deviations (SD) and Student's t-test combined with one-way ANOVA previously was performed to determine statistically signi cant differences between two groups. Results were considered statistically signi cant at p values 0.05(#), 0.01(##), or 0.001(###) for the control group versus the model group comparisons and p values 0.05(*), 0.01(**), or 0.001(***) for the model group versus the experimental group.

Analyzing the potential mechanism of ECT in the treatment of asthma by network pharmacology
The result of database search showed that a total of 450 compounds and 526 target genes were retrieved in Erchen tang, and 98 genes closely related to asthma among them (Supplementary material 1).
Functional enrichment indicated that ECT could regulate asthma (Supplementary material 2). The underlying mechanism was shown in Fig. 1, it could affect the secretion of IL-4, IL-13, TNF-α, eotaxin and so on, thereby inhibited the increase of a cells, and nally achieved regulation of tracheal in ammation and remodeling (Fig. 1).

Effect of ECT on serum TlgE and OVA sIgE levels
Results from Fig. 3 showed that the levels of TIgE (Fig. 3A) and OVA sIgE (Fig. 3B) in the model group were signi cantly higher than those in the control group (P < 0.001). Compare with the model group, dexamethasone and Erchen tang treatment signi cantly decrease the expression levels of TIgE and OVA sIgE (P < 0.01).

Effect of ECT on histopathology of bronchial tissue
HE staining of lung tissue (Fig. 4) showed that the bronchial epithelium of the control group was intact (Fig. 3D), there was no obvious in ammatory in ltration around, the lung tissue and the area of bronchial were normal. The lumen of the model group was narrowed, and many in ammatory cells in ltrated around the blood vessels and lung tissues, neutrophils and eosinophils were main types of them. Also, for model group, the basement membrane was thickened and irregularly shaped, and the connective tissue of the smooth muscle and bronchus of the wall was obviously proliferated. Compare with model group, the in ammatory in ltration of the dexamethasone group and the ECT group was signi cantly alleviated, the damage caused by the above model was signi cantly improved.
Statistical analysis showed that the ratios of WAi/Pi (Fig. 3A), WAm/Pi (Fig. 3B) and N/Pi (Fig. 3C) of model group were signi cantly higher than those in the control group (P < 0.01, P < 0.001). Both dexamethasone and ECT treated group signi cantly reduced the ratio of WAi/Pi, WAm/Pi and N/Pi (P < 0.05, P < 0.01).

Effect of ECT on pulmonary imaging
The ultra-microstructure of lung tissue (Fig. 5) was observed by transmission electron microscopy. The internal structure of the control group was normal, the mitochondria structure and basement membrane structure were intact, the sputum was clear with many visible lamellar bodies. For model group, EOS cells number increased signi cantly, the mitochondria were swollen and the sputum was blurred, also the number of lamellar corpuscles decreased signi cantly, and the number of voids increased with chromatin accumulated. With ECT treated, the basement membrane of ECT group is intact, and eosinophil aggregation with chromatin accumulated were both reduced compare with model group.

Effect of ECT on Eosinophil count and Tissue EOS activation markers' levels
Our data (Fig. 6) showed that marked EOS count was observed in BALF and plasma from asthma model group. Compare with control group, ECP and Eotaxin levels of model group were signi cantly increased (P < 0.05, P < 0.001) both in BALF (Fig. 6B/ 6C) and plasma (Fig. 6D/ 6E). The number of EOS in BALF and plasma both reduced signi cantly compare with model group with ECT treatment (P < 0.05). Also, ECP and Eotaxin were EOS activation markers in lung tissue. ECP and Eotaxin levels were signi cantly decreased compare with model group with ECT treatment (P < 0.05). These data suggested that ECT could reduce EOS number both in BALF and plasma, also ECP and Eotaxin levels in BALF, which is possible mechanism for asthma treatment.
The EOS cells separated from BALF were identi ed by Wright-Giemsa and observed under microscope.
The morphology and coloration of the cells were consistent. The Wright staining results was positive (Fig. 6A), which proved that the separated cells were the EOS cells. For EOS cells, purity testing showed that each group had EOS purity > 95%.

Discussion
Erchen Tang was constituted by Pinellia ternata (Thunb.) Makino, Smilax glabra Roxb., Glycyrrhiza aspera Pall., and Citrus reticulata Blanco. Pinellia ternata (Thunb.) Makino has protective effects against allergic airway in ammation in a model of asthma in mice [25]. The data supported that Smilax glabra Roxb. had immunomodulatory potential through activating macrophages and enhancing host immune system function [26]. Many reports have shown that Glycyrrhiza aspera Pall possess anti-in ammatory activity, suppress proin ammatory cytokine like TGF-β and inhibit the translocation of toll-like receptor 4. Citrus reticulata Blanco was suggested to be used as a therapeutic agent for patients with Th2-mediated or histamine-mediated allergic asthma [27]. Erchen decoction combined with Yu-Ping-Feng-San has been widely used for decades in treating asthma at the A liated Hospital of Nanjing University of Chinese Medicine [28].
Asthma is an immune allergic disease characterized by reversible airway obstruction, airway hyperresponsiveness and airway in ammation [29], among which airway in ammation is the most important pathological change [30] and determines the degree of airway obstruction [31] and airway hyperresponsiveness [32]. It was proved in our study that model group had sever in ammation of bronchus with results from histopathology. Many in ammatory cells in ltrated around the blood vessels and lung tissues. And all these bronchial tissue injuries were improved with ECT treatment, the in ammatory in ltration of the ECT group was signi cantly alleviated.
In ammatory cytokines IL-4 and IL-13 has been shown to play a vital role in allergic airway in ammation [33], which promote the airway epithelial cells to release eosinophil chemotactic factor-3 (Eotaxin-3), and then cause airway in ammation of eosinophilia [34]. At the same time, the increased lung LTs mediated by eosinophil, further induced increased IL-13 [35], which were secreted by airway epithelial cells, nally caused collagen deposition (i.e., pulmonary brosis) mice with respiratory airway in ammation. Our data suggested that the IL-4 and IL-10 levels decreased in asthma model, since IL-10 has been shown to suppress all the pro-in ammatory cytokines, lower IL-10 levels were reported to be associated with higher frequency of bronchial asthma [36], there was result showed signi cantly increased serum level of IL-10 in children with treatment [37]. ECT could signi cantly regulate serum in ammatory cytokine (IL-4, IL-10, IL-13, TNF-α) levels, with reduced EOS number both in BALF and plasma, which proved that EOS was related to the regulation of the internal environment balancemaintenance regulation induced by the in ammatory pathway in tissues [38].
It was found that both TGF-β level and pSTAT3/STAT3 would signi cantly increase either in Bronchial mucosal tissue [39] of asthma patients or in asthma mice [40], and these two proteins are both directly related to tissue brosis [41]. It had been indicated that TGF-β signaling was the master pathway regulating brosis pathogenesis, in which activator of STAT3 acted as the integrator of various pro-brosis signals [42]. Results from western-blot suggested the growing levels of protein in TGF-β/STAT3 pathway were inhibited with the ECT treatment. Since TGF-β induces integrin, matrix metalloproteinases, protease inhibitors, and regulators of small GTPases that participate in tissue remodeling and in uence cell-ECM interactions [43], the regulation of TGF-β/STAT3 pathway might be the mechanism of ECT on airway brosis.

Conclusions
In this study, network pharmacology was used to predict the potential mechanism of ECT in the treatment of asthma. And the results from the animal experiment indicates that ECT might have modulatory action on cytokines levels (IL-4, IL-10, IL-13, TNF-α) by regulating TGF-β and STAT3 levels, and thereby inhibit the increase of EOS cells, nally achieved regulation of tracheal in ammation and airway brosis.  Figure 1 The potential mechanism of ECT in the treatment of asthma. Red ve-pointed star represented the target of ECT regulation