Investigation on the Therapeutic Mechanism of Danbie Capsules for Endometriosis: A Network Pharmacology Approach

After retrieving the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) ( https://tcmspw.com/index.php ) and referencing relevant kinds of literature, we got information on the active substances and their corresponding target proteins in various TCMs present in Danbie Capsules. The search was performed with the following key words: “ Largehead Atractylodes Rhizome”, “Scutellaria Barbata”, “Radix Angelicae Sinensis”, “Herba Scutellariae Barbatae”, “Cortex Eucommiae”, “Radix Curcumae”, “Ramulus Cinnamomi”, “Seaweed”, “Rhizoma Sparganii”, “Radix Notoginseng”, “Semen Persicae”, and “Turtle Carapace”; screening criteria were set as oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18. For the TCM “Turtle Carapace”, due to no information available in the TCMSP database, the active substances were determined by referring to published literature, 6 and the corresponding target protein was obtained from the Drug Bank database using the chemical name as the search term. By using the STRING database to uniformly change and save the form of the target protein into the target gene, we obtained the target gene set of active substances in Danbie Capsules. Gene sample series for patients with endometriosis and healthy individuals were obtained from the GSE25628 dataset, retrieved from the GEO database ( https://www.NCBI.NLM.NIH.gov/GEO/ ). The R4.2.1 software installed R packages containing “GEOquery”, “limma”, “ggplot2” and “ComplexHeatmap”. We downloaded GSE25628 from the GEO database by using the GEOquery package, standardized the data by using the normalize Between Arrays function, and performed difference analysis between the two groups by using the limma package. Then Log2 (logFC) transformation was applied, and samples with a P-value 1 were considered genes that are statistically significantly differentially expressed. The gene volcano map of these samples was plotted, and the top 20 genes exhibiting the most significant upregulation or downregulation were selected to draw a heatmap. The R4.2.1 software was utilized to map the action targets of Danbie Capsules with the endometriosis-related targets using and obtaining the potential action targets (intersection targets) of Danbie Capsules in endometriosis treatment and drawing a Venn diagram. The active substances of Danbie Capsules and their corresponding targets were imported into Cytoscape version 3.9.1 software to construct a target network diagram of active constituents of Danbie Capsules anti-endometriosis. Then we used the “Network Analyzer” function to analyze the topological attributes of the network, calculating the three network topology parameters: degree of freedom (DOF), closeness centrality (CC), and betweenness centrality (BC) and analyzing the critical active substances of Danbie Capsules in endometriosis treatment according to the parameters. The targets of the active substances in Danbie Capsules, known for their anti-endometriosis properties, were queried in the STRING database ( https://string-db.org/ ). The search was limited to the species “Homo sapiens” to obtain protein-protein interaction relationships. Cytoscape version 3.9.1 software was employed to visualize the target protein-protein interaction network diagram. The “Network Analyzer” plug-in was utilized to analyze and calculate the metrics of each network node, including DOF, CC, and BC. The targets where all three indexes were above the average value were regarded as the critical targets of anti-endometriosis of Danbie Capsules’ active substances. The Metascape cloud platform ( https://metascape.org/ ) was adopted to analyze the function of Gene Ontology (GO) and the enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGGs) on the critical targets of Danbie Capsules in anti-endometriosis treatment, and the analysis results were visualized by Weishenxin cloud platform. The three-dimensional molecular structure formula of critical active substances was obtained from TCMSP and saved as a Mol2 format file. The anti-endometriosis target proteins of the critical active substances of Danbie Capsules were retrieved in the PDB database ( http://www.rcsb.org/ ). The water molecules, phosphate, and excess non-active ligands in the proteins were eliminated with the aid of PyMOL software. The treated target proteins were imported into AutoDock Tools software for hydrogenation, charge adding and torsion bonds setting. Then, the critical active substances and target proteins were converted into PDBQT format using AutoDock Tools and set as receptors and ligands respectively. The docking box was fine-tuned with AutoDock Tools to encompass all the protein structures. At the same time, set receptor protein as rigid docking. Run autogrid4 and autodock4 to get docking results, and then got the binding energy. Then used PyMol software to generate a local map of molecular docking. Samples were collected from normal endometrium and ectopic endometrium in the rectum of patients in the Sixth Affiliated Hospital of Sun Yat-Sen University for recent 3 years. Six normal endometrial specimens were set as the control group, and 6 ectopic endometrial specimens in the rectum were set as the experimental group. The tissues were made into glass slides with the following steps. Firstly, the slides were baked at 60 °C for 2 h. Next, dewaxing was performed with xylene for 15 min, with the process repeated three times. Then, these tissues were dehydrated with anhydrous, 95%, 90%, 80%, 70% ethanol, and distilled water for 5 min each. Subsequently, the slides were washed with phosphate-buffered saline (PBS) for 5 min, repeating this washing step three times. To repair the tissues, a boiling water bath containing citrate buffer was used for 15 min, followed by natural cooling to room temperature. The slides were washed with PBS for 5 min, repeating this step three times. For further processing, the slides were blocked with 3% hydrogen peroxide for 20 min, followed by washing with PBS for 5 min, repeating this step three times. Subsequently, the slides were blocked with normal fetal bovine serum at 37 °C for 20 min. Next, the primary antibody was incubated at 4 °C overnight, followed by rewarming for 20 min after overnight, washing with PBS for 5 min, and repeating this washing step three times. The slides were then incubated with the secondary antibody incubation, at 37 °C for 20 min, followed by washing with PBS for 5 min, repeating this washing step three times. To visualize the results, the DAB was used for color development, the slides were observed under the microscope, and the reaction was terminated in time. Counterstaining was performed with hematoxylin for 5 min, followed by rinsing with tap water. Differentiation was carried out with 1% hydrochloric acid alcohol (75%) solution for 30s, followed by rinsing with tap water for 5 min, and bluing. To prepare the slides for preparation, they were dehydrated with 70%, 80%, 90%, 95%, and anhydrous ethanol for 5 min each, and xylene for 15 min, repeating three times. A layer of neutral gum was applied to seal the slides. Finally, these gray values of the protein bands on the slides were analyzed using Image J software for further analysis.

In literature data and the TCMSP database, Danbie Capsules contained 2120 kinds of active substances, including 55 Largehead Atractylodes Rhizome, 94 Scutellaria Barbata, 125 Radix Angelicae Sinensis, 202 Herba Scutellariae Barbatae, 147 Eucommia ulmoides Oliv., 81 Radix Curcumae, 220 Ramulus Cinnamomi, 20 Seaweed, 975 Rhizoma Sparganii, 119 Radix Notoginseng, 66 Semen Persicae, and 16 Turtle Carapace. Then, 183 active substances were obtained under the conditions of DL ≥ 0.18 and OB ≥ 30%, including 7 from Largehead Atractylodes Rhizome, 29 from Scutellaria Barbata, 2 from Radix Angelicae Sinensis, 65 from Herba Scutellariae Barbatae, 28 from Cortex Eucommiae, 3 from Radix Curcumae, 7 from Ramulus Cinnamomi, 4 from Seaweed, 5 from Rhizoma Sparganii, 8 from Radix Notoginseng, 23 Semen Persicae, and 2 from Turtle Carapace ( Table 1 ). The DrugBank database was employed to predict the target of screened active substances. In the end, a total of 292 target proteins were obtained. Table 1 The Active Substances in Danbie Capsules Drug MOL_ID Molecule_Name OB DL Baizhu MOL000020 12-senecioyl-2E,8E,10E-atractylentriol 62.39646702 0.22294 Baizhu MOL000021 14-acetyl-12-senecioyl-2E,8E,10E-atractylentriol 60.3128707 0.30534 Baizhu MOL000022 14-acetyl-12-senecioyl-2E,8Z,10E-atractylentriol 63.37091823 0.29956 Baizhu MOL000028 α-Amyrin 39.51208978 0.7629 Baizhu MOL000033 (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R,5S)-5-propan-2-yloctan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol 36.22847056 0.78288 Baizhu MOL000049 3β-acetoxyatractylone 54.06671707 0.21906 Baizhu MOL000072 8β-ethoxy atractylenolide III 35.95091928 0.21079 Banzhilian MOL001040 (2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 42.36332114 0.21141 Banzhilian MOL012245 5,7,4’-trihydroxy-6-methoxyflavanone 36.62688628 0.26833 Banzhilian MOL012246 5,7,4’-trihydroxy-8-methoxyflavanone 74.23522001 0.26479 Banzhilian MOL012248 5-hydroxy-7,8-dimethoxy-2-(4-methoxyphenyl)chromone 65.81880606 0.32874 Banzhilian MOL012250 7-hydroxy-5,8-dimethoxy-2-phenyl-chromone 43.7169646 0.25376 Banzhilian MOL012251 Chrysin-5-methylether 37.2683358 0.20317 Banzhilian MOL012252 9,19-cyclolanost-24-en-3-ol 38.68565906 0.78074 Banzhilian MOL002776 Baicalin 40.12360996 0.75264 Banzhilian MOL012254 Campesterol 37.57681789 0.71486 Banzhilian MOL000953 CLR 37.87389754 0.67677 Banzhilian MOL000358 Beta-sitosterol 36.91390583 0.75123 Banzhilian MOL012266 Rivularin 37.94023355 0.3663 Banzhilian MOL001973 Sitosteryl acetate 40.38964165 0.85102 Banzhilian MOL012269 Stigmasta-5,22-dien-3-ol-acetate 46.44190225 0.85814 Banzhilian MOL012270 Stigmastan-3,5,22-triene 45.02668769 0.71047 Banzhilian MOL000449 Stigmasterol 43.82985158 0.75665 Banzhilian MOL000173 Wogonin 30.68456706 0.22942 Banzhilian MOL001735 Dinatin 30.97205344 0.27025 Banzhilian MOL001755 24-Ethylcholest-4-en-3-one 36.08361164 0.75703 Banzhilian MOL002714 Baicalein 33.51891869 0.20888 Banzhilian MOL002719 6-Hydroxynaringenin 33.22920875 0.24203 Banzhilian MOL002915 Salvigenin 49.06592606 0.33279 Banzhilian MOL000351 Rhamnazin 47.14113124 0.33648 Banzhilian MOL000359 Sitosterol 36.91390583 0.7512 Banzhilian MOL005190 Eriodictyol 71.7926526 0.24372 Banzhilian MOL005869 Daucostero_qt 36.91390583 0.75177 Banzhilian MOL000006 Luteolin 36.16262934 0.24552 Banzhilian MOL008206 Moslosooflavone 44.08795959 0.25331 Banzhilian MOL000098 Quercetin 46.43334812 0.27525 Danggui MOL000358 Beta-sitosterol 36.91390583 0.75123 Danggui MOL000449 Stigmasterol 43.82985158 0.75665 Danshen MOL001601 1,2,5,6-tetrahydrotanshinone 38.74538672 0.35791 Danshen MOL001659 Poriferasterol 43.82985158 0.75596 Danshen MOL001771 Poriferast-5-en-3beta-ol 36.91390583 0.75034 Danshen MOL001942 Isoimperatorin 45.46424674 0.22524 Danshen MOL002222 Sugiol 36.11353486 0.27648 Danshen MOL002651 Dehydrotanshinone II A 43.76228599 0.40019 Danshen MOL002776 Baicalin 40.12360996 0.75264 Danshen MOL000569 Digallate 61.84861803 0.25635 Danshen MOL000006 Luteolin 36.16262934 0.24552 Danshen MOL006824 α-amyrin 39.51208978 0.76221 Danshen MOL007036 5,6-dihydroxy-7-isopropyl-1,1-dimethyl-2,3-dihydrophenanthren-4-one 33.76525236 0.28585 Danshen MOL007041 2-isopropyl-8-methylphenanthrene-3,4-dione 40.86015408 0.22897 Danshen MOL007045 3α-hydroxytanshinoneIIa 44.92933597 0.44272 Danshen MOL007048 (E)-3-[2-(3,4-dihydroxyphenyl)-7-hydroxy-benzofuran-4-yl]acrylic acid 48.24363244 0.31229 Danshen MOL007049 4-methylenemiltirone 34.34867589 0.22726 Danshen MOL007050 2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-7-methoxy-3-benzofurancarboxaldehyde 62.78414726 0.39628 Danshen MOL007051 6-o-syringyl-8-o-acetyl shanzhiside methyl ester 46.6906586 0.71145 Danshen MOL007058 Formyltanshinone 73.444622 0.41736 Danshen MOL007059 3-beta-Hydroxymethyllenetanshiquinone 32.16103376 0.40894 Danshen MOL007061 Methylenetanshinquinone 37.07319368 0.36017 Danshen MOL007063 Przewalskin a 37.10650066 0.64901 Danshen MOL007064 Przewalskin b 110.3240001 0.43809 Danshen MOL007068 Przewaquinone B 62.24005962 0.41374 Danshen MOL007069 Przewaquinone c 55.7416731 0.40408 Danshen MOL007070 (6S,7R)-6,7-dihydroxy-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione 41.31045706 0.453 Danshen MOL007071 Przewaquinone f 40.30788399 0.45925 Danshen MOL007077 Sclareol 43.67068458 0.2058 Danshen MOL007079 Tanshinaldehyde 52.4747043 0.45196 Danshen MOL007081 Danshenol B 57.9508753 0.55764 Danshen MOL007082 Danshenol A 56.96524899 0.52172 Danshen MOL007085 Salvilenone 30.38365387 0.37639 Danshen MOL007088 Cryptotanshinone 52.34196226 0.39555 Danshen MOL007093 Dan-shexinkum d 38.88302101 0.55453 Danshen MOL007094 Danshenspiroketallactone 50.43128103 0.3067 Danshen MOL007098 Deoxyneocryptotanshinone 49.40034705 0.28555 Danshen MOL007100 Dihydrotanshinlactone 38.6847683 0.32227 Danshen MOL007101 DihydrotanshinoneI 45.04327919 0.36015 Danshen MOL007105 Epidanshenspiroketallactone 68.27315929 0.30549 Danshen MOL007107 C09092 36.06948986 0.2474 Danshen MOL007108 Isocryptotanshi-none 54.98193246 0.39449 Danshen MOL007111 Isotanshinone II 49.91602574 0.39674 Danshen MOL007115 Manool 45.04431636 0.20208 Danshen MOL007118 Microstegiol 39.61229457 0.27734 Danshen MOL007119 Miltionone I 49.68439433 0.32125 Danshen MOL007120 Miltionone II 71.02970321 0.43711 Danshen MOL007121 Miltipolone 36.55611206 0.36803 Danshen MOL007122 Miltirone 38.75698635 0.25418 Danshen MOL007123 Miltirone II 44.95106648 0.23537 Danshen MOL007124 Neocryptotanshinone II 39.46299114 0.23157 Danshen MOL007125 Neocryptotanshinone 52.48799701 0.32306 Danshen MOL007127 1-methyl-8,9-dihydro-7H-naphtho[5,6-g]benzofuran-6,10,11-trione 34.72082213 0.36634 Danshen MOL007130 Prolithospermic acid 64.37096207 0.31017 Danshen MOL007132 (2R)-3-(3,4-dihydroxyphenyl)-2-[(Z)-3-(3,4-dihydroxyphenyl)acryloyl]oxy-propionic acid 109.3805241 0.35119 Danshen MOL007140 (Z)-3-[2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3,4-dihydroxy-phenyl]acrylic acid 88.53602101 0.25869 Danshen MOL007141 Salvianolic acid g 45.56485578 0.60602 Danshen MOL007142 Salvianolic acid j 43.37604991 0.72497 Danshen MOL007143 Salvilenone I 32.43470856 0.22895 Danshen MOL007145 Salviolone 31.72415039 0.23568 Danshen MOL007149 NSC 122421 34.49292309 0.27645 Danshen MOL007150 (6S)-6-hydroxy-1-methyl-6-methylol-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-quinone 75.38587847 0.4551 Danshen MOL007151 Tanshindiol B 42.66581049 0.45303 Danshen MOL007152 Przewaquinone E 42.85485204 0.45301 Danshen MOL007154 Tanshinone iia 49.88730004 0.39781 Danshen MOL007155 (6S)-6-(hydroxymethyl)-1,6-dimethyl-8,9-dihydro-7H-naphtho[8,7-g]benzofuran-10,11-dione 65.25893771 0.44871 Danshen MOL007156 Tanshinone VI 45.63730602 0.29549 Duzhong MOL002058 40,957–99-1 57.20447445 0.61872 Duzhong MOL000211 Mairin 55.37707338 0.7761 Duzhong MOL000358 Beta-sitosterol 36.91390583 0.75123 Duzhong MOL000422 Kaempferol 41.88224954 0.24066 Duzhong MOL004367 Olivil 62.22859563 0.40642 Duzhong MOL000443 Erythraline 49.17676997 0.55031 Duzhong MOL005922 Acanthoside B 43.35308428 0.76689 Duzhong MOL006709 AIDS214634 92.42724327 0.54906 Duzhong MOL007059 3-beta-Hydroxymethyllenetanshiquinone 32.16103376 0.40894 Duzhong MOL000073 Ent-Epicatechin 48.95984114 0.24162 Duzhong MOL007563 Yangambin 57.52544673 0.80801 Duzhong MOL009007 Eucommin A 30.51335769 0.84815 Duzhong MOL009009 (+)-medioresinol 87.18865939 0.61875 Duzhong MOL009015 (-)-Tabernemontanine 58.66917753 0.60719 Duzhong MOL009027 Cyclopamine 55.42172002 0.82136 Duzhong MOL009029 Dehydrodiconiferyl alcohol 4, gamma’-di-O-beta-D-glucopyanoside_qt 51.44225525 0.39505 Duzhong MOL009030 Dehydrodieugenol 30.10301535 0.23906 Duzhong MOL009031 Cinchonan-9-al, 6’-methoxy-, (9R)- 68.2150183 0.40098 Duzhong MOL009038 GBGB 45.57744755 0.82668 Duzhong MOL009042 Helenalin 77.01051009 0.19049 Duzhong MOL009047 (+)-Eudesmin 33.28664314 0.62037 Duzhong MOL009053 4-[(2S,3R)-5-[(E)-3-hydroxyprop-1-enyl]-7-methoxy-3-methylol-2,3-dihydrobenzofuran-2-yl]-2-methoxy-phenol 50.75513649 0.3948 Duzhong MOL009055 Hirsutin_qt 49.81498455 0.37152 Duzhong MOL009057 Liriodendrin_qt 53.13736321 0.79961 Duzhong MOL000098 Quercetin 46.43334812 0.27525 Duzhong MOL002773 Beta-carotene 37.18433337 0.58358 Duzhong MOL008240 (E)-3-[4-[(1R,2R)-2-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-1-methylol-ethoxy]-3-methoxy-phenyl]acrolein 56.31706329 0.36095 Duzhong MOL011604 Syringetin 36.82222268 0.37414 Ezhu MOL000296 Hederagenin 36.91390583 0.75072 Ezhu MOL000906 Wenjine 47.92807652 0.272 Ezhu MOL000940 Bisdemethoxycurcumin 77.38200887 0.26088 Ezhu MOL001736 (-)-taxifolin 60.50621692 0.27342 Ezhu MOL000358 Beta-sitosterol 36.91390583 0.75123 Ezhu MOL000359 Sitosterol 36.91390583 0.7512 Ezhu MOL000492 (+)-catechin 54.82643405 0.24164 Ezhu MOL000073 Ent-Epicatechin 48.95984114 0.24162 Ezhu MOL004576 Taxifolin 57.84156034 0.27345 Ezhu MOL011169 Peroxyergosterol 44.39151838 0.82 Haizao MOL010578 N-[(1S)-1-(benzyl)-2-[[(1S)-1-(benzyl)-2-hydroxy-ethyl]amino]-2-keto-ethyl]benzamide 45.75831251 0.43303 Haizao MOL010580 Diglycol dibenzoate 59.21885418 0.27376 Haizao MOL005440 Isofucosterol 43.77639556 0.7576 Haizao MOL000098 Quercetin 46.43334812 0.27525 Sanleng MOL001297 Trans-gondoic acid 30.70294255 0.19744 Sanleng MOL000296 Hederagenin 36.91390583 0.75072 Sanleng MOL000358 Beta-sitosterol 36.91390583 0.75123 Sanleng MOL000392 Formononetin 69.67388061 0.21202 Sanleng MOL000449 Stigmasterol 43.82985158 0.75665 Sanqi MOL001494 Mandenol 41.99620045 0.19321 Sanqi MOL001792 DFV 32.76272375 0.18316 Sanqi MOL002879 Diop 43.59332547 0.39247 Sanqi MOL000358 Beta-sitosterol 36.91390583 0.75123 Sanqi MOL000449 Stigmasterol 43.82985158 0.75665 Sanqi MOL005344 Ginsenoside rh2 36.31951162 0.55868 Sanqi MOL007475 Ginsenoside f2 36.43174722 0.25282 Sanqi MOL000098 Quercetin 46.43334812 0.27525 Taoren MOL001323 Sitosterol alpha1 43.28127042 0.78354 Taoren MOL001328 2,3-didehydro GA70 63.29362943 0.49632 Taoren MOL001329 2,3-didehydro GA77 88.08054638 0.53017 Taoren MOL001339 GA119 76.36423404 0.49382 Taoren MOL001340 GA120 84.84963921 0.45279 Taoren MOL001342 GA121-isolactone 72.69926164 0.5371 Taoren MOL001343 GA122 64.79328513 0.49617 Taoren MOL001344 GA122-isolactone 88.11097358 0.5371 Taoren MOL001348 Gibberellin 17 94.64114807 0.49443 Taoren MOL001349 4a-formyl-7alpha-hydroxy-1-methyl-8-methylidene-4aalpha,4bbeta-gibbane-1alpha,10beta-dicarboxylic acid 88.59516065 0.46382 Taoren MOL001350 GA30 61.71773793 0.54002 Taoren MOL001351 Gibberellin A44 101.61317 0.54105 Taoren MOL001352 GA54 64.20664836 0.5349 Taoren MOL001353 GA60 93.16869233 0.53004 Taoren MOL001355 GA63 65.54355602 0.53773 Taoren MOL001358 Gibberellin 7 73.80061824 0.49609 Taoren MOL001360 GA77 87.89415596 0.52764 Taoren MOL001361 GA87 68.85254536 0.57188 Taoren MOL001368 3-O-p-coumaroylquinic acid 37.62790163 0.28636 Taoren MOL001371 Populoside_qt 108.8854875 0.20476 Taoren MOL000296 Hederagenin 36.91390583 0.75072 Taoren MOL000358 Beta-sitosterol 36.91390583 0.75123 Taoren MOL000493 Campesterol 37.57681789 0.71476 Biejia MOL005030 11-eicosenoic acid 30.7 0.2 Biejia MOL010861 Vitamin d 45.66 0.48 The Active Substances in Danbie Capsules By performing a comparative analysis between 6 normal samples and 8 disease samples available in the GEO database, a total of 12,548 differentially expressed genes were identified. Among these genes, 7379 were found to be up-regulated, while 5169 were down-regulated in disease samples compared to the normal samples. There were 559 up-regulated and 586 down-regulated genes after adjusting for P-value 1. As could be seen from the map of the gene volcano ( Figure 1 ), in the disease samples, the distribution of differential genes followed a normal distribution pattern, and there was a higher number of significantly down-regulated genes compared to significantly up-regulated genes. The top 20 genes exhibiting the most significant up-regulation and down-regulation are in Figure 2 . Figure 1 The map of the gene volcano highlights how genes are distributed across these disease samples. Green and red respectively highlight the up-regulated genes (logFC>0) and down-regulated genes (logFC 0) and down-regulated (logFC < 0), while white highlights the absence of significant differences. The first 6 samples were from healthy volunteers; the last 8 samples were from endometriosis patients. The map of the gene volcano highlights how genes are distributed across these disease samples. Green and red respectively highlight the up-regulated genes (logFC>0) and down-regulated genes (logFC 0) and down-regulated (logFC < 0), while white highlights the absence of significant differences. The first 6 samples were from healthy volunteers; the last 8 samples were from endometriosis patients. As shown in Figure 3 , there were a total of 24 intersection genes. The regulatory network of the TCM visually demonstrates the targeting relationship between the active substances of Danbie Capsules and the intersection of genes, providing insights into how these compounds interact and influence gene regulation. Figure 3 Venn diagram of targets of Danbie Capsules in endometriosis treatment. Venn diagram of targets of Danbie Capsules in endometriosis treatment. Figure 4 illustrates the network diagram of active substances of 11 TCMs in Danbie Capsules (without the involvement of Turtle Carapace) acting on 24 targets, including 60 nodes and 107 edges, showing the multi-component and broad-spectrum targeting action mechanism of Danbie Capsules. In the network diagram, the mean DOF of active substances was 4.28, the average of BC was 3.27×10 −2 and the average of CC is 3.27×10 −2 . Among them, the active substances whose topology parameters exceed the average value were: quercetin, β-sitosterol, and luteolin, suggesting that these ingredients might be the critical active substances of Danbie Capsules in anti-endometriosis. Figure 4 ( a and b ) TCM - Compound - Gene network: The network indicates the interplay and targeting relationship between active substances derived from TCMs and the intersection genes. The Dark colored circles in the middle represent the common components. (b) TCM - Compound - Gene network: The network indicates the interplay and targeting relationship between active substances derived from TCMs and the intersection genes. The blue rectangles indicate the components of Danbie Capsules, the red circles represent the common components, other colorful circles represent the active substances corresponding to each component of Danbie Capsules, and the dark blue diamonds represent the intersection of genes. ( a and b ) TCM - Compound - Gene network: The network indicates the interplay and targeting relationship between active substances derived from TCMs and the intersection genes. The Dark colored circles in the middle represent the common components. (b) TCM - Compound - Gene network: The network indicates the interplay and targeting relationship between active substances derived from TCMs and the intersection genes. The blue rectangles indicate the components of Danbie Capsules, the red circles represent the common components, other colorful circles represent the active substances corresponding to each component of Danbie Capsules, and the dark blue diamonds represent the intersection of genes. The PPI network was shown in Figure 5 , with a total of 22 nodes (target proteins PTGER3 and ADH1B were not involved in the interaction) and 214 interaction lines. The darker the color and larger the area of the circular nodes, the higher their DOF and importance. The mean DOF was 19.45, the mean BC was 2.90×10 −2 and the mean CC was 6.54×10 −1 . All seven targets exhibited DOM, CC, and BC values that exceeded the mean value, and they were considered the critical targets of Danbie Capsules in anti-endometriosis. The results were shown in Table 2 . Table 2 Intersection Genes (Sorted by DOF) Gene Name Degree Betweenness Centrality Closeness Centrality TTP53 38 0.149267812 0.913043478 AKT1 36 0.094165772 0.875 FOS 30 0.071929128 0.777777778 HSP90AA1 28 0.108127163 0.75 MAPK8 28 0.032446891 0.75 NOS3 24 0.043091757 0.7 MMP2 24 0.03843428 0.7 CDKN1A 26 0.017419939 0.724137931 CYCS 24 0.011324641 0.7 XIAP 22 0.010736961 0.677419355 RELA 22 0.00829743 0.677419355 CDK4 20 0.005214723 0.65625 HSPA5 20 0.027516112 0.65625 TGFB1 18 0.007301587 0.617647059 RB1 14 4.76E-04 0.583333333 BAX 14 5.95E-04 0.583333333 PLAU 12 0.008688388 0.567567568 NFATC1 8 0 0.525 PLAT 8 0.003061224 0.5 AKR1B1 6 0 0.525 NCOA2 4 0 0.5 KCNH2 2 0 0.4375 Note : The genes highlighted in red are the core genes ranked at the top. Figure 5 ( a and b ) PPI network from the STRING database; b: PPI network: The network shows the protein-protein interaction relationships of 22 target genes, and the darker the color and larger the area of the circular nodes, the higher their DOF. Intersection Genes (Sorted by DOF) Note : The genes highlighted in red are the core genes ranked at the top. ( a and b ) PPI network from the STRING database; b: PPI network: The network shows the protein-protein interaction relationships of 22 target genes, and the darker the color and larger the area of the circular nodes, the higher their DOF. GO enrichment analysis provides insights into the functional roles of genes at three distinct levels: biological process (BP), cellular component (CC), and molecular function (MF) ( Figure 6 ). BP primarily encompasses the gene’s participation in hormone response, cellular reactions to organonitrogen compounds, and cellular responses to nitrogen compounds. CC was mainly related to transcriptional regulator complexes, RNA polymerase II transcriptional regulatory complexes, and glutamatergic synapses. As for the MF level, the gene predominantly engages in interactions such as binding to ubiquitin-protein ligases, binding to kinases, and binding to ubiquitin-like protein ligases. Based on the KEGG enrichment analysis, the therapeutic mechanism of Danbie Capsules in endometriosis primarily revolves around the modulation of human T-cell leukemia virus 1, hepatitis B, and cancer pathways ( Figure 7 ). Figure 6 ( a – c ) GO enrichment analysis of Danbie Capsules in endometriosis treatment. In the BP ( a ), CC ( b ), and MF ( c ) columns, the horizontal axis indicates the proportion of genes enriched in each item, and the color indicates the enrichment degree based on P-values (log10 conversion). Figure 7 Bubble chart of KEGG. In the bubble chart of KEGG, the horizontal axis indicates the proportion of genes enriched in each entry, while the vertical axis represents the degree of enrichment based on the P-value (log10 conversion). ( a – c ) GO enrichment analysis of Danbie Capsules in endometriosis treatment. In the BP ( a ), CC ( b ), and MF ( c ) columns, the horizontal axis indicates the proportion of genes enriched in each item, and the color indicates the enrichment degree based on P-values (log10 conversion). Bubble chart of KEGG. In the bubble chart of KEGG, the horizontal axis indicates the proportion of genes enriched in each entry, while the vertical axis represents the degree of enrichment based on the P-value (log10 conversion). Among the identified pathways, the genes, BAX, AKT1, CDKN1, and TTP53, were found to be associated with the highest number of pathways ( Figure 8 ). Figure 8 Danbie Capsules signaling pathway - Anti-endometriosis critical target network. Danbie Capsules signaling pathway - Anti-endometriosis critical target network. The critical active substance luteolin was used for molecular docking with the critical targets TTP53 and AKT1. The affinity between the two target proteins and luteolin was < −5 kcal/mol and the amino acid residues docking with luteolin were shown in Figure 9 . Figure 9 ( a – d ), ( a and b ) Macromolecular docking model of luteolin and TP53; ( c and d ) Macromolecular docking model of luteolin and AKT1. ( a – d ), ( a and b ) Macromolecular docking model of luteolin and TP53; ( c and d ) Macromolecular docking model of luteolin and AKT1. TP53 group: The positive rate of TP53 protein expression in both the control group and the experimental group was 100% (both were highly positive); AKT1 group: The positive rate of AKT1 protein expression in the control group was 83.33% (5 low positive, 1 negative), while the positive rate of AKT1 protein expression in the experimental group was 100% (5 positive, 1 high positive). As shown in Figure 10 . Figure 10 Expression of P53 and AKT1 in normal endometrium and endometriosis. Expression of P53 and AKT1 in normal endometrium and endometriosis.

Endometriosis, as a chronic inflammatory disorder characterized by the ectopic localization of endometrial tissue outside the uterine cavity, can result in pelvic pain and infertility. This condition represents a substantial public health burden due to its profound influence on the quality of life of the affected women. 1 Endometriosis is recognized as one of the prevalent benign gynecological proliferative disorders among premenopausal women, with an estimated prevalence of 10–15% among women of childbearing potential. The exact biological mechanisms underlying endometriosis remain not fully understood. Despite being a prevalent disorder, the pathophysiology of endometriosis remains elusive. Furthermore, recent studies have suggested a lack of association between the severity of the disease and the manifestation of symptoms. Currently, there is a lack of widely used blood-based diagnostic tests specifically designed for endometriosis. Moreover, no universally effective treatment approach can guarantee the complete resolution of the condition. Endometriosis belongs to the “sticking mass” category in Traditional Chinese medicine. Traditional Chinese medicine believes that this disease is caused by the stagnation of qi and blood stasis and the stagnation of blood stasis. Danbie Capsules are composed of 12 drugs, including Herba Scutellariae Barbatae, Radix Notoginseng, Rhizoma Sparganii, Rhizoma Curcumae, Semen Persicae, Radix Angelicae Sinensis, Turtle Carapace, Seaweed, Largehead Atractylodes Rhizome, Cortex Eucommiae, Herba Scutellariae Barbatae, and Ramulus Cinnamomi. The combination of various drugs aims to promote blood circulation, dissipate blood stasis, and soften and disperse nodules. Hu Sisi et al found that Danbie Capsules can reduce the concentration of PGE2 and TNF- α in the blood of EMs rats, which may be one of the effective mechanisms for its treatment of endometriosis. 2 Liu Zhenming et al research showed that Danbie Capsules have a certain therapeutic effect on Endometriosis. According to the influence on cytokines and grafts, the Active substance in the prescription is separated, and its action target and specific action mechanism are studied according to the active substance. 3 Zhang Chunhong et al found that Danbie Capsules can exhibit inhibitory effects on the proliferation of ectopic endometrial tissue. This effect was attributed to the reduction in the levels of prostaglandin E2 (PGE-2) and tumor necrosis factor (TNF) in both the serum and peritoneal fluid of the experimental rat models. The anti-inflammation, analgesia, improvement of blood rheology and microcirculation, and other functions are very crucial in the treatment course of Endometriosis. It embodies the distinctive principles and features of traditional Chinese medicine (TCM), which is characterized by multiple components and multiple action steps. 4 But its precise mechanism of action is still elusive. Network pharmacology, an interdisciplinary field integrating pharmacology, bioinformatics, and other relevant disciplines, employs system network analysis to unravel the intricate mechanisms underlying the therapeutic effects of multi-component and multi-target drug treatments. By constructing comprehensive networks encompassing “disease-phenotype-gene-drug” interactions, this approach provides insights into gene distribution patterns, molecular functions, and signaling pathways. Notably, network pharmacology presents a particularly valuable methodology for investigating the complex mechanisms of action exhibited by TCM compounds. 5

The active substances of the Danbie Capsules are mainly Quercetin, Luteolin, and β- sitosterol. Seven critical target genes were identified, and two representative genes (TP53 and AKT1) have been verified in Macromolecular docking and immunohistochemical verification. Then we have some insights into the mechanism of the Danbie Capsules in Endometriosis therapy.

To further study the active substances and targets of Danbie Capsules in the Endometriosis therapy TCMSP and published kinds of literature screened and obtained 183 active substances of Danbie Capsules, combined and intersected with Endometriosis target genes collected and screened in the GEO database, obtained 24 target genes for Endometriosis treatment, and mapped the target network map of Danbie Capsules active substances against Endometriosis. With the help of Cytoscape version 3.9.1 to analyze this network, the research results obtained Quercetin β-3 critical active substances, sitosterol, and Luteolin. Quercetin and Luteolin are common Flavonoid in nature. They exhibit antioxidant, anti-inflammatory, and anti-tumor effects. Quercetin is widely acknowledged for its exceptional ability to scavenge reactive oxygen species (ROS) efficiently. It also acts as a potent inhibitor of several proinflammatory reactions, including the suppression of tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) production. The anti-tumor effects of Quercetin include promoting the loss of cell activity, apoptosis, and apoptosis. Autophagy is achieved by regulating PI3K/Akt/mTOR, Wnt/- catenin, and MAPK/ERK1/2 pathways. Its role in cancer metabolism can target molecular pathways involved in glucose metabolism and mitochondrial function. 7 Luteolin mediated targeting of protein network and microRNAs in different cancers: Focus on JAK-STAT, NOTCH, mTOR, and TRAIL-mediated signaling pathways. 8 As a well-known plant-derived nutrient with anticancer properties, β-sitosterol can help fight against a wide range of cancers, such as breast, stomach, colon, lung, prostate, and leukemia. Extensive research has proven that β-sitosterol can modulate numerous critical cell signaling pathways. It can exert influences on cellular processes such as cell cycle regulation, apoptosis induction, proliferation control, survival promotion, invasion inhibition, angiogenesis suppression, and metastasis prevention. What’s more, it also has demonstrated anti-inflammatory, anticancer, hepatoprotective, antioxidant, cardioprotective, and anti-diabetic properties during pharmacological screening without inducing severe toxicity. 9 Based on the STRING data analysis platform, PPI network analysis was conducted on 24 anti-endometriosis targets of Danbie Capsules, and seven critical targets (TP53, AKT1, FOS, HSP90AA1, MAPK8, NOS3, MMP2) were identified according to network metrics, including TP53 (regulating cell transcription and apoptosis), AKT1 (regulating cell proliferation), FOS (involving in signal transduction, cell proliferation, and differentiation), HSP90AA1 (facilitating the maturation, maintenance, and precise regulation of specific target proteins, ensuring their proper structural integrity and functionality), and MAPK8 (inducing cell proliferation, differentiation, migration, transformation, and programmed cell death). NOS3 is involved in promoting the relaxation of vascular smooth muscle, and MMP2 is involved in extracellular matrix breakdown. The critical component of Luteolin was Macromolecular docking with its two critical targets. In the visualization of the results, it can be seen that there are hydrogen bonding forces and other Intermolecular forces between the small molecule Luteolin and the surrounding amino acid residues, making it stably bound to the active pockets of each protein; The docking results showed that the affinity was less than −5kcal/mol, indicating that they were easy to combine and might contribute to the anti-endometriosis effects of Danbie Capsules. The analysis of GO function and KEGG pathway enrichment of 24 critical targets of the Danbie Capsules against Endometriosis was carried out. The analysis of the GO function enrichment revealed that the Endometriosis therapy with the Danbie Capsules was mainly involved in the response to hormones in vivo, the response of cells to organic nitrogen compounds, and the response of cells to nitrogen compounds. The analysis of the KEGG pathway enrichment revealed that the possible signal pathways of the Danbie Capsules in the Endometriosis therapy were mainly focused on the human T-cell leukemia virus 1 infection pathway, hepatitis B pathway, and cancer pathway. HTLV-1 primarily infects CD4+ T cells, which are the critical cells in the triggering and establishing of the adaptive immune response. 10 HBV, a hepatotropic virus, has the propensity to induce severe liver diseases, such as acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Epigenetic alterations, in conjunction with genetic alterations, have long been regarded as the pivotal drivers in the process of carcinogenesis. DNA methylation, histone modifications, and RNA-mediated regulation have been implicated in a multitude of cellular processes critical for the initiation and progression of cancer. A multitude of chromatin-associated and modifying proteins govern these intricate processes, subject to the regulatory influence of signaling pathways. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT) exerts regulatory control over a myriad of biological processes and is commonly dysregulated in human cancers. A growing body of evidence suggests that critical epigenetic modifiers are subjected to direct or indirect modulation by PI3K/AKT signaling. Therefore, it contributes to the oncogenicity of the PI3K cascade in cancers. 11 TP53 is the preeminent gene subject to mutations in human cancer, garnering over 100,000 literature citations in PubMed. This pathway holds a prominent position in cancer biology and oncology, tracing its roots back to p53 in 1979. With a myriad of inputs and downstream outputs that contribute to its tumor suppressor role, the p53 pathway constitutes an intricate cellular stress response network. 12 The heterogeneity of KSHV-associated malignancies arises from the interplay of multiple pathophysiologic mechanisms such as chronic antigenic stimulation, immunosup- pression, genetic abnormalities, cytokine release and dysregulation, and co-infection with HIV. There is an increasing acknowledgment that inflammatory manifestations in KSHV-associated malignancies (KSHV-MCD, KICS, and KS-IRIS) have been associated with a significant risk of mortality. 13 Pancreatic cancer is believed to be at least partially driven by the presence of somatic mutations in oncogenes and tumor suppressor genes. The most frequently affected genes in PDAC are the oncogene KRAS and the tumor suppressor genes CDKN2A, TP53, and SMAD4. 14 Genomic alterations in small‐cell lung cancer include TP53, RB1, TP73, NOTCH, MLL2, MYC, PI3K, BCL2, RICTOR. 15 Insulin resistance is significantly associated with metabolic syndrome, risk-related osteoporosis, and other factors. With the implementation of proper treatment using tyrosine-kinase inhibitors, regular monitoring, and favorable response to therapy, patients suffering from chronic myeloid leukemia can now attain a life expectancy comparable to that of individuals without the disease, offering them a near-normal quality of life. 16 The lack of research is due to experimental conditions and funding reasons, which led to the selection of immunohistochemical methods for semi-quantitative analysis of the target protein, which cannot be accurately quantified. Therefore, there was no difference in P53 between the two groups, considering the small sample size and limitations of immunohistochemical semi-quantitative methods. Despite these limitations, AKT1 showed some differences between the two groups.