Quantitative and bioinformatics integrated strategy to investigate the pharmacological mechanism of Ermiao Wan against eczema

Background Ermiao Wan (EMW) is used to treat eczema in China. However, its underlying pharmacological mechanisms against eczema remain unclear. Methods In this study, the components of EMW were quantitatively analyzed using HPLC. The role of the components, targets, and signaling pathways were predicted by network pharmacology. Moreover, molecular docking was used to verify the binding forces of the components with the target proteins. Results The results showed that the established HPLC method is simple and reliable, and can be used for the simultaneous determination of seven components in EMW. Moreover, 57 primary causal targets of EMW against eczema were identied. Among them, 10 hub targets were identied, including EGFR, AKT1, STAT3, MMP9, ICAM1, MAPK8, JUN, MAPK1, and VCAM1. The potential signaling pathways involved in the effect of EMW against eczema were identied, including ErbB, estrogen, and Epstein-Barr virus infection. Furthermore, palmatine, chlorogenic acid, and jatrorrhizine from EMW were shown to bind to the identied targets. Accordingly, EGFR, AKT1, and PTGS2 had good binding forces with EMW components. Our study revealed a possible pharmacological mechanism of EMW in treating eczema. This simple and effective method can help increase our understanding of the mechanisms of Chinese herbal formulations and further promote their research and development. berberine, 49.68 ± 1.38 mg/mL; and atractydin, 38.43 ± 0.92 mg/mL. The compounds present at high concentrations in EMW originated mainly from CP, including chlorogenic acid, phellodendrine, magnoorine, jateorhizine, palmatine, and berberine, whereas atractydin originated from RA. Alkaloids constituted the highest composition, followed by organic acids. The seven compounds represented the major bioactive components of EMW, and were therefore used for subsequent network pharmacology research in this study.


Introduction
Eczema is a common chronic, in ammatory skin disorder characterized by severe itching, with a complex pathogenesis and recurrent attacks [1]. It seriously affects the quality of life and economic outcomes of affected individuals, especially in those with moderate to severe eczema. In particular, with the outbreak of COVID-19, overzealous hand hygiene has caused an increased incidence of hand eczema among the general population [2]. Approximately 200 million people worldwide are affected by eczema, and the incidence is increasing, which can lead to tremendous medical and nancial pressure [3]. The relapsing and remitting nature of eczema can make it di cult to control are-ups. At present, although drugs for the treatment of eczema are effective in the short term, this disorder can easily occur repeatedly, accompanied by adverse reactions such as skin atrophy, hormone-dependent dermatitis, dry mouth, and dizziness [4]. Thus, the research and development of drugs that are effective against eczema in the long term has gained interest.
Although various treatments have achieved sustained control of eczema, their relative bene t remains unclear owing to the limited number of trials directly comparing the treatments. Traditional Chinese medicine (TCM) has the advantages of high safety and less side effects. In China, the TCM Ermiao Wan (EMW) is commonly used for the clinical treatment of eczema and included in the 2020 edition of the Chinese Pharmacopeia. The clinical application of EMW for treating eczema rst appeared in the book Danxi Xinfa, an ancient clinical TCM book from the Ming Dynasty of China. Owing to its e cacy in eliminating heat and dampness, EMW has been used to treat red and swollen skin, fever, and itching, which are the symptoms of eczema [5].
EMW is composed of equal amounts of Rhizoma Atractylodis (RA) and Cortex Phellodendri (CP). The alkaloids in EMW have been reported to have good biological activity [6,7], and the content of compounds in EMW is also an important factor affecting its e cacy. We believe that the main components of TCM should have high content and biological activity at the same time for its effective application. Although some components of EMW have been separated and shown to have pharmacological effects, the combined pharmacological action of the main compounds of EMW may be the main way to exert its e cacy [8]. However, the main components and pharmacological mechanisms of EMW in the treatment of eczema remain unclear. Moreover, owing to the multi-component and multi-target characteristics of TCM, it is di cult to clarify the action mechanism of EMW using conventional methods.
Network pharmacology is a novel discipline that integrates biology, pharmacology, and informatics [9]. According to this concept, multiple node targets of drugs in interrelated systems, rather than individual nodes, have been analyzed from a holistic point of view. The idea of network pharmacology is consistent with the action mechanism of TCM, which emphasizes synergistic effects and interconnection [10]. TCM formulations are composed of several herbs and compounds that exert synergistic effects by affecting multiple genes, proteins, and pathways [11]. Network pharmacology can elucidate the interaction between multiple compounds and disease targets, allowing the exploration of the actions of TCM. At present, network pharmacology has been widely used in the prediction of potential active components, targets, and action mechanism of TCM [12,13].
In this study, we aimed to elucidate the mechanism of action of EMW in the treatment of eczema using a combined method of quanti cation of components and network pharmacology. The method established in this study can help increase our understanding of the mechanisms of Chinese herbal formulations and promote their further research and development. Figure 1 shows the work ow of the study.

Materials and reagents
EMW was purchased from Guangdong Traditional Chinese Medicine Co., Ltd. (Guangdong, China). Reference standards, including berberine, palmatine, jateorhizine, phellodendrine, magno orine, atractylodin, and chlorogenic acid, were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile was of high-performance liquid chromatography (HPLC) grade and purchased from Merck (Darmstadt, Germany). The water used was distilled water, and other reagents were of analytical grade.

Quanti cation of EMW components
EMW was ground to powder and weighed (1.0 g). Next, 50 mL of ethanol-water (1:1, v/v) was added to the powder and stirred. The samples were then placed in an ultrasound machine for 30 min for extraction, followed by centrifugation (2500r/min,15min.) to obtain the supernatants. Subsequently, 5 μL of the supernatant was injected into an HPLC system to determine the components of EMW.
The method was validated as follows. (1) The reference substances of chlorogenic acid, phellodendrine, magno orine, jatrorrhizine, palmatine, berberine, and atractylodin were precisely weighed, and 50% ethanol was added to prepare a series of concentrations of the reference solution. A standard curve was drawn according to the chromatographic conditions with the peak area (Y) and reference substance concentration (X), and a linear regression equation was obtained. (2) The same sample solution was injected continuously six times according to the chromatographic conditions, the chromatographic peak area was recorded, and the precision of the instrument was veri ed. (3) Six samples of test solution were prepared and used for the chromatographic separation, the content of the components was calculated, and repeatable experimental results were obtained. (4) The content of the components in the same solution was determined within 24 h to obtain experimental stability data. (5) Recovery was tested by comparing the peak responses of spiked analytes at pre-extraction with those at post-extraction.

Network pharmacology studies
All protein targets of the seven components of EMW were obtained from the TCMSP database (http://tcmspw.com/tcmsp.php). Eczema-related targets were obtained by using 'eczema' as the keyword in the DisGeNET (https://www.disgenet.org/) database. A Wayne diagram of EMW active ingredient targets and eczema targets were drawn using the Venny 2.1 database (https://bioinfogp.cnb.csic.es/ tools/venny /index. html). The targets at the intersection were considered as the key targets of EMW in the treatment of eczema.

Protein-protein interaction network
The key targets were introduced into the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network model, with the protein species set as "Homo sapiens". The results were then imported into the Cytoscape 3.7.2 software to obtain the protein-protein interaction network.

Compound-target network
The active components and key targets were imported into the Cytoscape3.7.2 software to build a compound-target network. Simultaneously, visualization and network topology analyses were carried out to determine the critical degree of EMW targets.

Gene ontology (GO) and pathway enrichment analysis
To further elucidate the function of the screened target protein genes and their role in signaling pathways, the top 10 targets were introduced into the DAVID 6.8 database (https://david.ncifcrf.gov/). A list of target gene names was entered into the database, with the species set to "Homo sapiens". The names of all target genes were corrected to their o cial names (o cial gene symbol). Targets were searched and transformed in the above database, and the threshold was set to less than 0.5. Next, GO biological process enrichment analysis and KEGG signaling pathway enrichment analysis were carried out, and the top 10 items were determined. The R-language packages 'ClusterPro ler', 'ReactomePA', 'org.Hs.eg.Db', and 'GOplot' were used for analysis and visualization of the top 10 hub targets and top 10 terms. GO data were analyzed using 'org.Hs.eg.Db', with cutoffs for enrichment being P<0.05 and q<0.05. The outputs were bubble charts and Circoscircle charts. The 'pathview' package was used to create pathway diagrams for correlative targets of the enriched KEGG pathways; this step allowed for further analysis of the results [14,15].

Molecular docking analysis
Molecular docking analysis was conducted to examine the binding of the seven components of EMW to the top 10 targets.
Unfortunately, as there are no ligands in the protein structure of the 10th target, only nine targets were docked. The molecular structure of the seven components was drawn using ChemDraw and imported into Sybyl X2.1 for transformation of the three-dimensional (3D) structure, optimization of minimizing energy. The 3D structures of EGFR (ID:5GTY), AKT1 (ID:6HHH), PTGS2 (ID:5F1A), STAT3 (ID:6SMB), MMP9 (ID:2OW0), ICAM1 (ID:3E2M), MAPK8 (ID:3PZE), JUN (ID:2NO3), and MAPK1 (ID:5NHF) were downloaded from the PDB database (https://www.rcsb.org/). After removing the water molecule residues, hydrogenation, and side chains, the Sur ex-Dock program was used to dock and score the core compounds using the Sybyl X2.1 docking software. The higher the score, the more stable the binding between the ligand and the receptor.

Fingerprint and quanti cation of major components in EMW
HPLC ngerprinting was performed to identify the main chemical compounds in EMW. Seven chemical constituents of EMW were identi ed according to the spectrograms and retention times of the standard substances ( Fig.2A). Fingerprints of EMW were identi ed from six batches, with satisfactory similarity (Fig.2B), which suggested product stability. The seven components of EMW were measured quantitatively using HPLC, and their chemical structures are shown in Fig.2C.

PPI network
The chemical components and eczema-related targets were screened according to a previous study [16]. A total of 788 and 295 targets were acquired for eczema and EMW, respectively, and 57 common targets were screened (P < 0.001, according to Fisher's exact test) and identi ed as the potential targets of EMW in treating eczema (Fig.3A). A PPI network was constructed to scienti cally summarize the interfaces of EMW targets associated with eczema treatment. The network showed 57 possible protein target nodes connected by 318 edges, with an average node degree of 11.2. and an average local clustering coe cient of 0.548. The P-value of PPI enrichment was < 1.0 e −16 (Fig.3B). These results suggest that the key proteins are closely related to each other. The top 10 predicted hub genes included EGFR, AKT1, PTGS2, STAT3, MMP9, ICAM1, MAPK8, JUN, MAPK1, and VCAM1. As shown in Fig.3B, the darker the color of the node, the larger the size, which indicates that the protein may be more crucial for the treatment of eczema.

Compound-target network
Based on the results of target interaction analysis, a compound-target network was built as described in Fig.4. The network showed that most in ammation-related targets and immune-related targets were interrelated, indicating that the action mechanism of EMW was associated with in ammation and immune mechanisms. Furthermore, in terms of the compound structures, alkaloids corresponded to more targets than other compounds.
3.5. GO and pathway enrichment analysis GO analysis results revealed 76 terms of biological processes, and the core terms of EMW targets against eczema were mainly involved in the negative regulation of apoptotic process, positive regulation of vasoconstriction, positive regulation of nitric oxide biosynthetic process, positive regulation of smooth muscle cell proliferation, cellular response to mechanical stimulus, aging, response to drug, positive regulation of transcription from RNA polymerase II promoter, signal transduction, and regulation of sequence-speci c DNA binding transcription factor activity ( Fig.5A and 5

Compound-target-pathway network
On the basis of the compound-target network and pathway enrichment analysis results, we created a compound-targetpathway network consisting of the top 10 KEGG pathways (Fig.6A). The network showed that EMW and eczema shared 10 KEGG pathways, representing their combined anti-eczema targets. The ErbB signaling pathway, estrogen signaling pathway, and Epstein-Barr virus infection were the three pathways connected with the key targets and associated with the effect of EMW on eczema. Hence, their network relationships were isolated and further analyzed through annotation of the KEGG pathway. The network highlighted the hub genes EGFR, AKT1, MMP9, ICAM1, MAPK8, JUN, and MAPK1 (Fig.6B).

Molecular Docking
EMW components were chosen for molecular docking analysis based on their high content and network pharmacology results. Moreover, the importance of the top 10 hub genes in eczema treatment was validated through network pharmacology analysis. The docking results showed that EGFR, AKT1, and PTGS2 bound well with all components. Palmatine, phellodendrine, chlorogenic acid, and jatrorrhizine bound well with all docking targets ( Fig.7 and Table.5). The Sur ex-Dock scores (total scores) represent binding a nities. Generally, a docking score greater than 4.25 indicates certain binding activity, whereas a docking score greater than 7.00 indicates strong binding activity [17]. The comparable binding score between the compounds may be due to their similar molecular structures, which is consistent with a previous nding [18]. However, among the seven chemical components of EMW, atractylodin only exhibited 3 (33.33%) binding e ciencies with the docking targets. Among the nine targets, ICAM1 only exhibited 2 (28.57%) binding e ciencies with the components of EMW. These ndings imply that not every component has a good binding e ciency with every target, but the interaction between each component and each target is consistent with the multi-component and multi-target characteristics of TCM [19]. The 3D mode and schematic 2D representation of chlorogenic acid in the active site of MAPK8 are shown in Fig.8A.

Discussion
EMW is often used to treat eczema in clinical setting, but its speci c mechanism is unclear. Thus, in this study, we aimed to elucidate the mechanism of EMW in the treatment of eczema. The ngerprints of EMW were obtained by HPLC, and seven components with high content and excellent activity were quantitatively analyzed. Owing to its simplicity and reliability, the established method can provide a reference for evaluating the quality of EMW. Subsequently, compounds with high content in EMW were used for predicting eczema-related targets by network pharmacology. The results revealed the complex mechanism of EMW in treating eczema, which involves the interrelation of multiple components, protein targets, and pathways. Further, molecular docking analysis was performed to verify the binding force of the drug with the target proteins.
The pathogenesis of eczema is determined by many factors, such as poverty, environment, infection, and immunity. In fact, recent research has revealed a close relationship between eczema and in ammatory factors. EGFR, TH-17, IL-17, TNF-α, MMP, and other in ammatory factors interact with each other, forming a large and complex network [20,21]. Thus, owing to the complex pathogenesis of eczema, it is di cult for single-target drugs to achieve a good curative effect. Therefore, multi-component and multi-target drugs have become a trend for the treatment of eczema. Pharmacological studies have shown that EMW has anti-in ammatory and anti-allergic effects [22,23]. EMW can relieve the symptoms of eczema in rats by stimulating the inactivation of NF-κB and mitogen-activated protein kinases (MAPKs) in cells and reducing the level of IκBα [24]. Moreover, EMW signi cantly inhibits NO and prostaglandin E 2 production as well as inducible NO synthase and COX-2 expression in cells to regulate immunity and in ammation [25]. The immunomodulatory, anti-in ammatory, and anti-allergic effects of EMW are similar to the current treatment strategies for eczema.
Some evidence indicates that chlorogenic acid can inhibit the levels of pro-in ammatory cytokines (TNF-α and IL-2) and elevate the expression levels of anti-in ammatory cytokines (IL-4 and IL-13). Thus, chlorogenic acid can reduce the production of in ammatory factors and effectively inhibit the occurrence and development of in ammation [26]. In addition, magno orine has a considerable anti-in ammatory effect; it can inhibit in ammation induced by excess production of NO, and the possible mechanisms are associated with the inhibition of Toll-like receptor 4-mediated activation of the NF-κB and MAPK signaling pathways. Moreover, magno orine exerts antifungal and immunomodulatory activities by promoting the phosphorylation of JNK, ERK, p38 MAPK, and AKT [27]. Berberine, palmatine, and jatrorrhizine elevate the production of in ammatory factors mediated by TNF-α and IL-1b [28,29]. Furthermore, berberine has been widely used to treat eczema in China [30]. Atractylodin can also treat eczema through its anti-in ammatory and immunomodulatory effects [31]. The ndings of these pharmacological studies are consistent with those of our current study.
Combined with network topology analysis and related literature reports, this study predicted that EGFR, AKT1, PTGS2, STAT3, MMP9, ICAM1, MAPK8, JUN, MAPK1, and VCAM1 may be the key targets of EMW in the treatment of eczema. EGFR, AKT1, PTGS2, and JUN have anti-in ammatory pharmacological effects, and certain nonsteroidal anti-in ammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways [32,33]. The allergic in ammatory response has been associated mainly with the activation of MAPKs, which include the extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK. MAPKs are involved in the activation of the transcription of in ammatory and allergy-related mediators. Therefore, regulation of the MAPK pathway is considered vital for eczema prevention [34]. Accumulating evidence has shown that the skin barrier function is regulated via the IL-13/IL-4-JAK-STAT6/STAT3 axis; furthermore, the regulation of STAT3 can repair the skin barrier function and reduce allergic in ammation, thereby playing a therapeutic role in eczema [35]. MMP9 may be directly related to the pathogenesis and development of eczema, as it may affect the susceptibility to eczema by degrading COL5A3 [36]. Moreover, the downregulation of ICAM-1 and AKT expression via activation of MAPK8 and JNK has been shown as one of the mechanisms in the treatment of eczema [37]. These targets play an important role in the pathogenesis of eczema; thus, drug interventions targeting these genes can be effective for eczema treatment. This nding suggests that EMW may alleviate the anaphylactic reaction and immunologic and in ammatory responses by adjusting the network at different nodes, which exerts a synergistic effect on eczema [38].
Pathway enrichment analysis results showed that the effect of EMW in treating eczema may be related to the ErbB signaling pathway, estrogen signaling pathway, and Epstein-Barr virus infection. ErbB family members represent important biomarkers and drug targets for modern precision therapy, and they play a pro-in ammatory role by activating the MMP, PTGS2, and MAPK signaling pathways [39,40]. The components of EMW may regulate the ErbB signaling pathway to treat eczema by acting on its key target proteins, such as MMP, PTGS2, and MAPK. Estrogen affects the expression of MMP9 and EGFR through the classical endoplasmic reticulum pathway, which plays important anti-in ammatory role and regulates cell survival and cell cycle [41]. In the treatment of eczema, EMW regulates the levels of MMP9 and EGFR proteins by acting on the estrogen signaling pathway, which plays an anti-in ammatory and anti-allergic role. Epstein-Barr virus (EBV) infection is associated with various human cancers. The expression of EGFR, AKT1, JUN, and MAPK can be inhibited by inhibiting the expression of EBV [42]. The occurrence and development of eczema is closely related to these pathways. The enrichment analysis results showed that each pathway contained key targets of the active components of EMW, and each pathway played a complex role in the treatment of eczema, suggesting that these pathways have potential application in future research. But the mechanism of EMW treat in T2DM is complicated, network pharmacology only shows that EMW has hypoglycemic effect in theory, we will verify the results of this research in the future stage.

Conclusion
In this study, seven components with the highest content in EMW were quantitatively determined. Network pharmacology revealed 57 crossover genes between eczema and these seven components. EMW was shown to effectively treat eczema by regulating the key pathways of the anti-in ammatory system, including the ErbB signaling, estrogen signaling, and Epstein-Barr virus infection pathways, which involve EGFR, AKT1, STAT3, MMP9, ICAM1, MAPK8, JUN, and MAPK1. These ndings were supported by molecular docking results, which provide insights into the mechanism of EMW against eczema.
We successfully applied an integrated strategy of network pharmacology and content determination to explore the pharmacological mechanism of the major components of EMW in eczema treatment. The method established in this study can help increase our understanding of the mechanisms of Chinese herbal formulations and promote their further research and development.

Declarations
Ethics approval and consent to participate Not applicable

Consent for publication
Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no con ict of interest. The work ow of this study.  Compound-target network of EMW and Eczema. Ingredients -target-pathway network. Labels: Green squares, ingredients; Pink diamonds, protein targets.