Investigation of Pharmacological Mechanisms of Schisandrin a for the Treatment of Asthma Determined by Network Pharmacology and Experimental Validation


 Traditional Chinese medicines (TCM) are increasingly applied and accepted in asthma prevention and treatment. In the present investigation, we aimed to evaluate the effects of schisandrin A against asthma and examine its underlying mechanism. Here, 68 intersection targets between schisandrin A and asthma were identified by network pharmacology. Further enrichment analysis demonstrated that the nuclear factor-kappaB (NF-κB) signaling pathway may be a major signaling pathway and cyclooxygenase 2 (COX-2/PTGS2) may be a key target in the anti-asthmatic mechanism of schisandrin A. Then, the relevant mechanisms were verified. In vitro, we found that schisandrin A knock down the expression of COX-2 and iNOS (inducible nitric oxide synthase) in 16 HBE cells and RAW264.7 cells in a dose-dependent manner. While, it ameliorated the epithelial barrier function injury, and reduced the activation of NF-κB signaling pathway effectively. Additionally, OVA-induced asthma mice model showed that inflammatory cell infiltration, mucus secretion as well as airway remodeling could be availably suppressed by schisandrin A treatment. In conclusion, our data suggested that schisandrin A can reduce asthma symptoms by inhibiting inflammation production, including lowering the Th2 cell ratio, which provides a basis for further understanding of the treatment of asthma with schisandrin A.


Introduction
Asthma is a common chronic in ammatory disease characterized by airway hyperreactivity, airway in ammation, and airway remodeling [1]. The airways of asthmatic patients often show in ammatory cell in ltration, goblet cell hyperplasia, increased mucus secretion and epithelial cell tight junction changes [2]. T helper type 2 (Th2) lymphocytes activate eosinophils and mast cells in response to allergens by inducing the release of cytokines, which play an essential role in airway in ammation and excessive mucus secretion [3]. It is well-known that nuclear factor-kappaB (NF-κB) signaling pathway is an important pathway to regulate innate and adaptive immune responses [4,5]. Simultaneously, it is associated with asthma [6]. Cyclooxygenase 2 (COX-2), also known as prostaglandin-endoperoxide synthase 2 (PTGS2) and inducible nitric oxide synthase (iNOS) are involved in the activation of NF-κB [7].
On the other hand, the phosphorylation of NF-κB can increase the expression of iNOS and COX-2 [8,9].
Most of the current clinical treatments for asthma use western medicine treatments, such as corticosteroids and β2 receptor agonists, but not all patients respond and larger doses of corticosteroids cause many side effects [10,11]. In recent years, traditional Chinese medicine (TCM) has been increasingly selected to treat asthma.
Schisandrin A is a bioactive lignan isolated from the fruit of Schisandra chinensis, which has the effects of anti-in ammatory and anti-oxidation, that appears to be associated with the NF-κB signaling pathway [12,13]. Schisandrin A has potential effects on osteoarthritis by inhibiting IL-1β-induced in ammation and cartilage degradation through inhibition of MAPK and NF-κB signaling pathways [14]. In addition, Schisandrin A inhibits LPS-induced in ammatory and oxidative responses in RAW264.7 cells by inhibiting NF-κB, MAPK and PI3K/Akt pathways, indicating its potential role in over-activation of macrophages [15].
Studies have indicated that that schizandrin may be an effective novel therapeutic agent for the treatment of asthma, that appears to be mediated in part by a reduction in oxidative stress and airway in ammation [16][17][18]. Therefore, the purpose of our current study was to explore the protective effect of schisandrin A on asthma by in vitro and in vivo models. The target gene information were normalized using the Uniprot database (https://www.uniprot.org/) after removing duplicated genes.

Target collection and screening of bronchial asthma
Using "asthma" as the keyword in the GeneCards database (http://www.genecards.org/) and the OMIM database (Online Mendelian Inheritance in Man, http://www.omim.org/), asthma related target genes were acquired. The resulting target genes were combined, de-duplicated, and then normalized for target gene information using the Uniport database.
1. Construction of protein-protein interaction (PPI) network and acquisition of core targets of schizandrin A in the treatment of bronchial asthma The intersection target genes between schisandrin A and asthma were obtained by using Venny plot analysis (v2.1, http://bioinfogp.cnb.csic.es/tools/venny/index.html). The obtained intersection target genes were uploaded to the STRING database (https://string-db.org/) to obtain the protein-protein interaction (PPI) network, the species was de ned as "Homo sapiens", and TSV le was saved. The PPI network was visualized using the Cytoscape (version 3.6.2) software, and the topological analysis of the PPI network was carried out by using the NetworkAnalyzer plugin for Cytoscape, thereby obtaining the topology indices of the network such as degree, betweeness centrality, closeness centrality, and average path length. Then, the hub genes from the PPI network were selected using the MCC algorithm of the cytoHubba plugin.

GO functional enrichment and KEGG of core targets
For functional enrichment analysis of the intersection target genes, we carried out Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the DAVID database (the Database for Annotation, Visualization and Integrated Discovery, http://david.abcc.ncifcrf.gov/). Furthermore, the results were screened using P value < 0.05, and the top ranked results were visualized.

Western blot assay
Total protein lysates were extracted from each group of cells using ice-cold RIPA lysis buffer. The protein concentration of each sample was determined by BCA kit. The supernatant of each sample was boiled with 1/4 volume of boiling 5x loading buffer in a metal bath at 100 ℃ for 5 min. For loading, a 20 mug total protein per sample was calculated and loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by wet transfer to a PVDF membrane. The membrane was blocked with 5% Bovine Serum Albumin (BSA, Sangon, China) 1 h at room temperature; primary antibodies were used: anti-iNOS (1:1000), anti-COX-2 (1:1000), anti-p-p65 (1:1000), anti-p65 (1:1000), anti-E-cadherin (1:1000), anti-β-catenin (1:1000), and anti-GAPDH (1:1000), overnight at 4℃. All antibodies were purchased from Abcam (USA). Then, the corresponding secondary antibodies (1:4000) were added for 1 h at room temperature. Gray scale expression of protein bands was analyzed by Image J analysis software, and relative protein expression was obtained using GAPDH as the corresponding pair internal reference.

Immuno uorescence
Cells cultured on glass slides were xed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 15 min, and then blocked with goat serum for 1 h at room temperature. Primary antibodies: anti-E-cadherin (1:200), anti-β-catenin (1:200), and anti-COX-2 (1:200) were added dropwise and incubated overnight at 4℃. Then, the cells were incubated with corresponding uorescent secondary antibody (1:1000) for 1 h at room temperature in the dark. DAPI was used for nuclear staining for 10min. Immuno uorescence photography was performed using a super-resolution confocal microscope.

Animals
BALB/c male mice (8-12 weeks, 18-22 g) were obtained from the Laboratory Animal Center of Guangdong Medical University. Before the experiment, the mice were kept in a standard laboratory animal facility for 1 week. All mouse experiments were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University.

OVA-induced asthma mouse model and drug treatment
The mice were randomly divided into three groups: control group, OVA group and OVA + schisandrin A (OVA + Sch A) group (n = 7 per group). On days 0, 8, and 15, the OVA and OVA + Sch A groups were sensitized with an intraperitoneal injection (i.p.) of 0.2 ml of sensitizing solution containing 50 µg of ovalbumin (OVA, Sigma, USA), 100 ul of aluminum hydroxide (Thermo Scienti c Pierce, USA) and 100 µl of saline. The control group was replaced with an equal volume of normal saline. Then, using an ultrasonic nebulizer (Omron Automation) on days 16-22, the mice in OVA and OVA + Sch A groups were airway challenged with 3% OVA solution (dissolved in PBS) for 30 min each time. The control group inhaled PBS instead. The OVA + Sch A group was intraperitoneal injection with schisandrin A (40 mg/kg, in 0.5% carboxymethylcellulose sodium aqueous solution) half an hour before nebulization, and the control and OVA groups were orally administered with an equal volume of carboxymethylcellulose sodium solution.

Blood collection and ow cytometry
Anesthetized mice were bled by retro-orbital puncture using heparinized capillary tube. Blood samples were placed in heparin sodium treated EP tubes, centrifuged at 3000 g for 5 min at 4 ℃, and the supernatant was discarded. The cells were re-suspended in RPMI 1640 medium containing 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin, mixed well and cultured for 5 h in the 37 ℃ and 5% CO2 incubator. Cell pellets were treated with 1X red blood cell lysis buffer for 10 min on ice to remove any red blood cells and washed once with PBS. The cells were incubated with Fc-receptor blocking antibody (anti-CD16/32, Biolegend, USA) to reduce non-speci c binding. Subsequently, the cells stained with the FITC anti-mouse CD3, PE anti-mouse CD4, Alexa uor 700 anti-mouse CD8a and Brilliant Violet 650 anti-mouse CD25 antibodies for 20 minutes at 4 ℃ in the dark. Then, the cells were xed and permeabilized, and further stained with PE anti-mouse IL-4 and PerCP/Cyanine5.5 anti-mouse IFN-γ antibodies for 20 minutes at 4 ℃ in the dark. All antibodies were purchased from BioLegend (USA). The percentages of CD3 + CD4 + IFN-γ Th1 and CD3 + CD4 + IL-4 + Th2 cells were detected through ow cytometry.

Collection and processing of bronchoalveolar lavage uid (BALF)
Twenty-four hours after the last OVA inhalation, mice were sacri ced after anesthesia by pentobarbital (50 mg/kg i.p.). BALF was collected three times from each mouse by endotracheal intubation with 0.8 ml of ice-cold PBS. Cell numbers were counted using a hemocytometer. In order to distinguish differential cells, BALF was centrifuged at 800g for 5 min using a centrifuge, and the supernatant was taken, resuspended with 100 ul PBS for precipitation, and dropped onto a glass slide. The cells were xed with xative and stained with Wright-Giemsa staining solution. 200 white blood cells were read under the microscope. The number of various white blood cells was counted and the percentage of various white blood cells were calculated.

Lung tissue staining
Lung tissue samples were made from the left lung which were harvested and xed in 10% neutral formalin, dehydrated, embedded in para n. Lung tissues were cut into 5µm para n sections. The para n embedded tissue sections were baked at 60 ° C for 1 hour, depara nized and rehydrated in xylene and graded ethanol solutions. Hematoxylin and eosin (H & E) staining was used to determine in ammatory cell in ltration and periodic acid Schiff (PAS) was used to evaluate mucus production.

Immunohistochemistry
After baking, depara nization, and hydration, para n sections were microwaved in citrate buffer (pH 6.0) for 10 min, followed by cooling at room temperature. 5% goat serum was added dropwise to block the sections for 20 min at room temperature, and then α-SMA primary antibody (1:400, Abcam, USA) was incubated overnight at 4 ° C. Next, the corresponding secondary antibody was incubated for 1 hour at room temperature after 3 washes with PBS for 5min. The sections were developed with diaminobenzidine solution (DAB) for 7 min, counterstained by hematoxylin for 2min, mounted, and visualized under a microscope.

Data Presentation and Statistical Analysis
All ata was presented as means ± standard deviation (SD) of three independent experiments and analyzed by two-tailed Student t-test or one-way analysis of variance (ANOVA), followed by Tukey tests for multiple comparisons, when appropriate (p < 0.05 was considered as statistically signi cant).

Prediction of action targets of schisandrin A and screening of common targets
The 2D and 3D structures of schisandrin A were obtained by searching PubChem database, as shown in Fig. 1A. In order to ensure the integrity of the target collection, we used Swisstarget Prediction and other databases to predict the potential targets, and combined with relevant literature reports, we obtained 99 targets of Schisandrin A (after deleting the repeat targets). 8162 targets of asthma were obtained from OMIM database and GeneCards database. The targets of Schisandrin A were mapped to the targets of asthma, and the Venn map (Fig. 1B) was drawn. After mapping, 68 intersection targets were obtained. The results show that the compound Schisandrin A may synergistically treat asthma through multiple potential action targets.

Protein interaction network
The PPI network (Fig. 1B) was obtained from STRING database analysis, including 68 nodes and 373 edges. After excluding the genes that are not associated with other genes, the obtained tsv le was imported into Cytoscape for network topology analysis, as shown in Fig. 1C. The analysis results show that the network contains 62 nodes and 373 edges, with clustering coe cient of 0.561 and network centralization of 0.440. Each edge is short, and each gene has an average length of 12. These data indicate that the target genes are all closely related. At the same time, through the above analysis, it is speculated that these cross genes may be potential therapeutic target genes of schisandrin A in the treatment of asthma (Fig. 1C), re ecting the comprehensive regulation characteristics of schisandrin A multi-target. Cytohubba is ranked according to the attributes of nodes in the network, and MCC is a new method, which has better performance in the prediction of key proteins in PPI network. As shown in Fig. 1D, through the MCC algorithm in the cytohubba plug-in, we obtained the top 20 key genes, namely TP53, AKT1, CASP3, TNF, IL6, PTGS2, STAT3, RELA, NFKBIA, IL1B, MMP9, TLR4, BCL2L1, SOD2, EGFR, NOS2, CCND1, IKBKB, MPO and NQO1. The detailed information is shown in Table 1. These genes may play a key role in the mechanism of schisandrin A in the treatment of asthma, which can be considered as the core targets of schisandrin A in the treatment of asthma.

Results of gene enrichment analysis
The 68 potential targets of schisandrin A in the treatment of asthma were entered into DAVID database, and P < 0.05 was used as the screening condition. A total of 239 BP (biological processes) enrichment results were mainly enriched in drug response, camp catabolism, negative regulation of apoptosis, positive regulation of nitric oxide biosynthesis, aging, lipoxygenase pathway, cell response to organic compounds, cGMP catabolism, in ammation and response to lipopolysaccharide. The chord diagram was drawn according to "term" and "gens", as shown in Fig. 2A. The enrichment results of CC (cell composition) and MF (molecular function) were 18 and 51, respectively. CC was mainly enriched in cytoplasm, membrane, organelle membrane, plasma membrane, voltage-gated calcium channel complex, and MF was mainly enriched in 3',5'-cyclic-nucleotide phosphodiesterase activity, 3',5'-cyclic-AMP phosphodiesterase activity, cAMP binding, 3',5'-cyclic-GMP phosphodiesterase activity, drug binding, identical protein binding, iron ion binding, oxidoreductase activity, enzyme binding, heme binding and so on. KEGG pathway is enriched in 81 signal pathways with statistical signi cance, as the top 20 pathways shown in the bubble diagram in Fig. 2C. KEGG is mainly enriched in hepatitis B, toxoplasmosis, apoptosis, legionellosis, tuberculosis, small cell lung cancer, pertussis, Chagas disease (American trypanosomias), TNF signaling pathway, NF-κB signaling pathway, cAMP signaling pathway, Pathways in cancer, Pancreatic cancer, HIF-1 signaling pathway, Leishmaniasis, Serotonergic synapse, MicroRNAs in cancer, Prostate cancer, Morphine addiction, Measles and others. The above analysis results suggests that Schisandrin A may be used to treat asthma through these processes and pathways.

Effects of Schizandrin A on the HDM-induced in ammatory response in 16HBE cells
To investigate the in ammatory response of schisandrin A on HDM-induced 16 HBE epithelial cells, we evaluated the effect of schisandrin A at concentrations lower than 10 µM. As shown in Fig. 3, HDM stimulation induced up-regulation of iNOS and COX-2 expression in 16 HBE epithelial cells. However, pretreatment with schisandrin A attenuated the expression of both in a concentration-dependent manner.
In addition, HDM phosphorylated and activated NF-κB p65 in 16 HBE epithelial cells, and pretreatment with schisandrin A attenuated the phosphorylation of NF-κBp65 in a concentration-dependent manner.

SchizandrinA has a protective effect against HDMinduced epithelial mucosal barrier damage in 16 HBE cells
The integrity of the epithelial barrier, is the rst line of defense against chronic airway in ammation.
Western blotting analysis showed that HDM treatment did not affect the expression of E-cadherin or βcatenin in normal 16 HBE cells (Fig. 4A-C). Immuno uorescence showed that HDM promoted delocalization of E-cadherin and β-catenin in 16 HBE cells and exhibited discontinuous diffusion from adjacent cell boundaries to the cytoplasm, this delocalization was reversed to varying degrees by the intervention of schisandrin A (Fig. 4D-4E).

Effect of schizandrin A on in ammatory responses in LPS-stimulated RAW264.7 macrophages
To investigate the in ammatory response of schisandrin A on LPS-induced RAW246.7 macrophages, we evaluated the effect of schisandrin A at concentrations lower than 10 µM. As shown in Fig. 5, LPS stimulation induced the expression of iNOS and COX-2 in RAW246.7 macrophages. However, pretreatment with schisandrin A attenuated the expression of both in a concentration-dependent manner.
In addition, immuno uorescence results showed that COX-2 uorescence intensity in RAW264.7 macrophages was upregulated by LPS, while pretreatment with schisandrin A effectively inhibited this effect.
3.7 Effects of schisandrin A on OVA-exposed asthmatic mice.
After 22 days of OVA sensitization and challenge, we assessed the severity of airway in ammation in the lungs (Fig. 6A). OVA inhalation signi cantly increased the number and percent of eosinophils in BALF as compared to the healthy control, and schisandrin A treatment effectively reduced the increase in in ammatory cells (Fig. 6B). At the same time, schisandrin A could obviously inhibit the increase of Th2 cell ratio induced by OVA (Fig. 6C). In addition, OVA induction occurred corresponding pathological changes, including extensive in ammatory cell in ltration in the airways, excessive mucus secretion, and airway remodeling. The level of in ltration of in ammatory cells was assessed by HE staining of the lung, and the level of airway mucus secretion was measured by PAS staining. Compared with the control group, OVA treatment signi cantly increased in ammatory cell in ltration and mucus secretion, while schisandrin A effectively reduced in ammatory cell in ltration and the overproduction of mucus (Fig. 6D). Similarly, the expression of α-SMA, a major protein of airway remodeling in asthma, was analyzed using immunohistochemistry. We observed airway thickening and increased expression of α-SMA in OVA-treated airways. However, schisandrin A signi cantly alleviated these pathological changes ( Fig. 6D).

Discussion
Fructus Schisandrae Chinensis (FSC) is the dried and ripe fruit of Schisandra chinensis, which is a traditional Chinese herb originally recorded in Shen Nong Ben Cao Jing, and has antibacterial, liver protection, anti-tumor, antioxidant, sedative and hypnotic effects [19]. Schisandrin A belongs to the lignans class, the main characteristic active component of FSC, which has therapeutic effects on in ammatory diseases such as osteoarthritis, mastitis, and neuronin ammation [14,20,21]. The mechanism of action of schisandrin A has been found to be associated with inhibition of the NF-κB signaling pathway and inhibition of the expression levels of iNOS and COX-2 [14,15,20]. The pathogenesis of asthma is closely related to exposure to allergens, so chronic in ammation is aggravated once the airways are exposed to allergens [22]. Airway epithelial shedding and mucin production are increased in asthmatic patients, and exposure to allergens leads to increased NF-κB activation, further exacerbating epithelial barrier dysfunction and increasing the expression of proin ammatory factors [23,24]. In our study, we found that schisandrin A has a protective effect on dust mite-induced 16 HBE epithelial cell barrier damage, in addition to an inhibitory effect on the phosphorylation of NF-κB p65.
Prostaglandin-endoperoxidase (PTGS) is a key enzyme in the prostaglandin biosynthetic pathway, of which, PTGS2 is the main isoenzyme responsible for the production of in ammatory prostaglandins and plays an important role in the in ammatory response [25,26]. The iNOS and COX-2 induce the generation of a large number of proin ammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) during in ammation [27]. In the pathogenesis of asthma, the expression of COX-2 and PGE2 was upregulated, and at the same time, COX-2 has a regulatory effect on the differentiation of T lymphocytes [28][29][30]. In addition, it was found that NF-κB-induced iNOS and COX-2 are important mediators in the development of pulmonary in ammation [7,31,32]. In patients with asthma, increased production of macrophage-derived nitric oxide re ects the severity of asthma, and increased iNOS derived NO may contribute to the imbalance of oxidant and antioxidant pathways [33]. It has been shown that LPS induction can lead to activation of Th2 responses and asthma [34]. In our study, schisandrin A exerted an excellent effect by inhibiting the production of iNOS and COX-2 in 16 HBE cells and activated murine macrophages.
It is well-known that Th1/Th2 imbalance is one of the key factors contributing to the pathogenesis and severity of asthma [35,36]. The most common type of asthma, allergic asthma, is mediated by Th2 cells characterized by the accumulation of eosinophils, bronchial hyperreactivity, and airway remodeling [37]. Allergic asthma patients usually present with clinical manifestations such as cough, wheezing and dyspnea [38,39]. Our in vivo study con rmed that schisandrin A signi cantly reduced the OVA-induced increase of in ammatory cells, such as the proportion of Th2 cells, and the number and proportion of eosinophils, as well as signi cantly alleviated in ammatory cell in ltration in lung tissue. Hypersecretion of mucus in the airways is another important feature of asthma. In severe asthma, goblet cells are signi cantly increased, and metaplasia and hyperplasia occur, which leads to mucin overproduction in the airways [40,41]. In this study, schisandrin A signi cantly alleviated OVA-induced mucus hypersecretion and goblet cell hyperplasia. In addition, studies have shown that α-SMA is associated with airway remodeling and severity in asthma [42,43]. Schisandrin A was also effective in reducing α-SMA production. According to these results, we believe that schisandrin A has a therapeutic effect on asthma.

Conclusion
In this study, we proved that schisandrin A signi cantly inhibited the in ammatory response of 16 HBE epithelial cells induced by HDM and RAW264.7 cells induced by LPS, respectively, and signi cantly reduced OVA-induced airway in ammation and mucus hypersecretion of airway goblet cells in mice. In conclusion, the results suggested that Schisandrin A can reduce asthma symptoms by inhibiting in ammation production, including lowering the Th2 cell ratio, which provides a basis for further understanding of the treatment of asthma with Schisandrin A.    Effects of Schizandrin A on the HDM-induced in ammatory response in 16HBE epithelial cells. (Fig. A-E) 16 HBE cells were pretreated with schisandrin A (1, 5, 10 μM) for 0.5 h and then treated with 50 μg/mL HDM for 24 h. Protein expression of iNOS, (COX) -2, p-p65, and p65 were determined by western blotting, and quanti ed using Image J software. All the experiments were independently repeated at least three times. (*p<0.05, **p <0.005, ***p<0.0005, ****p<0.0001, one-way ANOVA t-test) . Immuno uorescence showed that the distribution of E-cadherin and β-catenin protein was diffusely distributed from the membrane to the interstitial space (original magni cation x200; scale bar = 100 μm;

Abbreviations
representative images from three experiments). All the experiments were independently repeated at least three times. (ns p>0.05, one-way ANOVA t-test) .