Exploring the Modulatory Mechanism of Eleng Capsule in the Treatment of Endometriosis Based on Integrated Strategy of Transcriptomics Combined With Network Pharmacology

Endometriosis causes severe chronic pelvic pain and infertility. Chinese medicine plays an active role in the treatment of endometriosis. ELeng Capsule(ELC) is a Chinese medicine formula used for the treatment of endometriosis for several years. The previous studies have shown that ELC inhibits endometriosis. However, the mechanisms of action of ELC have not been characterized. In this study, network pharmacology and mRNA transcriptome analysis were used to study various therapeutic targets in ELC. Ultra-performance liquid chromatography/quadrupole time-of-ight mass spectrometry analysis(UPLC-Q-TOF/MS) was used to identify the compounds in ELC.And network pharmacology was used to analyze the network of targets and identied compounds of ELC. Apoptosis was assessed using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL) assay.The protein expression of VEGFA,VEGFB,VEGFC and α-SMA in the ectopic endometrium were identied by immunohistochemistry(IHC).The level of VEGFA,VEGFB and IL1-β in serum were used by ELISA. Further, RNA-sequencing was used to identify differentially expressed genes (DEGs) in ELC. Biological functions and pathways were determined through the Gene Ontology(GO) and Kyoto encyclopedia of genes and genomes pathway(KEGG) analyses. In addition, Gene Set Enrichment Analysis (GSEA) analysis was used to further analyze the genetic network and modular genetics. in ectopic lesions(P(cid:0)0.05). of endometriosis inammation,epithelial-mesenchymal transition(EMT) RNA-sequencing. in the of ELC actin and cytoskeleton,EMT,focal adhesion,and inammatory immunity on the DEGs analysis. In additon, GSEA analysis of ELC was associated with the Notch signaling pathway, Hippo signaling pathway, Vernier caliper endometriosis SD were randomly divided into four groups: the ELeng Capsule low-dose group(group A, 0.5g/kg/d of ELeng Capsule), the ELeng Capsule high-dose group (2g/kg/d of ELeng Capsule), the middle group (1g/kg/d of ELeng Capsule), the low group (0.5g/kg/d of ELeng Capsule), and the model group (group E,10ml/kg/d of 0.9%Nacl). The mixed suspension was orally administered once daily.In addition, ten SD rats were selected as the control group and fed routinely.Based on the previous research basis, we choose the equivalent and human dose to RNA-sequence. At the end of ELC administration, eutopic endometria from the control group and ectopic endometria from the other groups were collected and xed in paraformaldehyde,and parts of the tissues were maintained at −80°C for future research.The volumes of ectopic endometrial lesions in each group pre-(V1) and post-(V2) treatment were measured. The tissues were used for histopathology analyse,immunohistochemistry, RNA-sequence and quantitative-polymerase rate (FDR) method. GO enrichment and KEGG pathway enrichment analyses of differentially expressed genes (DEGs) were respectively performed using R studio based on the hypergeometric distribution. Fisher’s exact and chi-squared tests were applied to classify signicant GO categories, and the FDR was used to correct p-values. The GO analysis provides three structured networks of dened terms to describe gene product attributes: cellular compartment (CC), biological process (BP), and molecular function (MF). Pathway analysis was applied to determine the signicant pathways of DEGs according to the Kyoto Encyclopedia of Genes and Genomes(KEGG), MapSplice, and Reactome Functional Interaction Network and external interaction databases(Reactome databases). Fisher’s exact test was used to identify signicantly enriched pathways, and the threshold of signicance was dened as P<0.05 and FDR<0.05. These result showed that “endodermal cell differentiation”, “regulation of signaling receptor activity”, “positive regulation of myoblast differentiation”, “response to cytokine”, “chemokine-mediated signaling pathway”, “positive regulation of smooth muscle cell migration”,etc. The KEGG pathways of GSEA showed that the peroxisome proliferator-activated receptor signaling, tumor necrosis factor signaling, MAPK signaling, apelin signaling, hypoxia-inducible factor-1 signaling, PI3K-Akt signaling pathway, and focal adhesion are related to endometriosis(Figures 7C). ELC associated with endometriosis rat by RNA-sequencing.


Background
Endometriosis causes severe chronic pelvic pain and infertility. Currently, the treatment of endometriosis is mainly based on surgery and pharmacological treatment, and long-term treatment management programs are continuously optimized [1]. Endometriosis is a disease under the in uence of multiple factors.
TCM plays an active role in the treatment of endometriosis such as endometriosis, chronic pelvic pain, abnormal uterine bleeding, dysmenorrhea, and infertility, which is associated with pain and infertility, by regulating in ammation, immunity, and angiogenesis [1,4].
The Chinese preparation ELeng Capsule (ELC), as a hospital preparation, has been used in the Guangdong Provincial Hospital of Chinese Medicine (Guangzhou, China) to relieve the symptoms of endometriosis-associated pain and dysmenorrhea for nearly 20 years. The previous clinical experience, modern clinical practice and animal experiments of ELC have shown that ELC could reduce endometriosis-associated pain and inhibit the development of endometriosis through inhibits adhesion and regulate in ammation and immunity [5,6]. However, the mechanisms of action of ELC have not been characterized. It is signi cance to discover the multi-target regulatory mechanism of ELeng capsule with new methods.
Traditional Chinese medicine (TCM) compounds exist in complex mixtures and may contain thousands of compounds. Therefore, it is di cult to explain the principle of compatibility of Chinese medicine ingredients and analyze relevant results. Network pharmacology can predict the pro les of targets and pharmacological actions of herbal compounds to reveal "drug-genes/targets-disease". This approach could be used to determine the herbal ingredients and their related properties, as well as compound-target and target-disease relationships,which will improve current drug discovery strategies [7][8][9]. At the same time, the development of multi-omics also provides new tools for research on TCM. The expression of mRNA examined through microarray or high-throughput RNA-sequencing (RNA-seq) has been used to reveal molecular mechanisms and explore biomarkers for diagnosis and treatment [10,11]. Transcriptome-based studies of endometriosis could provide the basis for clarifying the therapeutic mechanisms of drugs [12].
In the study, we rst predicted the potential targets of ELC involved in endometriosis. Then, endometriosis model rats were used to validate some of the potential targets in ELeng capsules. Further,the RNA-sequencing were performed to identify genes whose expression is regulated by ELC treatment in endometriosis model, and nd the potential multi-targets regulation of ELC.We aim to provide a reliable way for subsequent experimental veri cation and new drug research and development.

Methods
The online search tool for recurring instances of neighboring genes (STRING, Version 9.1) (http://www.stringdb.org/) to predict the protein-protein interactions.The compounds-targets networks were constructed using Cytoscape software 3.7.2(http://cytoscape.org/). Related parameters were calculated to detect signi cant nodes [14].

Animal model and treatments
This study used female Sprague-Dawley (SD) rats (age: 8±1 weeks, weight: 220-230 g). The rats are from the Experimental Animal Center of Guangdong Province(Guangzhou,Guangdong,China),and certi cate number is 44007200054328.The rats were housed at 20±2°C on a 12-h light/dark cycle, with ad libitum access to food and water,raised in the Laboratory Animal Center of the The Second Clinical College of Guangzhou University of Chinese Medicine(Guangzhou,Guangdong,China). The rats were housed ve per cage. Animals, and the protocols protocols were approved by the Guangdong Provincial Hospital of Chinese Medicine Committee on the Use of Live Animals for Teaching and Research (SZY2016007). And disposal methods were in accordance with animal ethics standards. Surgical operation A model of endometriosis was established through allotransplantation in rats. All operational procedures were conducted under sterile conditions. The rats were anesthetized with 3% pentobarbital sodium prior to performing a vertical incision in the abdomen for tumor transplantation. The right uterus of each rat was removed and immediately placed in a saline solution. The endometria were separated from the myometria and cut into 0.5×0.5 cm pieces. The endometria were sutured onto the peritoneum close to blood vessels in each abdominal wall using a 5-0 absorbable suture. The incision was closed and disinfected, and the animals were allowed to recover from anesthesia. After transplantation (28 days), the growth of the ectopic endometrium was observed via gross and microscopic examination. Endometriosis rats model established are following criteria that viable and well-vascularized endometrial explants [15].The volume of ectopic endometrium were detected by Vernier caliper with volume formula (length × width × height×0.52).
The 40 endometriosis SD rats were randomly divided into four groups: the ELeng Capsule low-dose group(group A, 0.5g/kg/d of ELeng Capsule), the ELeng Capsule high-dose group (2g/kg/d of ELeng Capsule), the middle group (1g/kg/d of ELeng Capsule), the low group (0.5g/kg/d of ELeng Capsule), and the model group (group E,10ml/kg/d of 0.9%Nacl). The mixed suspension was orally administered once daily.In addition, ten SD rats were selected as the control group and fed routinely.Based on the previous research basis, we choose the equivalent and human dose to RNA-sequence. At the end of ELC administration, eutopic endometria from the control group and ectopic endometria from the other groups were collected and xed in paraformaldehyde,and parts of the tissues were maintained at −80°C for future research.The volumes of ectopic endometrial lesions in each group pre-(V1) and post-(V2) treatment were measured. The tissues were used for histopathology analyse,immunohistochemistry, RNA-sequence and quantitative-polymerase chain reaction (qPCR) validation.

Euthanasia
Intraperitoneal injection of three times the anesthetic dose of 1% sodium pentobarbital solution.
Hematoxylin and Eosin(H&E) and Masson's trichrome staining Sections from different groups were stained with hematoxylin and eosin. Masson's trichrome staining was used for the detection of collagen bers in tissues. The stained areas of the sections were observed under an optical microscope(Nikon Japan) and NIS-Elements.Fibrosis analysis was performed using Image J software to analyze the proportion of blue staining ( brosis staining).

Electron Microscopic Analysis
These tissue samples were xed immediately with 1% glutaraldehyde and 4% formalin for 6 hours at 4°C and rinsed in 0.1 M cacodylate buffer overnight.
Ultrathin sections were prepared with an Ultratome Nova, doublestained with uranyl acetate and lead citrate, and examined under an electron microscope.
Terminal Deoxynucleotidyl Transferase-mediated DigoxigenindUTPNick-End Labeling Assay (TUNEL) The para n-embedded tissue sections were dewaxed in xylene and rehydrated after remaining in a 60°C warm case for 1 h. The sections were washed three times, for 5 minutes per wash, with TBS. Apoptosis was detected using terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) apoptosis detection kit (C1086,Beyotime Biotechnology,China) according to the manufacturer's instructions.The labeled apoptotic cells expressed green uorescence under uorescence microscopy. The Image J software was used for assessing the ratio.

Monoclonal antibody and microvessel density (MVD)
In this study, vascular endothelial cells were labeled with a CD34 monoclonal antibody and microvessel density (MVD) was counted(n=6 each group). The dilution ratio of anti-CD34(1:500, ab185732,ABcam, USA) were used. Three microvessel dense areas were selected for each slice, and the microvessels were counted by double-blind method under high power (200×).

Immunohistochemical Staining
The sections were stained by IHC to detect the expression of factors in the VEGF family and α-SMA. After the antigen was repaired, primary antibodies were added at 4°C for overnight, and then secondary antibodies were added at room temperature for 1 hours, avoiding light. Diaminobenzidine (DAB) was used for staining, and neutral gum was used to seal pieces. The antibodies were anti-VEGFA (1:000, ab81289, Abcam, USA), anti-VEGFB (1:000, ab81289, Abcam, USA) and anti-VEGFC (1:000, ab81289, Abcam, USA) ,α-SMA(1:1000, 14395-1-AP, Proteinech, USA)was used to evaluate. The Image J software was used for assessing the mean optical density.

ELISA assay
To expression of VEGF family and IL1-β in blood serum, we used an ELISA kit. The samples and standard samples were diluted with distilled water and applied to ELISA plates. The VEGFA,VEGFB and IL1-βconcentrations were determined according to the manufacturer's instructions. Absorbance levels were measured at 450 nm using an ELISA reader.

RNA-sequencing of ELC
The design of RNA-sequencing analysis Dose of 1 mg/kg ELC was choose for further biological experiment because of its optimal effect in our previous study. We randomly selected transcriptomes from endometrial tissues and ectopic endometria for analyses between the model and ELC groups (n=4, each group eutopic endometrium (Con_Euto), (Model_Euto) and (ELC_Euto); n=4 model group ectopic endometrium (Model_Ecto); n=5, ELC group ectopic endometrium (ELC_Ecto)). A crossover comparison was performed in the following three paired groups to identify genes that were differentially regulated in the endometriosis rat model and ELC treatment rat model: The publicly available GSEA software package (www.broad.mit.edu) was used for leading edge analysis to examine genome-wide expression pro les [17]. In this study, 1000 genes of permutations were set to generate a null distribution for the enrichment score in the hallmark gene sets and functional annotation gene sets. Nominal P<0.05, FDR<0.25, and gene set size >100 were de ned as the cut-off criteria. The aim of this analysis was to determine whether the members of the identi ed gene ontology and KEGG pathways were randomly distributed throughout the ranked gene list or concentrated at the top or bottom.

Trend modular analysis(STEM analysis)
To identify genes that were differentially regulated under the conditions of endometriosis model and ELC_Ecto groups, a crossover comparison was performed in the following 3-paired groups:control group, model endometriosis rats groups and ELC treatment groups,as the Venn diagram to analysis demonstrates that they are coexpressed, respectively, in each pair of groups. Short Time-series Expression Miner (STEM) the rst software program speci cally designed for the analysis of short time series microarray gene expression data [18],which could also be used to analyze trends in genes in different groups.STEM software were used to analyze the trend of ectopic endometrium in control rats-model_Ecto endometrirum-ELC_Ecto endometrium group.

Construction of the protein-protein interaction (PPI) network
The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database provides comprehensive information regarding interaction between proteins, including both predicted and experimental interactions. Subsequently, the PPI network was visualized using Cytoscape (Version 3.7.2; National Institute of General Medical Sciences, Bethesda, MD, USA [14]. The PPI network was used to lter modules based on the Molecular Complex Detection plug-in (MCODE) in Cytoscape with the following conditions: degree cut-off=2; k-core=2; node score cut-off=0.2; and max depth=100. The interaction of each gene with other genes was determined using the GeneMANIA software (www genemania.org) [19].

Connectivity map(CMAP) analysis
CMAP is a systematic approach developed for the discovery of functional connections among diseases, genes, and drugs through the analysis of genomewide transcriptional expression data.We used these probesets to query the CMAP O2 database (https://portals.broadinstitute.org/cmap/, updated September 12, 2017) [20]. The CMAP database was searched to identify small molecule compounds in TCM. The small molecule drugs were ltered according to the pvalues (P<0.05).
Quantitative reverse transcription-PCR (qPCR) qRT-PCR was performed to validate the gene expression data obtained from deep sequencing. Total mRNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer. The rst strand of cDNA was synthesized using primers designed in our laboratory. The RT product was ampli ed using SYBR Green on a 7500 Real-Time PCR System (Thermo Fisher Scienti c Inc. Waltham, MA, USA). All samples were run in triplicate, and relative gene expression was analyzed according to the 2 −ΔΔCt method. The sequencing accessions of the primers were myogenin (Myog) SET and MYND domain containing 1(Smyd1),SIX Homeobox 1(Six1),calcium voltage-gated channel subunit alpha1 S (Cacna1s), eukaryotic translation elongation factor 1 alpha 2 (Eef1a2), ryanodine receptor 1 (Ryr1), actinin Alpha 2(Actn2), myogenic differentiation 1 (Myod1), mitogen-activated protein kinase 12 (Mapk12), myosin heavy chain 4 (Myh4), .Gene expression levels were normalized to that of ACTB. The primer sequencings are shown in Table 3.

Statistical analysis
Data were analyzed using the Prism software (version 7.0;GraphPad prism, California San Diego,USA). All experimental data are presented as the mean ± standard error of the mean. The qPCR data were analyzed using two-tailed Student's t-test. The F-test was used to determine whether two groups possessed equal variances. Unless otherwise indicated, P<0.05 denoted statistical signi cance.

UPLC-Q-TOF/MSresults for ELC
In our previous study, GC-MS analysis was used to reveal the volatile oil components of ELC. A total of 14 compounds were identi ed, namely eucalyptol, Dcamphor and isoborneol, l(−)-Borneol, α-terpineol, β-elemene, γ-elemene, α-humulene, germacra, curcumenol, β-cyclocostunolide, curcumenone, pulmonary Zedeone, and ent-kaurene. In this study, the compounds of ELC were identi ed by UPLC-Q-TOF/MS. According to the multi-stage MS information combined with the data obtained from the literature and databases, 26 compounds were identi ed in the ELC compound preparation.Representative ngerprint chromatograms of ELC are displayed in Figure 2. The identi ed compouds of ELC were showed in Table 4. Combined with our previous study results, we have identi ed a total of 40 compounds in ELeng Capsule.These results provide identi ed compounds in ELC for network pharmacology analysis.

Network pharmacology analysis
The compounds associated targets of ELC Based on the UPLC-Q-TOF/MS data, we performed a network pharmacology analysis on the related compounds of ELC and predicted the targets of compounds. Based on the data obtained from the network pharmacology-related databases, we identi ed 27 core compounds with 194 targets based on STITCH TCMSP and UNPD datasets. The result showed that the major targets were involved in angiogenesis, in ammation, immunity, and other modules.Supplementary Material 1 showed the major compouds and targets of ELC.In addition,we had collected 1289 endometriosis-related targets from Genecard and Genbank. A total of 75 targets of ELC capsules coincided with those of genes associated with endometriosis. Following cytoHubba analysis, VEGFA,IL6,TP53,PTGS2,AKT1,MMP9,MAPK1,JUN,etc may be core targets of ELC.

The analysis of GO terms and KEGG pathways
We analyzed the GO terms and KEGG pathways affected by targets associated with ELC. The results suggest that the BP in GO terms are related to cell death, apoptosis, and proliferation. The results of KEGG pathways were mainly enriched in cancer pathway, in ammatory response, drug reaction, etc., which were consistent with the present pathologic mechanism of endometriosis.The main pathways include neuroactive ligand-receptor interaction, TLR signaling pathway, metabolism of xenobiotics by cytochrome P450, vascular endothelial growth factor (VEGF) signaling, calcium signaling, B cell receptor signaling, among others. GO and KEGG enrichment analysis suggest that the core components of ELC may be involved in the regulation of in ammation, immunity and angiogenesis modules.The major GO terms and KEGG pathways are shown in Figure 3A and 3B. The network of major targets and compounds from database is showed in Figure 3C.
Potential mechanism of ELC in Endometriosis rat model

Pathology of ectopic lesions
We successfully established a rat model of endometriosis. Figure 4A shows the changes in lesions after modeling. After the treatment,the average value of tissue in ELC group was lower than the model group, while the difference was not statistically signi cant (P 0.05)(supplementary material 3). Compared with the model group, the lesion volumes of before and after ELC treatment in ELC middle group was change signi cantly (P=0.028 0.05). These result showed that ELC may reduce the volume of ectopic lesions to a certain extent in endometriosis rats mode. The HE revealed the formation of local glands in the lesions of the model and ELC treatment rats( Figure 4B). Figure 4 C was showed the ELC on apoptosis of endometriotic tissues, as determined by TUNEL assay (×100, n = 4 each). The distribution of green uorescence included glandular epithelium and mesenchymal cells. The nuclei of positive staining apoptotic cells emitted green uorescent signals in the ectopic endometrium tissues. Compared with the model group, the number of apoptotic cells in ectopic lesions in high, medium and low dose of ELC were signi cantly increased. Compared with the model group, the apoptotic area in the middle dose group of ELC increased signi cantly(P < 0.05).And there was no statistically signi cant difference in the dipsion area between the high-dose group and low-dose group compared with the model group(P > 0.05). This result suggested that ELC could participate in the process of cell apoptosis.

Ultrastructural Features of Ectopic Endometrium in a Rat Model
The glandular epithelial cells in the model group were relatively neat and had microvilli.In the ectopic tissue cells in ELC group,the microvilli were reduced, and the mitochondria were swollen in the cytoplasm, and autophagy and apoptotic bodies were observed( Figure 4D). Figure 4D(c,d) showed the apoptotic body,and gure 4D(e,f) showed the autophagosome.This result suggested that ELC treatment may be related to the regulation of autophagy and apoptosis.
The effect of Eleng capsule on angiogenesis Figure 5A showed that CD34 was positively expressed in both the model group and the ELeng capsule group. Among them, the expression of MVD in the ectopic lesion was signi cantly higher than that in the eutopic endometrium, and angiogenesis was observed(n=6, P=0.015 0.05). The expression of CD34 in high, medium and low dose groups of ELeng capsule was signi cantly lower than the model group expression (P 0.05). GO and KEGG enrichment in the endometriosis rat model ectopic endometrium versus control rat eutopic endometrium As shown in GO terms, the up-regulated genes were most signi cantly enriched in the CC of extracellular region, the BP of muscle contraction, and MF of actin lament binding, bronectin binding, calcium ion binding, etc. The actin-associated GO terms may relate to the development of ectopic lesions in the endometriosis rat model( Figure 7A). The KEGG analysis of genes associated with endometriosis suggested that these genes are associated with the pathway of extracellular matrix-receptor (ECM-receptor) interaction, p53 signaling pathway, pathways in cancer, endocrine resistance, interleukin-17 (IL-17) signaling pathway, chemokine signaling pathway, cytokine-cytokine receptor interaction, etc( Figure 7B).
We performed a Venn analysis of DEGs and human endometriosis-related genes using Genecard datasets. The results showed that there were 113 upregulated and 28 down-regulated genes that were related to endometriosis. The proteins expressed by these genes are involved in EMT and may be involved in the process of brosis in endometriosis. The up-regulated genes associated with endometriosis in humans are related to cytokine-cytokine receptor interaction, phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway, pathway in cancer, MAPK signaling pathway, ECM-receptor interaction, Ras signaling pathway, toll-like receptor (TLR) signaling pathway, IL-17 signaling pathway, p53 signaling pathway, forkhead box protein O signaling pathway, focal adhesion, etc. Based on the above analyses, the rat model of endometriosis may be suitable for investigating the transcriptome level.
The enrichment score for this signaling pathway was positively correlated to endometriosis samples. Based on the path of GSEA enrichment [17], neuroactive  Figure 8A).
We also analyzed the KEGG pathways of the ELC ectopic endometrium(ELC_Ecto) versus the model ectopic endometrium(Model_Ecto). The results of KEGG pathway enrichment showed that down-regulated DEGs were mainly enriched in the following pathways: calcium signaling pathway, apelin signaling pathway, cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway, 5' adenosine monophosphate-activated protein kinase signaling pathway, hypoxia-inducible factor-1 signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, focal adhesion, etc( Figure 8B).These pathways could be related to the inhibitory effect of ELC in endometriosis treatment.
Through the GSEA of the KEGG analysis, we also found other signaling pathways, including the Notch signaling pathway, adherens junction, Hippo signaling pathway, basal transcription factors, regulation of actin cytoskeleton, etc., which were related to the treatment of endometriosis with ELC.The The major KEGG pathways from GSEA analysis were shown in Figure 8C(FDR<0.25). ELC may inhibit brosis and EMT by regulating the aforementioned pathways.
The core genes established in the network may be related to the regulation by ELeng Capsule treatment. Two modules obtained using the default criteria of MCODE for ELC_Ecto versus Model_Ecto were used to construct the network. We selected major modules for module network visualization ( Figure 8D). The core nodes continued to be associated with genes related to actin, cytoskeleton, and brosis.

STEM analysis of differential expression patterns
Temporal expression patterns and the signi cance of the genes were determined using the STEM software. This approach was used to pro le the DEGs, select signi cant gene clusters with parallel expression patterns, and identify the pro les of the "up to down" model from control to model to treatment with ELC. The results of the gene cluster analysis were statistically signi cant in pro les_14, pro les_11, pro les_10, and pro les_4 (P<0.05)( Figure 9A). After the development of endometriosis model, actin-associated DEGs in the Model_ecto and ELC_ecto groups were up-regulated; these may be the regulatory genes for the development of the endometriosis model (pro le_11 and pro le_4). The series test of pro le_14 and pro le_10 showed that the signi cant clusters were considered as potential pro les that could be affected by treatment with ELC (P<0.05). Several actin-related and micro lament proteins were up-regulated in the model group and down-regulated after treatment, suggesting that the overall regulation mechanism of ELC treatment in ectopic endometriun is related to the regulation of actin cytoskeleton. The results of the STEM analysis were further re ecting the process of the endometriosis rat model and intervention with ELC( Figure 9B).

Protein-protein interaction network
We constructed network relationships between the core genes of the two groups of DEGs, and analyzed the possible network relationships through relevant pathways. We selected the calcium signaling pathway, cGMP-PKG signaling pathway, apelin signaling pathway, and the DEGs to construct the network ( Figure   10A). The hub down-regulated genes closely related to treatment with ELC were as follows: Actn3, Actn2, Myom2, myoglobin, Ryr1, Myog, Myh7, Myod1, sarcalumenin, myosin light chain kinase 2, Smyd1, Map3k7, Mapk12, Myh4, Cacna1s, EEF1A2, and Cacng1.

CMAP analysis
We compared the DEGs in gene expression pro les between induced by ELC(ELC_Ecto) vs Model_Ecto groups,and used the CMAP database to better understand the therapeutic e cacy and identify potential targets of this agent. Based on the results, a network was constructed to link ELC with representative drugs and their therapeutic targets. The pro le of ELC was similar to that of classical pharmacotherapies (e.g.,sulfaphenazole, carbinoxamine, esculin, emetine, zoxazolamine, and ni umic acid). The result of CMap analysis are showed in Table 5. Carbinoxamine is a rst-generation antihistamine of the ethanolamine class. Zoxazolamine is a centrally acting myorelaxant used as an antispasmodic and uricosuric. Ni umic acid are used in the treatment of rheumatoid arthritis, dysmenorrhea, and osteoarthritis. Ni umic acid inhibited invasion and reduced apoptosis in CNE-2Z cells through regulation of the extracellular regulatory kinase/MAPK pathway. Thus, this agent may serve as a candidate of anticancer drug [21,22]. Based on the results of the CMAP analysis, we consider that ELC may similarly regulate in ammation, the cytochrome C P450 pathway, and MAPK pathway.

Real-time quantitative RT-PCR
Quantification was performed with a two-step reaction process: reverse transcription (RT) and PCR. The expression ratios of these DEGs, determined by qPCR, were consistent with those obtained from the mRNA transcriptome analysis( Figure 10B). The results for some genes exhibited a different trend; however, the differences were not statistically signi cant. The observed trend may be related to individual differences in the degree of gene expression in rats.The expression of veri ed genes in qPCR were showed in supplementary material 6.

Discussion
The mechanism of Traditional Chinese medicine in the treatment of endometriosis could related to inhibit in ammation, enhance the immune response, regulate angiogenesis-related pathways, and inducing apoptosis [23]. In this study, we identify potential molecular mechanisms involved network pharmacology and investigated the regulated genes of ELC directly associated with endometriosis rat models by RNA-sequencing.
The characteristic of endometriosis rat model In this study, we found that up-regulated DEGs were signi cantly enriched in several pathways, including focal adhesion, ECM-receptor interaction, calcium signaling pathway, and cytokine-cytokine receptor interaction. Moreover, up-regulated DEGs may play important roles in the development of endometriosisassociated BP and pathways (e.g., in ammation, immunity, EMT, wound healing, and stem cell behavior), and contributes pathologically to brosis and progression of cancer.
EMT and broblast-to-myo broblast transdifferentiation, as well as increases in cellular contractility, collagen production, and smooth muscle metaplasia lead to brosi [24,25].A study investigating a rat model of endometriosis showed that the up-regulated genes included those encoding cytokines, chemokines, growth factors, and cell adhesion molecules.Endometriosis may be triggered by infection, mechanical damage, and in ammation, as each of these mechanisms can induce EMT in the mesothelium [26].In endometriosis, the endometrial stroma and glands exhibit ectopic growth and are surrounded by dense brous tissue [27]. The results of the transcriptome analysis in endometriosis model rats also suggested endometriosis are realated to EMT.
Endometriotic tissue is often induced in rodents via transplantation through surgery or intraperitoneal injection of uterine tissue fragments.The time of collection in rat models is 8 weeks after modeling; hence, the lesion has begun to undergo brosis. The interstitial space is a possible source of brotic cells during serosal in ammation and tissue repair.Therefore, it may play an important role in peritoneal brosis and adhesion formation [28]. Actin, a member of the ezrin-radixin-moesin family, plays a role in cell movement by linking the actin cytoskeleton. The roles of actin and the cytoskeleton in the development of endometriosis, as well as the relationship with cell adhesion, invasion, and brosis, also attracted our attention [29].The result of RNA-seq showed that the rat models of endometriosis could represent the characteristics of endometriosis to a certain extent and could provide the basis for further pharmacological research [30].

Potential mechanism of ELC treatment
In this study,we had identi ed 40 coumpouds in ELC and established compounds and targets network, and further analysis of the potential mechanism involved in treatment with ELC by network pharmacology and RNA-sequence. Compounds in ELC could relieve endometriosis-associated pain and regulated the neuroactive ligand-receptor interaction, metabolism of xenobiotics by cytochrome P450, TLR signaling, VEGF signaling, calcium signaling pathway.We further analyze the mechanism of the core compounds in ELC.
The reported e cacy of servical compounds are related to the mechanism of ELC in endometriosis treatment.The coupoumds of Curcuma phaeocaulis and Sparganium stoloniferum are common compounds with anti-cancer properties. Sparstolonin B could serve as a potential therapeutic agent for the treatment of TLR-mediated in ammatory disorders [31],also alleviate neuropathic pain by selectively suppressing TLR2 and TLR4 [32]. β-elemene possesses broadspectrum antitumor activity and is effective against several types of tumors [33]. The active ingredients of Salvia miltiorrhiza include tanshinone I, tanshinone IIA, salvianolic acid, dihydrotanshinone, etc [34]. Tanshinone IIA could reduce the VEGF/VEGFR2 pathway and CD146 in vitro and in vivo, and regulates angiogenic function in human umbilical vein endothelial cells [35]. Rosmarinic acid is a potential natural compound with anti-cancer properties, as demonstrated in various human cancer cell lines [36]. It also could inhibit the proliferation of primary and T-HESCs and induce cell cycle arrest of the latter in the G2/M phase in vitro [37]. Paeonia lactifora has hematopoietic functions, anti-in ammatory activity, and immunological properties.In P. lactifora, paeoni orin could inhibit the plantar incision-induced microglia TLR4/MMP-9/2/IL-1β signaling pathway and suppresses postoperative pain [38].
Paeoni origenone could induce apoptosis and exerts an antiproliferative effect [39]. Citrus aurantium may possess anti-tumor activity. Naringenin could induce apoptosis and endoplasmic reticulum stress through regulation of the MAPK and Akt signal transduction pathways in End1/E6E7 and VK2/E6E7 cells [40]. Limonin could induce apoptosis, thereby affecting the growth of SNU449 and HCT-15 tumor cells [41]. These small molecule compounds may exert new synergistic regulatory effects. The mechanism in endometriosis treatment of above compounds need to be futher studied. ELC have the characteristics of multi-target regulation, which may regulate angiogenesis and induce apoptosis based on network pharmacology and experimental veri cation.
In order to further explore the regulatory mechanism of ELC in the transcription level, we further conducted mRNA transcriptomics analysis.The transcriptome results showed that ELC treatment could relieve endometriosis through regulating the biological processc of ytoskeleton, troponin, EMT,etc. After the intervention with ELC, the expression of genes related to troponin,cytoskeleton and the MAPK signaling pathwaywere down-regulated compared with that measured in the model group [42].The results of the GSEA suggest that the formation of a rat model of endometriosis and the intervention of ELC on endometriosis model rats are related to the cytoskeleton, EMT, brosis, and muscle brosis, which are more closely related to abdominal endometriosis and deep in ltration of endometriosis patients. And the result of Masson's trichrome staining and α-SMA expression also identi ed the inhibition effection of brosis of ELC.In addition, through the GSEA of the ELC_Ecto group and the Model_Ecto group, the results of the KEGG enrichment analysis revealed a relationship with the Notch signaling pathway and the Hippo signaling pathway. The hyperactivation of the ADAM17/Notch signaling pathway could result in an increase in brosis, which is associated with deep in ltrating endometriosis [43].
In other enriched signaling pathways of ELC regulation, 19 down-regulated DEGs were enriched in the calcium signaling pathway. Apelin,as a ligand of the APJ receptor,has functions in angiogenesis and cell proliferation, and is a vasoactive and regulatory peptide [44]. And apelin expression in the ectopic and ectopic endometrium changes periodicall [45].The DEGs in the Apelin signaling pathway are related to muscle contraction, calmodulin binding, and the myosin complex. Moreover,the kinase associated pathways are associated with endometriosis.A genome-wide association study analysis revealed that multiple pathways, new variants in MAP3K4, and several pathways linked to MAPK are associated with endometriosis[46]. The serine/threonine kinase Akt and extracellular regulatory kinase signaling pathways can synergistically support deep endometriosis by enhancing the proliferation and survival of endometrial stromal cells (ESCs) in the in vitro brotic microenvironment [47].These above pathways, as related pathways for endometriosis, may participate in the regulation process of ELC.
Further,the DEGs were veri ed through qPCR. These genes may be involved in the intervention with ELC. Alterations in key pathways (MAPK pathway) regulating cell cycle checkpoints, apoptosis, and EMT, appear to be closely associated to the development of chemoresistance in cancer. These genes, which are related to tumors, cytoskeleton, and cell potential, have numerous biological functions and may be involved in the development of endometriosis. EEF1A2 encodes an isoform of the alpha subunit of the elongation factor-1 complex, and may be critical in the development of ovarian cancer [48]. Targeting EEF1A2 and plitidepsin to release protein kinase R may trigger the extrinsic pathway of MAPK and nuclear factor-κB-dependent activation, leading to tumor cell death [49]. RYR1 is the core factor of the calcium signaling pathway. The ryanodine receptor calcium release channel is central to the cytoplasmic calcium signaling pathway [50].
Based on this research, we also have new discovery about the mechanism of action of Chinese medicine for removing blood stasis.At present, current research on the role of TCM in promoting blood circulation and removing phlegm is focused on apoptosis, in ammatory immunity, angiogenesis in endometriosis. These results of proteomic studies suggest that endometriosis is associated with EMT, and that there are differences in differentially expressed proteins among various syndromes in TCM [51]. The mechanism for the regulation of cytoskeleton and EMT through TCM is lacking. Several natural compounds suggested that treatment in cancer, in ammatory and brosing diseases through the regulation of EMT process [52].

Limitations
There are several limitations in this study. Firstly, only the small molecule compounds derived from plants in ELC were analyzed in the UPLC-Q-TOF/MS analysis,without the source animals were not analyzed. Secondly, although we have shown that rats/mice are a good animal model for studying endometriosis, they cannot re ect the natural course of the human disease. Further research on cells is warranted to clarify the mechanism involved in the intervention with ELC. Third, the relationship between the regulation of the cytoskeleton and troponin and the presence of endometriosis, and further optimization of the core components of ELC.

Conclusions
In this study, we have explained the treatment mechanism of ELeng capsules using transcriptome analysis and network pharmacology. According to the transcriptomic and network pharmacology analyses, we hypothesized that ELC may regulate in ammation and immunity, regulate cell adhesion, cytoskeletonrelated genes, in uence the process of EMT, and consequently affect the development of lesions. The use of network pharmacology combined with transcriptome could help us understand the associated targets and pathways networks, candidate genes in ELeng Capsule. Combined techniques may also offer an e cient method of drug discovery in herbal medicines.

Consent for publication
Not applicable.

Availability of data and materials
All data is contained within the manuscript and additional les. Citrus aurantium L., a dried, immature fruit of the cultivar.
Angelica laxifoliata (Danggui) Angelica laxifoliata root.         The STEM analysis of ELC treatment in endometroisis rats. (A)The overall STEM analysis. (B)The stem analysis results suggest that there are 4 statistically signi cant trends. The results of gene cluster analysis were statistically signi cant in pro le of 14,11,10 and 4(P < 0.05). The pro le_11 (Figure 9 B( )) and pro le_4 (Figure 9 B( )) could be think as the regulatory genes of endometriosis model development(P < 0.05).The series test of pro le_14 (Figure 9 B( )) and pro le_10 (Figure 9 B( )) showed that the signi cant clusters were considered as potential pro les that could be affected by ELC treatment (P< 0.05). The KEGG pathways of the STEM enrichment genes in pro le 14,pro le 11,pro le 10,and pro le 4.