Exploring Action Mechanism of Sanzi Yangqin Decoction in Treating Bronchial Asth

Yajie HUANG, Dayan CHEN, Wanhong GU, Xinhuan MA

Gansu Provincial Hospital of TCM, Lanzhou 730050, China

Abstract [Objectives]To explore the molecular mechanism of Sanzi Yangqin Decoction in the treatment of bronchial asthma based on network pharmacology and molecular docking.[Methods]The components of Fructus Perillae, Semen Raphani and Semen Sinapis three traditional Chinese medicine-related components and targets of Feiduqing Sanzi Yangqin Decoction were obtained using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the targets of bronchial asthma were obtained using Genecards and OMIM databases.Sanzi Yangqin Decoction "drug-active ingredient-target-disease" network was established with the aid of Cytoscape 3.7.2 software and network topology analysis was carried out.The gene ontology(GO)function enrichment analysis and the KEGG pathway enrichment analysis were performed by DAVID.The top 3 components and targets in the network topology analysis were respectively molecularly docked.[Results]Through network analysis, 4 key active components were obtained, mainly luteolin, arachidonic acid, β-carotene, etc.; 5 key targets, mainly NCOA2, PGR, PTGS2, etc. Through GO analysis, 523 items(P<0.05)were obtained, including 396 items in biological process(BP), 53 items in cell composition(CC), and 74 items in molecular function(MF).KEGG analysis generated 144 signal pathways(P<0.05), involving PI3K-Akt signal pathway, human cytomegalovirus infection, Kaposi’s sarcoma-associated herpes virus infection, proteoglycans in cancer, prostate cancer, etc. The results of molecular docking showed that core active compounds such as luteolin and β-carotene in Sanzi Yangqin Decoction had good affinity with NCOA2, PGR, PTGS2 and other target genes, which were similar to clinically recommended chemical drugs.[Conclusions]The active compounds luteolin and carotene in Sanzi Yangqin Decoction may mainly bind to targets such as NCOA2, PGR, PTGS2, and regulate multiple signaling pathways such as PI3K-Akt to play a role in treating bronchial asthma.It is intended to provide new ideas for the clinical application and research of Sanzi Yangqin Decoction.

Key words Sanzi Yangqin Decoction, Bronchial asthma, Network pharmacology, Molecular docking, Action mechanism

1 Introduction

Bronchial asthma is a common clinical disease.Asthma belongs to the category of "wheezing disease" in traditional Chinese medicine, and wind phlegm obstruction of the lungs is its pathogenesis.Sanzi Yangqin Decoction, originated fromHanShiYiTong, has the effects of dispelling wind and phlegm, lowering breath and relieving asthma.Pharmacological study[1]has shown that Sanzi Yangqin Decoction has anti-inflammatory, anti-cough, anti-asthmatic, and phlegm dispelling pharmacological effects, and is a common prescription for the treatment of bronchial asthma.Network pharmacology is a new discipline developed in recent years.It has been widely used in the screening of active components of traditional Chinese medicine, the prediction of targets, the action mechanism of traditional Chinese medicine and the speculation of the pathogenesis mechanism of disease[2-4].Molecular docking technology is applied to theoretically verify the binding mode and affinity between the receptor and the drug molecule[5-8].On the basis of the network pharmacology and molecular docking technology, we explored the potential therapeutic effects of Sanzi Yangqin Decoction in the treatment of bronchial asthma, to provided new ideas for the clinical application and research of Sanzi Yangqin Decoction.

2 Materials and methods

2.1 Collection, screening and component target prediction of active components of Sanzi Yangqin DecoctionThrough the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)(http://tcmspw.com/tcmsp.php), we searched the chemical composition of each Chinese medicine with the key words of " Fructus Perillae", " Semen Raphani" and "Semen Sinapis", and usedOB≥30%,DL≥0.18 as a screening condition, we obtained the active components[5-8].At the same time, we obtained the target proteins corresponding to the active components under the Targets Information item of the TCMSP database and converted them into the gene name with the help of the Uniprot(https://www.uniprot.org/)database.

2.2 Disease target predictionThrough GeneCards database(http://www.genecards.org/)and OMIM database(https://omim.org/), we obtained the target genes of bronchial asthma.

2.3 Network constructionUsing R software, we obtained common targets of diseases and components.We imported the common targets into the STRING online software, selected the species as homosapiens, set the highest confidence to 0.9, removed the free nodes, and obtained the PPI network diagram.Using Cytoscape 3.7.2 software, we performed topological analysis, determined the key targets with the degree value of the species greater than the median, and explored the potential material basis of Sanzi Yangqin Decoction and the key targets for the treatment of bronchial asthma and constructed the Sanzi Yangqin Decoction drug-component-target-disease network.

2.4 GO and KEGG analysisUse David[9](https://david.ncifcrf.gov/), we performed GO and KEGG analysis on common targets in the network.In the GO analysis, we selected three modules, namely, biological process(BP), cellular component(CC)and molecular function(MF)to plot a bar graph, withP<0.05 as the critical function and pathway.Through the R software, we installed the corresponding installation package to make the biological pathway diagram into a bubble diagram.

2.5 Component-target molecular dockingWe downloaded the 3D structures of the key targets and compounds screened from the RSCB PDB database(https://www.rcsb.org/)and the Pub Chem database(https://pubchem.ncbi.nlm.nih.gov/), respectively, and converted them to Pdb format.Then, using AutoDock software, we performed operations such as hydrogenation and electron addition.For the molecular docking, we set the protein structure to be a rigid macromolecule, the molecule was flexible, and the algorithm was Genetic Algorithm.Using Pymol, we ploted the docking results.With reference to the literature, we took components with binding energy ≤-5.0 kJ/mol[5-8]as the screening basis for the active components of Sanzi Yangqin Decoction for the treatment of bronchial asthma.

3 Results and analysis

3.1 Screening of active components of Sanzi Yangqin Soup

Through searching the TCMSP database, combined withOB≥30% andDL≥0.18, we obtained a total of 22 active compounds in Fructus Perillae, Semen Raphani, and Semen Sinapis, of which 16 were from Fructus Perillae, 3 were from Semen Raphani, and 3 from Semen Sinapis(Table 1).

3.2 Prediction results of the active components of Sanzi Yangqin DecoctionUsing Prel processing of the targets of Fructus Perillae, Semen Raphani, and Semen Sinapis retrieved from TCMSP database, we obtained 201 active targets of Sanzi Yangqin Decoction, in which 179 from Fructus Perillae, 18 from Semen Sinapis, and 4 from Semen Raphani.

3.3 Prediction results of disease targetsThrough retrieving in the GeneCards and OMIM databases, we obtained 2 210 targets related to bronchial asthma.

3.4 Results of network constructionWith the aid of R software, we constructed a Venn diagram(Fig.1)of the intersection of the "component-target" network and the "disease target".There were a total of 93 intersection targets, which are candidate targets for Sanzi Yangqin Decoction for the treatment of bronchial asthma.Among them, NCOA2, ADRA1A, ADRA1B, AR, CHRM1, CHRM2, CHRM3, OPRM1, PTGS1, PTGS2 were common target genes of Fructus Perillae and Semen Sinapis.NCOA2, NR3C2, and PGR were common target genes of Fructus Perillae and Semen Raphani.With the aid of Cytoscape 3.7.2 software, we constructed Sanzi Yangqin Decoction drug-component-target-disease network, which includes 106 nodes and 249 edges, as shown in Fig.2.In network topology analysis, there was one medicinal material(Fructus Perillae)with network median value higher than median(median value of Chinese medicine degree=6, median value of component degree=19, median value of target value=6), 4 components and 5 targets.The top 3 compounds were MOL000006-luteolin, MOL001439-arachidonic acid, and MOL002773-β-carotene, with degree values of 44, 25, and 22, all from Fructus Perillae.The top 3 targets were NCOA2, PGR, and PTGS2, with degree values of 12, 11, and 10 respectively, which are key nodes in the network(Table 2).

Fig.1 Venn diagram

Fig.2 Drug-active component-target-disease network of Sanzi Yangqin Decoction

Table 1 Basic information of active components of Sanzi Yangqin Decoction

3.5 Target pathway analysisUsing the DAVID online tool, we performed GO and KEGG analysis on the 93 key targets of Sanzi Yangqin Decoction in the treatment of bronchial asthma(P<0.05).Through GO analysis, we obtained 523 items, inclu-ding 396 biological process(BP), 53 cell components(CC), and 74 molecular function(MF), accounting for 76%, 10%, and 14%, respectively.In biological processes, negative regulation of apoptotic process, positive regulation of nitricoxide biosynthetic process, and positive regulation of vasoconstriction ranked high.Among cell components, membrane rafts, extracellular space, cytosol,etc.ranked first; in molecular functions, it mainly included enzyme binding and identicalprotein binding, and protein heterodimerization activity,etc.We selected the top 20 items and plotted a bar graph(Fig.3).

Note: A is biolohgical process(BP), B is cell component(CC), and C is molecular function(MF).

KEGG pathway analysis results show that they were enriched in 144 signal pathways, and the high-level bubble chart and histogram drawn with the top 20 signal pathways(P<0.05)are shown in Fig.4.The results showed that the KEGG pathway of Sanzi Yangqin Decoction in the treatment of bronchial asthma is associated with PI3K-Akt signaling pathway, human cytomegalovirus infection, and Kaposi sarcoma-associated herpes virus infection, proteoglycans in cancer, and prostate cancer, among which the PI3K-Akt signaling pathway involves CHRM1, CHRM2, RELA, CCND1, MAPK1 andetc.Human cytomegalovirus infection involves genes such as PTGS2, RELA, CCND1, MAPK1, CASP3; Kaposi’s sarcoma-associated herpes virus infection involved genes such as PTGS2, RELA, CCND1, MAPK1, and CASP3; proteoglycans in cancer involve PLAU, CCND1, MAPK1, CASP3, COL1A2 andetc.Prostate cancer involved PLAU, RELA, CCND1, MAPK1, EGF(Fig.4-5).These pathways were closely related to the formation mechanism of bronchial asthma.Studies have shown that these pathways directly or indirectly mediate and participate in the occurrence and development of bronchial asthma.The above results indicate that the targets of the main chemical components of Sanzi Yangqin Decoction involved diversified biological processes and were distributed in different metabolic pathways, reflecting its multi-component, multi-target, and multi-channel therapeutic characteristics.

Fig.4 KEGG enrichment analysis of 20 pathways(bubble chart and histogram)

The three core active compounds, luteolin, arachidonic acid, and β-carotene, were molecularly docked with three key targets NCOA2, PGR, and PTGS2, respectively.Using binding energy ≤-5.0 kJ/mol as the screening criterion, it can be seen that the binding energy of luteolin and β-carotene to key targets in Sanzi Yangqin Decoction were all lower than-5.0 kJ/mol, the conformational energy is low, and the structure is stable.The binding activity is high(Table 2 and Fig.6).

Fig.5 PI3K-Akt signal pathway

Fig.6 Molecular docking patterns of luteolin, β-carotene, and arachidonic acid with NCOA2, PGR, and PTGS2

Table 2 Binding energy of luteolin, β-carotene, arachidonic acid with NCOA2, PGR, PTGS2

4 Discussion

4.1 Active components of Sanzi Yangqin Decoction in the treatment of bronchial asthmaIn this study, a total of 22 active components in Sanzi Yangqin Decoction that can be orally absorbed were screened, of which 16 were from Fructus Perillae, 3 were from Semen Raphani, and 3 were from Semen Sinapis.Among compounds of Fructus Perillae, flavonoids include luteolin, fatty acid compounds include arachidonic acid, cis-11-eicosenoic acid, and eicosatrienoic acid, and sterols include β-sitosterol and stigmasterol, spinsterols, and aliphatic compounds include 2,6,10,14,18-pentamethyl-2,6,10,14,18-pentene, 24-methylisoamylphenol andetc.Studies have shown that the volatile oil components, flavonoids and phenolic acid compounds ofPerillafrutescensleaves are the main material basis for its anti-inflammatory activity, and luteolin has been identified as the active component.The results of the study on the dose-effect relationship between Sanzi Yangqin Decoction and anti-asthmatic compatibility[10]show that Fructus Perillae is the main traditional Chinese medicine for anti-asthmatic in the prescription, and the anti-asthmatic effect is related to the dosage.Another study has shown that[11], Sanzi Yangqin Decoction has better antiasthmatic effects than any single medicine, Fructus Perillae has stronger antiasthmatic effects than Semen Sinapis, and Semen Sinapis has stronger expectorant effects than Fructus Perillae and Semen Raphani has stronger antitussive effect.These are consistent with the results screened in this study and consistent with the traditional efficacy of Sanzi Yangqin Decoction.They are the main pharmacodynamic material basis for the traditional efficacy of Sanzi Yangqin Decoction and also the main way and important basis for screening the quality markers of Sanzi Yangqin Decoction.

4.2 Potential targets of Sanzi Yangqin Decoction in the treatment of bronchial asthmaThis study shows that nuclear receptor coactivator 2(NCOA2), PGR, and PTGS2 are the top 3 targets in the Sanzi Yangqin Decoction for the treatment of bronchial asthma.Among them, NCOA2 and PTGS2 are the common targets of Fructus Perillae and Semen Sinapis; NCOA2 and PGR are common target genes of Fructus Perillae and Semen Raphani.NCOA2 is the common target of three traditional Chinese medicines, and PTGS2 is the common target of luteolin, arachidonic acid, and β-carotene.These indicate that the different components of Sanzi Yangqin Decoction have common or similar targets, which is consistent with the idea of compatibility of Chinese medicine and these targets are the key targets of Sanzi Yangqin Decoction in the treatment of bronchial asthma.NCOA2 is a member of the p160 steroid receptor co-activator(SRC)family and plays an important role in many physiological and pathological processes, including development, endocrine regulation, and tumorigenesis.PGR is the progesterone receptor.Clinical studies have found that elevated estrogen and reduced progesterone in menstrual asthma patients can enhance airway hyperresponsiveness, which may be the cause of menstrual asthma[12].Progesterone and estrogen can reduce the expression of let-7fmicroRNA and increase the expression of IL-23/IL-23 signal and IL-17A in patients with severe asthma.It may also be the reason why there are more women with severe asthma than men[13].Prostaglandin-endoperoxide synthase(PTGS), also known as cyclooxygenase, has two isoenzymes, constitutive PTGS1(also known as COX1)and inducible PTGS2(also known as COX2), are different in the regulation of expression and tissue distribution.COX-2 participates in the metabolism of arachidonic acid into various PGI products and can induce acute asthma attacks[14].COX-2 can also induce the secretion of IL-18, prostaglandin and other inflammatory mediators, aggravate the inflammatory response, induce asthma or make asthma worse.Inflammatory cytokines, growth factors,etc.can induce the expression of COX-2[15].

4.3 Mechanism of Sanzi Yangqin Decoction in the treatment of bronchial asthmaAsthma is related to inflammation and cytokines.Studies have found that luteolin can reduce the production of PGE2 by inhibiting the expression of COX-2[16]and can significantly reduce the inflammatory response of activated macrophages and down-regulate the expression of inflammatory factors such as IL-6, IL-1β and TNF-α.Luteolin can reduce the production of reactive oxygen species and reactive nitrogen, inhibit the expression of inflammatory cells factors[17-18]and inflammatory mediators, thereby alleviating the inflammatory response.In the pathophysiological process of asthma, studies have shown that PI3K plays an important role by affecting the recruitment, activation, apoptosis and cytokine production of inflammatory cells.In the inflammatory process of asthma, T cells play an important role.The pathogenesis of asthma is closely related to the imbalance in the number, activation and function of Th1/Th2 cells.According to some study, it can be inferred that the PI3K signaling pathway is involved in the pathogenesis of asthma by participating in the imbalance of Th1/Th2[19].The results of this study show that the most important pathway involved in the treatment of bronchial asthma by Sanzi Yangqin Decoction is PI3K-Akt signaling pathway, which is consistent with the literature report.

4.4 Molecular dockingFrom the results of network pharmacology analysis and molecular docking, it can be known that the affinity with key targets NCOA2, PGR, and PTGS2.The levels of luteolin and β-carotene in Sanzi Yangqin Decoction are lower than those of salbutamol and aminophylline, which are commonly used in clinical medicine, indicating that the structure is stable and the binding activity is higher.They have a good effect on the treatment of bronchial asthma and may be the potential material basis.In summary, we found that luteolin and β-carotene, the core active components in Sanzi Yangqin Decoction, act on multiple targets such as NCOA2, PGR, PTGS2, and regulate multiple signaling pathways such as PI3K-Akt, thereby playing the role of treating bronchial asthma.We are intended to provide ideas and scientific basis for further research on the pharmacological and pharmacological mechanism of Sanzi Yangqin Decoction.