Research into the mechanism of intervention of Wenjing decoction in endometriosis based on network pharmacology and molecular docking technology

In this study, due to the traffic restrictions as a result of COVID-19, we were unable to perform work on more samples and the study was completed in less than the allotted time. Due to our limited financial sources, we were unable to conduct analysis and chromatography on the protein fractions extracted from WJD. Further work should address these issues.

By retrieving the component data of the medicines in the TCMSP and screening the active ingredients and action targets of WJD, this study demonstrated that WJD can treat EMs. Because of the numerous chemical components, action pathways, and action targets of traditional Chinese medicine compounds, it is difficult to carry out detailed and in-depth exploration from a macro-level to the molecular level. Nonetheless, this study explored the mechanism of WJD in the treatment of EMs from the perspectives of network pharmacology and bioinformatics, and provided a new theoretical support for the participation of WJD in the treatment of EMs.

Endometriosis (EMs) is defined as a common disease in women of reproductive age that occurs in endometrial tissue (glands and interstitium) in the endometrium of the uterine cavity and extrauterus, growth, infiltration, and repeated bleeding, manifested as secondary dysmenorrhea, menstrual disorders, ovarian endometriosis cysts, infertility, and other symptoms, [ 1 ] the occurrence of EMs has been on the upward thrust in recent years, seriously affecting women’s physical, mental health and quality of life, [ 2 ] EMs has a long history, however its actual pathogenesis is not yet regarded. Western medicine treatment strategies recognition on estrogen suppression, including gonadal hormone analogues and progesterone, or surgical treatment if necessary. [ 3 ] However, estrogen suppression has many side effects, and easy recurrence, and casting off the lesion by means of surgical operation does not offer a treatment, which is invasive. [ 4 ] Traditional Chinese medicine (TCM) treatment does not interfere with the normal menstrual cycle, has an obvious healing effect, as an example relieves ache, controls the improvement of lesions, and regulates menstruation, and does no longer affect being pregnant. [ 5 ] EMs is not diagnosed in TCM specifically, rather, the diagnosis is based on clinical signs of certain categories, such as “dysmenorrhea,” “infertility,” “irregular menstruation,” and “fistula.” The pathogenesis is blood stasis blockade of the uterine rush, and the clinical treatment is mainly based on TCM formulas that activate blood circulation and get rid of stasis, that has definite efficacy in relieving ache, slowing disorder development and preventing disorder recurrence after surgical treatment. [ 6 ] The clinical application of Wen Jing decoction (WJD) in the treatment of dysmenorrhea in EMs can significantly reduce the pain level and improve the quality of life of patients, the overall efficiency is over 90%. [ 7 ] WJD became first produced by using Zhang Zhongjing “Jin Kui Yao Lue,” which is composed of Evodia rutaecarpa (Wu zhuyu), Cinnamomum cassia Presl (Gui zhi), Angelicae Sinensis Radix (Dang gui), Chuanxiong Rhizoma (Chuan xiong), Moutan Cortex (Mu danpi), Peaoniae Radix Alba (Bai shao), and Dipsaci Radix (Xu duan) 7 forms of Chinese herbal medicine. [ 8 ] These formulas have the effects of activating blood stasis, regulating menstruation and relieving pain. [ 9 ] Clinical studies have found that the treatment of EMs with WJD can significantly reduce pain, and down-regulate serum levels of prostaglandin E2 (PGE2), interleukin-17 (IL-17), tumor necrosis factor-a (TNF-a), vascular endothelial growth factor (VEGF) and carbohydrate antigen (CA125). [ 10 ] WJD can shrink ectopic endometrial lesions in mice with endometriosis, which may be related to the regulation of TLR4/NF-kB signaling pathway for anti-inflammation, regulation of cell adhesion, and inhibition of vascular neogenesis. [ 11 ] But the mechanism of actions are relatively complicated, whether at the cellular or molecular level, it is difficult to carry out comprehensive and systematic studies, [ 12 ] this study uses network pharmacology to explain the mechanism of action of compound TCM formulas through the analysis of common genes and molecular pathways of drugs-genes-diseases.

Conceptualization: Linhui Huang. Data curation: Linhui Huang, Wei Yang, Minxue Su. Formal analysis: Linhui Huang, Wei Yang, Minxue Su. Investigation: Linhui Huang. Methodology: Linhui Huang. Project administration: Linhui Huang. Resources: Linhui Huang. Software: Linhui Huang. Supervision: Linhui Huang. Validation: Linhui Huang. Visualization: Linhui Huang. Writing – original draft: Linhui Huang, Wei Yang, Minxue Su. Writing – review & editing: Linhui Huang, Wei Yang, Minxue Su.

Through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database ( http://tcmspw.com/tcmsp.php ), [ 13 ] “Evodia rutaecarpa (Wu zhuyu), Cinnamomum cassia Presl (Gui zhi), Angelicae Sinensis Radix (Dang gui), Chuanxiong Rhizoma (Chuan xiong), Moutan Cortex (Mu danpi), Peaoniae Radix Alba (Bai shao), and Dipsaci Radix (Xu duan)” was searched, based on the ADME (such as drug absorption, distribution, metabolism, excretion) model, [ 14 ] the screening criteria were set as oral bioavailability (OB) ≥ 30% and drug likeness ≥ 0.18 to obtain the active ingredient monomer and target of WJD. Using BATMAN-TCM (bionet.ncpsb.cn/batman-tcm/index.php), [ 15 ] the active ingredient and target information of WJD were supplemented, and the search condition was “Score cutoff ≥ 20, P -value cutoff < 0.05.” The UniProt database was used to add gene target names to the screening results, and the parts without relevant gene names and target duplicates were excluded. Search in GeneCards ( https://www.genecards.org/ ), OMIM ( https://omin.org/ ), TTD ( https://db.idrblab.net/ttd/ ), Kyoto encyclopedia of genes and genomes (KEGG) ( https://www.genome.jp/kegg/ ) and GAD ( https://integbio.jp/ ), and use “EMs” and “Endometriosis” as search terms to obtain potential targets for EMs. The EMs targets obtained in the above database were combined after deleting repetitive genes. The intersection targets of WJD in the treatment of EMs were obtained by merging the ingredient targets of WJD and the disease targets of EMs by Venny 2.1 online software ( https://bioinfogp.cnb.csic.es/tools/venny/ ). By using Cytoscape 3.8.2 software, the active ingredients and intersection targets of WJD in the treatment of EMs were collected, and the active ingredient-intersection target network was constructed, [ 16 ] and the Analyze Network plug-in was used to calculate the topological parameters of nodes in the network, and the main targets of vital compounds and formulas in the network were screened. The common target obtained by WJD and EMs was introduced into the String online platform ( https://string-db.org ), [ 17 ] the species was set to “Homo sapiens,” and the remaining parameters were set to default values, resulting in a PPI network for potential therapeutic targets. This network was imported into Cytoscape 3.8.2 for visualization, using the Analyze Network plug-in to calculate the topological parameters of nodes in the network and filter out proteins that play an essential role in the network. In the constructed PPI network, the node size and color change reflect the degree value, and the larger the degree value, the larger the node size. Combined with Metascape and DAVID databases, the common target of Venn graph was imported, and the screening analysis was carried out beneath the conditions of P 1.5. Microbiotic letter mapping ( http://www.bioinformatics.com.cn ) was used to obtain bar charts and bubble charts of GO and KEGG pathway enrichment analysis. [ 18 ] The target-signaling pathway network diagram was constructed by using Cytoscape 3.8.2 software to further illustrate the role of targets and signaling pathways in the treatment of EMs. Light red nodes represent the signaling pathway, orange nodes represent the target, and green nodes represent the drug component. The binding activity of active compounds and key targets is confirmed by molecular docking. The choice of molecular docking procedure is AutoDock Vina (Vina), which adopts semiflexible docking with a docking accuracy of 78%. [ 19 ] The mol2 format files of the key ingredients of WJD were downloaded from the TCMSP database, and the pdb format files of the core targets were downloaded in the Protein Data Bank (PDB) database ( http://www.rcsb.org/ ). AutoDock Tools was used to separate proligands from protein structures, hydrogenate protein structures, remove water molecules, and convert them into PDBQT format for docking. Acquire small molecule ligand 3D structures using the RCSB PDB online database ( https://www.rcsb.org ). Process protein crystals and active compounds with AutoDock Tools. After docking with Vina, the scores of each combination were counted, and 30 molecular docking model results are shown by heat maps using the R language software, and the virtual docking of tremendously active target proteins with compounds were visualized with PyMOL 2.4.

By searching the TCMSP database and utilizing the criteria OB ≥ 30%, drug likeness ≥ 0.18, 78 active ingredients of WJD were screened, including 30 in Evodia rutaecarpa (Wu zhuyu), 7 in Cinnamomum cassia Presl (Gui zhi), 2 in Angelicae Sinensis Radix (Dang gui), 7 in Chuanxiong Rhizoma (Chuan xiong), 11 in Moutan Cortex (Mu danpi), 13 in Peaoniae Radix Alba (Bai shao), and 8 in Dipsaci Radix (Xu duan) (in descending order of OB). The active ingredient and target information of WJD were supplemented by BATMAN-TCM. The UniProt database was used to add gene target names to the screening results. Table 1 shows the details of the active ingredients. Main components of WJD. DL = drug likeness. The herbs-compounds-genes multinetwork included 7 herbs, 78 compounds, and 108 target genes of WJD was constructed by utilizing Cytoscape 3.8.2 software (Fig. 1 ). A total of 108 targets corresponding to the active ingredients of WJD were obtained, The GeneCards, OMIM, TTD, KEGG, and GAD were searched to obtain 2626 EMs-related targets, 124 intersection targets were obtained (Fig. 2 ). The herbs-compounds-genes multinetwork. Overlapping target genes between drug and EMs. After obtaining the TSV file of the target interaction using the String online database, the intersection target PPI network diagram was drawn with Cytoscape 3.8.2 software, which contains 434 nodes and 1235 edges (Fig. 3 ). The network was built with Cytoscape 3.8.2 software and the PPI network sorted by Betweenness value was plotted, as shown in Figure 4 . Figure 5 shows the top 30 targets by degree value. These targets are hubs that join different nodes in the network, play a key role in the PPI network, and may be crucial targets for the treatment of EMS. Therefore, it is speculated that AKT1, EGFR, IL6, VEGFA, TP53, and PTGS2 play an important role in the biological network of endometriosis intervention. Intersectional target interaction network. The more a target is connected to other targets, the more important the target is. Core target screening. Note: Picture above is the core target, where the larger the node, the higher the degree value. Top 30 targets of WJD in the treatment of EMs. The GO enrichment study is divided into 3 sections: cellular component (CC), molecular function (MF), and biological process (BP). A bar graph was made out of the GO term BP, CC, MF three-in-one histogram (Fig. 6 ). GO functional enrichment analysis yielded a total of 709 BPs involving humans positive regulation of transcription from RNA polymerase II promoter, positive regulation of gene expression, positive regulation of transcription, signal transduction, positive regulation of cell proliferation, negative regulation of apoptotic process, inflammatory response, apoptotic process, response to drug, negative regulation of transcription from RNA polymerase II promoter and so on; 54 CC, involving nucleus, cytoplasm, cytosol, plasma membrane, nucleoplasm, extracellular space, extracellular region, extracellular exosome, macromolecular complex, mitochondrion, and so on; 131 MF, involving protein binding, identical protein binding, enzyme binding, protein homodimerization activity, DNA binding, transcription factor activity, sequence-specific DNA binding, polymerase II core promoter proximal region sequence-specific DNA binding, RNA polymerase II transcription factor activity, sequence-specific DNA binding, protein kinase binding, zinc ion binding, and so on. The longer the length of the histogram, the greater the number of enriched genes, and the redder the color, the more significant the enrichment. GO analysis results. Note: Left side of the picture above demonstrates the top 10 significantly enriched BP, CC, and MF categories, while below the picture shows the number of enriched genes for these terms ( P < .05). GO = gene ontology, BP = biological process, CC = cellular component, MF = molecular function. A bubble map was created using KEGG pathway enrichment analysis, the top 20 KEGG pathways were screened out according to P -value (Fig. 7 ). The target-signal pathway network diagram was constructed with Cytoscape 3.8.2 software to obtain a network containing 292 nodes and 1333 edges, and the targets of the most important value mainly included AKT1, IL6, TP53, VEGFA, and PTGS2. In the figure, light red is the signal pathway, orange is the target, green is the drug component, dark red is the disease, and the larger the node, the larger the target degree value. A total of 185 pathways were obtained by KEGG pathway enrichment analysis, and their common targets were mainly enriched in pathways in cancer, lipid and atherosclerosis, PI3K-Akt signaling pathway, hepatitis B, human cytomegalovirus infection, Kaposi sarcoma-associated herpesvirus infection, chemical carcinogenesis-receptor activation, AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, hepatitis C, proteoglycans in cancer, prostate cancer, human T-cell leukemia virus 1 infection, chemical carcinogenesis-reactive oxygen species, human papillomavirus infection, MAPK signaling pathway, IL-17 signaling pathway, TNF signaling pathway, influenza A, coronavirus disease – COVID-19, and so on. The larger the bubble chart node, the greater the number of enriched genes, and the redder the color, the more significant the enrichment. Bubble map of 18 KEGG pathway. Note: The y -axis demonstrates the top 18 significantly enriched KEGG pathways, while the x -axis shows the number of enriched genes for these terms ( P < .05), the colors and the sizes indicate different P -value ranges; the redder and bigger it is, the more significantly enriched it is. KEGG = Kyoto Encyclopedia of Genes and Genomes. Cytoscape 3.8.2 software was used to build a disease-drug-active ingredient-target-signaling pathway interaction network, resulting in a network containing 277 nodes and 760 edges (Fig. 8 ). In the figure, the target-signaling pathway network diagram was constructed by using Cytoscape 3.8.2 software to further illustrate the role of targets and signaling pathways in the treatment of EMs. Light red represents the signaling pathway, orange represents the target, and green represents the drug component. Disease-drug-active ingredient-target-signaling pathway interaction network, resulting in a network containing 277 nodes and 760 edges. Note: Light red represents the signaling pathway, orange represents the target, and green represents the drug component. From the outcomes of PPI, it can be seen that the genes AKT1, EGFR, IL6, VEGFA, TP53, and PTGS2 play an important role in the biological network of warm menstrual decoction in the intervention of endometriosis dysmenorrhea, so Vina was used to conduct molecular docking experiments at the above genes and their corresponding compounds to confirm the binding activity of the compounds and targets. [ 18 ] Since the binding energy is less than −1.2 kcal/mol or less than −5 kJ/mol, the docking result can be considered feasible. Therefore, according to the size of the binding energy, 30 molecular docking model results are shown by heat maps (Fig. 9 ). It is known that the active compounds of WJD are especially made by hydrogen bonding and hydrophobicity interactions with π-π and π-alkyl groups with target targets binding. These interactions form a junction of the active compound with the protein of interest bonding, and the bonding gives sturdy bonding. Table 2 shows the top 30 molecular binding energy information. Furthermore, AKT1 with beta-sitosterol and paeoniflorin, and their binding energy is −10.1 kcal/mol and −9.9 kcal/mol, PTGS2 with kaempferol and quercetin, and their binding energy is −9.6 kcal/mol and −9.5 kcal/mol. They displayed the most powerful combined effects, as shown by the specific docking diagrams in Figures 10 – 13 . Binding capacity of core compounds to core proteins (kcal·mol −1 ). According to the size of the binding energy, 30 molecular docking model results are shown by heat maps. Visualization result of molecular docking AKT1(PDB:3MVH)-paeoniflorin. Visualization result of molecular docking AKTI(PDB:3MVH)-kaempferol. Visualization result of molecular docking AKTI(PDB:3MVH)-quercetin. Visualization result of molecular docking EGFR(PDB:3GKW)-quercetin.

WJD is a classic gynecological treatment for improving blood circulation and stasis, which has a great effect on a variety of diseases including endometriosis. Evodia rutaecarpa (Wu zhuyu), Cinnamomum cassia Presl (Gui zhi) have the efficacy of inducing sweating and relaxing muscles, warming and unblocking meridians, and supporting yang and transforming qi, they belong to the category of monarch medicine. The main efficacy of Angelicae Sinensis Radix (Dang gui), Chuanxiong Rhizoma (Chuan xiong) include promoting blood flow, casting off blood stasis, nourish blood and modifying menstruation; Moutan Cortex (Mu danpi) does not only reinforce the effect of other drugs in promoting blood circulation and removing blood stasis, but also clears heat, which is a common medicine. [ 20 ] Peaoniae Radix Alba (Bai shao) is sour and slightly cold, nourishes blood and yin, and softens the liver and relieves ache; Dipsaci Radix (Xu duan) has a lukewarm, bitter taste and is tough. It can tonify the liver and kidney, retain muscles and bones, and adjust blood veins. The combination of Angelicae Sinensis Radix (Dang gui), Chuanxiong Rhizoma (Chuan xiong), and Moutan Cortex (Mu danpi) nourishes the blood and liver, nourishes the yin and dries dampness, and clears the deficiency and heat. The warmth of evodia and laurel nobilis branches are adjuvants. The mixture can not only effectively relieve the clinical symptoms of patients with endometriosis dysmenorrhea, but also achieve pharmacological effects such as anti-inflammatory, anticoagulant, and endocrine regulation. [ 21 ] According to the quality control index of the Chinese Pharmacopoeia. [ 22 ] The quality control index of WJD in Chinese Pharmacopoeia screened out that for Evodia rutaecarpa (Wu zhuyu), impurities must not exceed 7% (General Principle 2301), moisture content must not exceed 15.0% (General Principle 0832 Law II), total ash content must not exceed 10. 0% (General Principle 2302). For Cinnamomum cassia Presl (Gui zhi) moisture content must not exceed 12.0% (General Principle 0832 Law IV), total ash content must not exceed 3. 0% (General Principle 2302). For Angelicae Sinensis Radix (Dang gui), moisture content must not be more than 15.0% (General Principle 0832, Fourth Law), total ash content must not exceed 7.0% (General Principle 2302), acid insoluble ash must not exceed 2.0% (General Principle 2302), heavy metals, and harmful elements are measured by lead, cadmium, stele, mercury, and copper (General Principle 2321 Atomic Absorption Spectrophotometry or Inductively Coupled Plasma Mass Spectrometry), lead must not exceed 5 mg/kg; cadmium must not exceed lmg/kg; stele must not exceed 2mg/kg; mercury must not exceed 0.2 mg/kg; copper must not exceed 20mg/kg. For Moutan Cortex (Mu danpi), moisture content must not exceed 13.0% (General Principle 0832 Law IV), total ash content must not exceed 5.0% (General Principle 2302). For Peaoniae Radix Alba (Bai shao), moisture content must not exceed 14.0% (General Principle 0832 Law II). Total ash content must not exceed 4. 0% (General Principle 2302). For Dipsaci Radix (Xu duan), moisture content must not exceed 10.0% (General Principle 0832 Law II), total ash content must not exceed 12. 0% (General 2302). EMs is a benign disease, but similar to malignant tumors, it has characteristics such as implantation, invasion, and distant metastasis. [ 23 ] The incidence has continued to rise in recent years, although the etiology is unknown. It has been reported that the incidence of women of childbearing age is 10% to 15%, of which 36% to 87% of ectopic lesions are located in the ovaries, the infertility rate is as high as 40%, [ 24 ] seriously affecting the quality of life of patients, it has been found that the pathogenesis of EMs is related to endocrine, inflammatory immunity, invasive adhesion, and angiogenesis. These are related to a variety of factors, and combination therapy from anti-inflammatory, anti-adhesion, anti-invasion, antiangiogenesis, and other aspects are required. [ 25 ] Western medicine still has certain shortcomings in the treatment of EMs, and cannot reap high-quality efficacy. [ 26 ] Surgical treatment, although it has achieved proper healing outcomes in clinical practice, is an invasive surgery, and there are still some patients with poor efficacy and easy recurrence. [ 3 ] According to the above mechanism of action and the results of this study, it was discovered that WJD has a particularly effect on endometriosis dysmenorrhea mainly through the pharmacological effects of anti-inflammatory, antioxidant, angiogenic inhibition, and other active ingredients, including quercetin, ellagic acid, and kaempferol; PPI network analysis showed that the targets of WJD in the treatment of endometriosis dysmenorrhea mainly included AKT1, EGFR, IL6, VEGFA, TP53, PTGS2, etc. Among them, AKT1 is one of the 3 subtypes of AKT, a serine/threonine kinase that has been widely studied in many biological phenomena, [ 27 ] and has become one of the most essential regulators of cell life activities in regulating cell proliferation, apoptosis, and glucose metabolism. This versatile serine/threonine kinase is a vital link in many physiological processes. [ 28 , 29 ] EGFR is a member of the epidermal growth factor receptor (HER) family, which plays an important regulatory role in cell physiological processes. EGFR signaling pathways are important in physiological processes such as cell growth, proliferation, and differentiation. [ 30 ] IL-6 is a lymphokine secreted by activated T lymphocytes and fibroblasts, which stimulates the activation of B cell precursors to provide antibodies for anti-inflammatory and immunomodulatory effects. Studies have found elevated IL6 levels in peritoneal fluid in patients with endometriosis, demonstrating that IL6 is related to the onset of endometriosis. [ 31 ] VEGFA, a main member of the VEGF family that promotes basement membrane decomposition, is a highly specific provascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and vascularization, and induces inflammation. [ 32 ] TP53, a gene located on chromosome 17, is a tumor suppressor gene that encodes a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomeric domains involved in cell proliferation, growth, and damage repair. TP53 codon 72 polymorphisms are related to the risk of endometriosis. [ 33 ] PTGS2, also known as cyclooxygenase (COX), is a key enzyme in prostaglandin (PG) biosynthesis, acting as both a dioxygenase and a peroxidase. PTGS2 is expressed in smooth muscle and many tissues stimulated by growth factors, cytokines or hormones, and is involved in biological processes such as cell growth, PG production and regulation of ovulation, and research have determined that rehabilitation can play an anti-inflammatory role by lowering the expression of PTGS2. [ 34 ] Therefore, the targets of EMs in the treatment of WJD are particularly associated with hormone secretion, anti-inflammatory, cell proliferation, invasion and regulation of analgesic mechanisms. The functional enrichment analysis showed that the biological processes mainly involved in the treatment of endometriosis include cell proliferation, inflammatory response and apoptosis, the cell components mainly included nucleus, cytosolic cyst cavity and exosomes, and the MFs included protein binding, enzyme binding and RNA polymerase transcription factor activity. The signaling pathways of warm menstrual decoction in the treatment of endometriosis dysmenorrhea mainly inflammation-related signaling pathways, such as the IL-17 signaling pathway and the TNF signaling pathway. Among them, IL-17 is a cytokine formed by the differentiation of transforming growth factor β (TGF-β) and IL6, which can mediate the pathological process of inflammatory signaling pathways such as NF-κB and p38 MAPK. [ 35 ] TNF is an inflammatory cytokine, divided into TNF-α and TNF-β, which induces the occurrence of EMs by participating in the immunomodulatory mechanism. [ 36 ] TNF-α interacts with NF-κB signaling pathway to participate in immune regulation, inflammatory response, and cell proliferation and differentiation, thus promoting the development of endometriosis. [ 37 – 39 ] The signaling pathways of cell proliferation, including MAPK and PI3K-AKT signaling pathways, promote the proliferation and migration of ectopic endometrial cells in patients with ectopia, and promote neoangiogenesis. [ 40 ]