However, these interactions have not been defined. This study used association rule mining and systems pharmacology to discover a method by which potential antiendometriosis herbs can be investigated. We analyzed various combinations and mechanisms of action of medicinal herbs to establish molecular networks showing interactions with multiple targets. The results showed that endometriosis treatment in Chinese medicine is mainly based on methods of supplementation with blood-activating herbs and strengthening qi. Furthermore, we used network pharmacology to analyze the main herbs that facilitate the decoding of multiscale mechanisms of the herbal compounds. We found that Chinese medicine could affect the development of endometriosis by regulating inflammation, immunity, angiogenesis, and other clusters of processes identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The antiendometriosis effect of Chinese medicine occurs mainly through nervous system–associated pathways, such as the serotonergic synapse, the neurotrophin signaling pathway, and dopaminergic synapse, among others, to reduce pain. Chinese medicine could also regulate VEGF signaling, toll-like reporter signaling, NF-κB signaling, MAPK signaling, PI3K-Akt signaling, and the HIF-1 signaling pathway, among others. Synergies often exist in herb pairs and herbal prescriptions. In conclusion, we identified some important targets, target pairs, and regulatory networks, using bioinformatics and data mining. The combination of data mining and network pharmacology may offer an efficient method for drug discovery and development from herbal medicines.Endometriosis is a common benign disease in women of reproductive age. It has been defined as a disorder characterized by inflammation, compromised immunity, hormone dependence, and neuroangiogenesis. Unfortunately, the mechanisms of endometriosis have not yet been fully elucidated, and available treatment methods are currently limited. The discovery of new therapeutic drugs and improvements in existing treatment schemes remain the focus of research initiatives. Chinese medicine can improve the symptoms associated with endometriosis. Many Chinese herbal medicines could exert antiendometriosis effects  Endometriosis is a common benign condition in women of reproductive age, which is characterized by inflammation, compromised immunity, hormone dependence, and neuroangiogenesis, etc. . The maiData mining approaches and network pharmacology have been utilized to discover the underlying pathogenesis of the condition . Data miIn this study, data mining was used to analyze the application rules of TCM in the treatment of endometriosis. We also proposed a computational systems pharmacology method and statistical analysis to determine the associated molecular mechanisms. We analyzed an endometriosis biomarker network and identified biologically relevant functional modules of Chinese medicine targets in endometriosis. These findings would provide a better understanding of the mechanism of multitarget regulation of endometriosis in Chinese medicine and shed light on the screening of core compounds. This in turn has facilitated the discovery of novel effective drugs.http://stitch.embl.de/) (The research framework of this study is presented in mbl.de/) , etc. Thmbl.de/) .We conducted an electronic search of the Chinese and English databases from their inception to 31/12/2018. We searched the Chinese literatures in China National Knowledge Infrastructure (CNKI) database and WanFang database. The English databases included Embase and the PubMed database. All types and formulations of herbal medicines were considered, including extracts, decoctions, pills, and natural compounds. The outcomes of clinical studies were not considered.The following search terms were used either individually or in combination: “Chinese medicine,” “traditional medicine,” “herbal medicine,” “oriental medicine,” “herb,” “plant,” “prescription,” “decoction,” and “endometriosis,” among others. All prescriptions for endometriosis treatment were selected and the drug records were complete. Formulations of pills, powders, and decoctions were all evaluated. The name of each herbal formula was extracted, as well as its composition of medicinal herbs, its origin (name of article or ancient literature), author, publication year, and internal/external application. Exclusion criteria included the following: missing prescription; the application of acupuncture, massage, or other external treatments. The method of literature searching is presented in https://mpns.science.kew.org/mpns-portal/), the Plant List (http://www.theplantlist.org), and the Plants of the World Online(http://plantsoftheworldonline.org/). Chemical ingredients were compiled from the TCM Systems Pharmacology (TCMSP) Database (http://tcmspw.com/tcmsp.php), TCM Integrative Database (TCMID) (http://www.megabionet.org/tcmid/), universal natural product database (UNPD) (http://mesh.tcm.microbioinformatics.org/), STITCH database (http://stitch.embl.de), and DrugBank (https://www.drugbank.ca). Finally, the structures of relevant natural compounds in the medicinal plants were retrieved from the NCBI Pubchem dataset (https://www.ncbi.nlm.nih.gov/pccompound/).The names of Chinese herb included the Chinese pinyin and full plant name with taxonomic validation (species name/scientific name). The validated information of herbs/plants names with taxonomic validation were collected from Kew Royal Botanic Garden (e (UNPD) , TCM-MeSThe absorption, distribution, metabolism, excretion, and toxicity (ADMET) modeling as a tool for rational drug design has significant effect in new drug discovery . The druhttps://www.malacards.org/), literature on PubMed, Genbank, and the OMIM database (http://www.omim.org/). (B) Disease-related drug targets were compiled from the TTD (http://bidd.nus.edu.sg/group/cjttd/), DrugBank databases , PharmGKB (https://www.pharmgkb.org), The Drug-Gene Interaction database (DGIdb) (http://dgidb.org/search_interactions), as well as the European Society of Human Reproduction and Embryology (ESHRE) guidelines (https://www.uniprot.org).We also collected targets of endometriosis. To obtain the most reliable results, the following steps were taken: (A) endometriosis-associated genes were integrated from three different sources: the Genecard database, Malacards database : The compatibility rules of the couplet medicinal/group medicinal were extracted according to association rule analysis. The strength of the association rule was measured in terms of its support, confidence, and lift. ARM was used to demonstrate the prescription .ARM and RNNs analysis could further reflect the compatibility rules. Based on the above, combined with the results of previous literature reviews, further screening of TCMs, drug pairs, and compound pharmacological analyses could be performed.http://www.genome.jp/kegg/) is a database for large-scale systematic analysis of molecular interaction networks of genes or proteins . Significant pathway terms of KEGG were mapped into a bubble graph. The big and higher bubbles represent those highly significantly enriched pathway terms.To better understand the processes associated with endometriosis and Chinese medicine treatment, we performed GO terms and KEGG pathway enrichment analyses. The GO enrichment analysis provides three structured networks of defined terms to describe gene attributes. Enriched GO terms are classified according to biological process (BP), molecular function (MF), and cellular component (CC). The KEGG (proteins . The DAVhttp://www.string-db.org) to predict the interactions. The Cytoscape software 3.7.2 (http://cytoscape.org/) was used to visualize networks. To identify crucial relationships in the PPI network, potentially overlapping modules that were densely connected were subsequently identified using Molecular Complex Detection (MCODE) plugin in the Cytoscape program. The MCODE plugin was used to re‐analyze the clusters among the network according to the k‐core = 2. A value of P < 0.01 was considered the significant threshold. And Cytoscape plugin cytohubba (Protein-protein interaction (PPI) networks included information on the biological processes and molecular functions of cells . We usedytohubba  was usedhttps://www.genome.jp/kegg/tool/map_pathway1.html). Based on the mechanism of endometriosis, multiple pathways were integrated and overlapped according to cross-talk targets in these maps. Based on the cross-talk of the pathways, we further constructed a targets-pathways network of Chinese medicine treatment.To better analysis the holistic mechanism of Chinese herbs in endometriosis treatment, the subnetworks pathway was compiled by following the procedures: All targets of Chinese herbs in endometriosis treatment were submitted to an online tool KEGG Search Pathway (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25628) gene expression profiles comprised 22 samples (GPL571 Affymetrix Human Genome U133A 2.0 Array), with eight ectopic samples and eight eutopic endometrium samples from several affected women in the proliferative phase, and normal health donors as the control. The limma package in the R software was used to compare the differentially expressed genes (DEGs) between eutopic endometrium samples group and ectopic endometrium samples group, and the DEGs between eutopic endometrium samples group and endometrium in normal health women group. In addition, these two sets of DEG profiles were used to predict compounds by the Connectivity Map O2 (CMap)   to determine the natural compounds for further consideration.The Connectivity Map (CMap) is an online pharmacogenomic database using gene-expression signatures from cultured cells treated individually with various chemicals to connect small molecules, genes, and disease. The exp2, 2017) . The repWe established a network analysis of the relevant compounds targets. The components of the target networks for the herbs were constructed using the Cytoscape software 3.7.2. The nodes in each network were evaluated based on three indices: degree, node betweenness, and node closeness. Degree indicated the number of edges between a single node and other nodes in a network. Node closeness represented the inverse of the sum of the distance from one node to other nodes. The importance of a node in a network was indicated by the values of these indices, with higher values indicating greater importance.in vitro, 210 literatures were in vivo, and one literature was both in vitro and in vivo). The major TCM literatures were clinical observational studies. Fifteen literatures were clinical randomized controlled studies. Some literatures contain two or three prescriptions according to different syndromes in TCM. A total of 615 medicinal prescriptions were selected from the literature screened (Guizhi Fuling Wan (GFW), Jiawei Sanleng Wan, Neiyi Fang, and Shaofu Zuyu decoction.In the past, we have studied the prescriptions of Chinese medicine for endometriosis through literature monographs. A total of 551 literatures were screened: 513 literatures in Chinese databases (including CNKI and WanFang databases) and 38 literatures from the PubMed and Embase database. Among those screened, 315 literatures were clinical studies, and 234 literatures were experimental studies , Sparganium stoloniferum (Buch.-Ham. ex Graebn.) Buch.-Ham. ex Juz.[Typhaceae] (Sanleng), and Corydalis yanhusuo (Y.H.Chou & Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu(Yanhusuo) are both effective in promoting blood circulation to alleviate pain and activate qi to resolve stagnation in Chinese theory. The treatment of endometriosis with basic theory of Chinese medicine has always been based on promoting blood circulation to minimize blood stasis and alleviate pain.The total 231 kinds of Chinese herbs were selected in literatures. The top 20 herbs were determined using frequency statistics and are presented in The characteristics of four properties, five tastes, and channel tropism were evaluated. The analysis of herbal properties is presented in S. stoloniferum (Sanleng)-C. phaeocaulis(Ezhu); C. phaeocaulis-Paeonia lactiflora Pall(Chishao); and C. yanhusuo (Yanhusuo)-Angelica sinensis (Oliv.)Diels (Danggui), among others. The main roles of these herbs were to promote blood circulation and relieve pain. Frequent itemset mining is a crucial data mining task with numerous applications in discovery, including recommendation and classification, among others . By consTo uncover latent knowledge, unsupervised machine learning and RNNs were applied to generate the knowledge graph, by calculating the distance between knowledge element vectors, according to preset categories. We set various parameters, and obtained multiple knowledge graphs. We obtained a ratio of nine clusters/four clusters of knowledge maps according to the clinical medication rules of Chinese medicine. Different clusters were formed by setting parameters and applying the combination rules of TCM. These clusters may reflect the law of compatibility and combination of clinical prescriptions in TCM. We collected a total of 1,289 endometriosis-related targets from Genecard, Genbank, as well as the OMIM records of genomic databases of human diseases. The Venn diagram in The majority of the pathways were related to cytokine-cytokine receptor interactions, the PI3K-Akt signaling pathway, focal adhesion, the TNF signaling pathway, and HIF-1 signaling pathway, among others (cytoHubba in Cytoscape to find the hub genes. The hub genes showed in We used the Typha angustifolia L. [Typhaceae] (Puhang) and Faeces Togopteri (Wulingzhi) represented one herbal pair (HP). The source of F. Togopteri is Trogopterus xanthipes feces; thus, we did not analysis this HP. The single herbs, HPs, and prescriptions were evaluated, to determine the characteristics of Chinese medicine more comprehensively. In order to further corroborate our predictions, a literature review was conducted to determine whether the compounds were already experimentally validated for any associated therapeutic effects. We analyzed gene enrichment for the major Chinese herbs, as hub herb associated targets. Compounds and targets from of major Chinese herbs for endometriosis treatment are listed in Chinese herbal medicines each contain dozens or even hundreds of various ingredients. Thus, the compilation of an ingredient database is crucial. Some Chinese medicines are animal-derived compounds that were not included in the network pharmacological analysis. Salvia miltiorrhiza Bunge were found, including lipophilic diterpenoids, water-soluble phenolic acids, and other constituents. The active ingredients of S. miltiorrhiza include tanshinone I, tanshinone IIA, salvianolic acid, and dihydrotanshinone, among others . Compounds such as ursolic acid, rosmarinic acid, ferulic acid, caffeic acid, tanshinone IIA, protocatechuic acid, and tetramethylpyrazine could evidently regulate pain-associated targets to reduce pain, based on the network pharmacological analysis.More than 200 compounds from g others . A totalAngelica sinensis var. wilsonii (H.Wolff) Z.H.Pan & M.F.Watson is predominantly known as a treatment for intractable gynecological disorders. Ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) showed that A. sinensis contains eight components, including ferulic acid, senkyunolide A, butylphthalide, ligustilide, butylidenephalide, senkyunolide I, senkyunolide H, and levistolide A  could effectively attenuate acute inflammatory and neuropathic pain. The main alkaloid contents and composition of C. yanhusuo includes protopine, α-allocryptopine, tetrahydrocolumbamine, coptisine, palmatine, berberine, dehydrocorydaline D, L-tetrahydropalmatine, tetrahydroberberine, corydaline, and tetrahydrocoptisine, as determined by the high performance liquid chromatography-diode array detector (HPLC-DAD) method. A total of 165 targets were possibly related to C. yanhusuo, and 47 targets were associated with endometriosis genes. The KEGG enrichment analysis revealed pathways associated with cancer, calcium signaling, neuroactive ligand-receptor interactions, VEGF signaling, apoptosis, T cell receptor signaling, and B cell receptor signaling pathway, among others.Conioselinum anthriscoides ‘Chuanxiong’ is used to regulate menstruation and relieve pain in multiple diseases. A total of 174 ingredients of L. chuanxiong and 22 compounds demonstrated favorable bioavailability  Engl., the Commiphora species, known as “myrrh,” are characterized by resinous exudates from the bark of plants. They are used in the treatment of trauma, arthritis, and fractures, and exert antiproliferative, antioxidant, antiinflammatory, and antibacterial effects -germacrone-1,4-diepoxide, aromadendrene, hederagenin, epoxycaryophyllene, and calarene showed relatively higher levels of OB and DL  J.Presl [Lauraceae](Guizhi); 78 targets in Paeonia suffruticosa Andrews [Paeoniaceae](Mudanpi); 135 targets in Paeonia lactiflora Pall.(Chishao); 67 targets in Prunus persica (L.) Batsch(Taoren); and 55 targets in Poria cocos(Schw.)Wolf(Fuling). In Cinnamomum cassia, taxifolin; beta-sitosterol; sitosterol; catechin; ent-epicatechin; and peroxyergosterol all showed effective OB and DL levels. Protocatechuic acid, coumarin, cinnamyl alcohol, 2-methoxy cinnamic acid, cinnamic acid, and cinnamaldehyde are considered the core components of C. cassia, and the core herbs in such prescriptions. The target proteins in C. cassia were focused on neurological disease, inflammatory disease, cancer, cellular growth and proliferation, cell signaling, and molecular transport. P. lactiflora is used for pain and blood stasis, and has hematopoietic functions, antiinflammatory activity, and immunological properties. And a total of 94 of its targets were associated with endometriosis treatment. Furthermore, 70 targets in P. suffruticosa, 16 targets in P. cocos and 10 targets in P. persica could be associated with endometriosis genes.GFW was the core prescription used in data mining for endometriosis treatment, as well as the classical prescription in Chinese medicine. A total of 565 herbal compounds could be identified from databases. There were 230 targets in In the present study, we obtained 521 targets , adrenoceptor beta, HTR2A prostaglandin endoperoxide synthase (PTGS), brain-derived neurotrophic factor (BDNF), serotonin receptors  are all related to the regulation of neural receptors. The regulatory mechanism of these targets can directly affect various neurotransmitter receptors and the synthesis of neurotransmitters to modulate pain. Chinese medicine could also indirectly affect pain through downstream immune inflammatory factors , and immuno-inflammatory pathways, such as cytokine-cytokine receptor interactions, the TNF signaling, VEGF signaling, HIF-1 signaling, toll-like receptor signaling, and PI3K-Akt signaling pathway. Estrogen could also regulated of the central serotoninergic system in pain.The second module was associated with cell growth and death, invasion, adhesion, and angiogenesis-related signaling pathways in endometriosis. Chinese medicine could regulate invasion and adhesion-related signaling pathways, induce apoptosis, and inhibit cell proliferation, through processes such as apoptosis, P53 signaling, PI3K-Akt signaling, Wnt signaling, ECM-receptor interaction, and focal adhesion, among others. The angiogenesis and the tumor-related functions also showed considerable overlap, which included HIF signaling and the VEGF signaling pathway.The third module was associated with inflammation and the immune system. Related processes included cytokine-cytokine receptor interaction, toll-like receptor signaling, T cell receptor signaling, B cell receptor signaling, NF-κB signaling, and IL-17 signaling pathway, which are also important in regulating inflammation and immune responses.Moreover, kinase signaling pathways in endometriosis could affect the proliferation and differentiation of endometriosis cells, which could be potential targets for non-hormonal therapeutics. The associated module could be related to the canonical IKKβ/NFκB pathway, MAPK pathways, the PI3K/AKT/mTOR pathway, and the AMPK signaling pathway . These rIn order to clarify the similarities and differences between the related targets of Chinese medicine and conventional drugs for endometriosis, we considered the targets of conventional therapeutic drugs for comparison. At present, the treatment options for endometriosis include NSAIDs and hormonal drugs . A total of 85 protein targets of these drugs were identified from DrugBank and the TTD database of conventional drugs. The drug targets of endometriosis conventional treatment are listed in AR, BCHE, BCL2, CYP19A1, CYP1A2, CYP2C8, CYP3A4, ESR1, NR1I2, NR3C2, PGR, PLAT, PPARA, PPARG, PTGS1, PTGS2, and THBD) coincided with the targets of conventional drugs for endometriosis treatment. The enrichment of overlapping genes was mainly focused on the regulation of hormones and pain relief. The mechanism of action of Chinese herbs was similar to that of current conventional treatments. Furthermore, Chinese medicine and its compounds provided more possibilities for multitarget therapy.A total of 17 Chinese medicine associated targets . A total of 67 upregulated and four downregulated DEGs with ectopic endometrium and eutopic endometrium were detected among the patients with endometriosis.We used expression profile data of CMap analysis for drug discovery. By querying CMap, we screened compounds in likely drug targets showing a similar gene expression profile with the desired and chemopreventive conditions. The DEGs were downloaded from the GSE25628 expression file. This dataset contained three sets of sample data, and we chose the comparison of two sets of samples for analysis. The endometriosis eutopic groups were compared with the non-endometriosis eutopic group , to include 52 upregulated and 169 downregulated DEGs  is an important associated pathology in Chinese medicine. Based on the data mining results, most of the common prescriptions for the treatment of endometriosis originate from classic prescriptions in ancient Chinese treatment of dysmenorrhea. These discovered herbs combinations and prescriptions could provide guidance for clinical treatment. The use of data mining methods to screen the core prescriptions of TCM also provides a new method for discovery of TCM.Framework for Automated Knowledge Graph Construction Towards Traditional Chinese Medicine” in our work could also aid the discovery of pharmaceutic rule of Chinese medicines. These tools are crucial to establish the effectiveness of Chinese medicine/herbs in endometriosis clinical treatment.Machine learning and deep learning methods provide a new method to extract informative medicinal and pharmaceutical research . Data miWe selected some commonly used herbs and HPs to provide a source for network pharmacology. To provide better clinical evidence, the quality of modern clinical research in Chinese medicine is in urgent need of improvement. The mechanisms of animal-derived Chinese medicine, as well as some herbal Chinese medicines, need further analysis and exploration.In this study, we discovered potential compounds for the treatment of endometriosis. Studies on phytochemicals have been mostly focused on polyphenolic compounds, sesquiterpenes, terpenoids, flavonoids, alkaloids, polysaccharides, and steroid glycosides. The molecular biological approach has further helped to elucidate the mechanisms of HPs and their pharmacological actions. TCM has advantages in the regulation of endometriosis pain, can directly regulate neurotransmitters, and indirectly regulate pain by modulating inflammation and immunity.in vivo and in vitro experiments are consistent with the analysis of network pharmacology. This suggests that TCM could reduce ectopic lesions and inhibit the development of endometriosis by intervening inflammation, immunity, and inducing apoptosis.Chinese herbs may interfere with the development of endometriosis, during the processes of invasion, adhesion, angiogenesis, immune inflammation, and oxidative stress, among other processes, such as inhibition of the epithelial-mesenchymal transition. The results of the current The potential effective compounds based on network pharmacology have also been validated in experiments. The effects of compounds in endometriosis treatment typically induce apoptosis to reduce lesions through various pathways. For example, nerolidol could reduce the average volume of lesions in rats with endometriosis . Beta-caTo analyze the role of various modules, we discuss only the main modules of Chinese medicine treatment in endometriosis.Increased levels of proinflammatory cytokines have been observed from endometriotic lesions, based on examination of the peritoneal fluid of women with endometriosis. These cytokines are also responsible for excessive sensory innervation and the development of chronic pelvic pain . The resC. anthriscoides ‘Chuanxiong’ and C. yanhusuo could reduce pain via HTR3A, HTR2C, HTR2A. Furthermore, PTGS, also known as cyclooxygenase, is the key enzyme in prostaglandin biosynthesis. It acts both as a dioxygenase and a peroxidase, and may be a core target of TCM for pain. Prostaglandin E2 (PGE2) production by PTGS2/cyclooxygenase 2 (COX-2) is known to be a critical inflammatory factor in endometriosis-associated pain  and Curcuma phaeocaulis Valeton alleviate mechanical hyperalgesia in models of chronic inflammatory and neuropathic pain in mice , vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinases (MMPs)] associated with endometriosis  by upregulating VEGF and activating extracellular signal-regulated kinase (ERK) . Tanshinare also active compounds that are present in several other types of Chinese herbs. These compounds could form complexes with NF-κB and IκB in the cytoplasm  occurs in women with endometriosis, and this can promote survival, growth, invasion, differentiation, angiogenesis, and immune escape of the endometriotic lesions . Rutin, ytoplasm . Curcumiytoplasm .Imbalance of protein kinases can lead to uncontrolled cell proliferation by stimulating the tumor formation process, leading to kinase-dependent tumor growth. Chinese medicine could play an active role in regulating the kinase signaling pathways. These phenomena are also related to the occurrence and development of endometriosis. The kinase signaling pathway represents a viable target for the treatment of endometriosis . Major sThe PI3K-Akt-mTOR pathway is one of the major signaling pathways in endometriosis. Furthermore, mTOR is a key kinase downstream of PI3K-Akt, which regulates tumor cell proliferation, growth, survival and angiogenesis. The expression of the PI3K-Akt pathway  is related to the severity of endometriosis . Naturalvia the c-Jun N-terminal kinase (JNK) 1/2 and NF-κB signaling pathways in breast cancer cells , as a basic HP was first recorded as Sanleng Wan in “Jingyan Liangfang” during the Qing dynasty. The pairs possess extensive activities, including anticancer activity, antiinflammatory activity, and antithrombotic activity , and monoterpene glycosides. Paeoniflorin could inhibit the plantar incision-induced microglia TLR4/matrix metalloproteinase (MMP-9)/MMP2/IL-1β signaling pathway and suppress postoperative pain in mice   , metabol design) , mass sp design) , and new design) , which wEndometriosis is a common and difficult disease in gynecology. The mechanism of TCM in the treatment of endometriosis is realized through functional modules, such as inhibiting inflammation, enhancing the immune response, regulating angiogenesis-related pathways, inhibiting the epithelial-mesenchymal transition, and inducing apoptosis. The use of data mining combined with bioinformatics techniques could help us understand the associated targets and pathways networks, candidate genes in endometriosis, and the mechanisms of action of Chinese medicine. Combined techniques may also offer an efficient method of drug discovery and development from herbal medicines.in vitro or in vivo experiments should be performed to validate the predicted herbs/compounds and targets.However, there are still several potential limitations in the study and need to improve. First,the mechanism of endometriosis needs to be further clarified based on molecular biology and multiomics technology. Moreover,the antagonistic or agonistic effects of compounds–targets pairs should be further clarified. It is essential to improve the methods in data mining and network pharmacology. Further All datasets generated for this study are included in the article/All authors were responsible for the study concept and design: WZ and JWu have contributed equally for this work. WZ drafted the paper. JWu help drafted the paper. WZ, JWu, and JWa participated in the literature search. WZ, JWu, and HW participated in data mining. WZ, JG, and TW participated in network pharmacology analysis. LC and XL supervised the study. All authors approved the final paper.This study is supported by the National Natural Science Foundation of China (81574008), the National Natural Science Foundation of China (61871141), Special Research Project for Traditional Chinese Medicine of Guangdong Hospital of TCM (NO. YN2016ML05), and Situ Yi Renowned Medical Heritage Studio.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ginkgo biloba extract (EGb) can prevent and treat aging-related diseases, but its pharmacological effects need to be further clarified. This study aimed to propose a network pharmacology-based method to identify the therapeutic pathways of EGb for aging. The active components of EGb and targets of sample chemicals were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. Information on aging-related genes was obtained from the Human Ageing Genomic Resources database and JenAge Ageing Factor Database. Subsequently, a network containing the interactions between the putative targets of EGb and known therapeutic targets of aging was established, which was used to investigate the pharmacological mechanisms of EGb for aging. A total of 24 active components, 154 targets of active components of EGb, and 308 targets of aging were obtained. Network construction and pathway enrichment were conducted after data integration. The study found that flavonoids  and beta-sitosterol may be the main active components of EGb. The top eight candidate targets, namely, PTGS2, PPARG, DPP4, GSK3B, CCNA2, AR, MAPK14, and ESR1, were selected as the main therapeutic targets of EGb. Pathway enrichment results in various pathways were associated with inhibition of oxidative stress, inhibition of inflammation, amelioration of insulin resistance, and regulation of cellular biological processes. Molecular docking results showed that PPARG had better binding capacity with beta-sitosterol, and PTGS2 had better binding capacity with kaempferol and quercetin. The main components of EGb may act on multiple targets, such as PTGS2, PPARG, DPP4, and GSK3B, to regulate multiple pathways, and play an antiaging role by inhibiting oxidative stress, inhibiting inflammation, and ameliorating insulin resistance.Aging is a main risk factor for a number of debilitating diseases and contributes to an increase in mortality. Previous studies have shown that  Aging is inevitable, but pathological aging poses a serious threat to human health, reduces the quality of life of elderly individuals, and promotes the development of related diseases , 2. AginGinkgo biloba (EGb), a well-known medicinal plant, is used to treat many aging-related diseases, such as cardiovascular and neurodegenerative aging-related diseases [The extract of diseases . Due to diseases . EGb prediseases , 11. Prediseases , 12–14. http://lsp.nwsuaf.edu.cn/tcmsp.php). TCMSP is an integrated systems pharmacology platform of Chinese herbal medicines that covers chemicals, targets, and drug targets and pharmacokinetic properties for compounds involving oral bioavailability (OB), drug likeness (DL), blood-brain barrier, intestinal epithelial permeability, and aqueous solubility [The active constituents of EGb were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (lubility . In thislubility . In addihttp://www.uniprot.org/). The UniProtKB is the central hub for the collection of functional information on proteins, with accurate, consistent, and rich annotation. The target protein names were inputted into UniProtKB, with the organism restricted to “Homo sapiens,” eventually gaining the official symbol.The validated target proteins of the active components were obtained from the TCMSP database. The target protein name of the active ingredient was converted to the standard target gene name through the UniProt Knowledge Base  and JenAge Ageing Factor Database . HAGR is a collection of databases and tools for studying human aging through modern approaches, such as functional genomics, network analysis, systems biology, and evolutionary analysis [Aging-related genes were obtained by retrieving data from the Human Ageing Genomic Resources (HAGR) database  provided a comprehensive set of functional annotation tools to understand the biological meaning behind a large list of genes. DAVID tools were able to identify enriched biological themes, particularly GO terms that involved biological process (BP), cell component (CC), and molecular functions (MFs) and visualized genes on KEGG pathway maps . The tarP < 0.05 , 22.https://pubchem.ncbi.nlm.nih.gov/), and target proteins were retrieved from the Protein Data Bank (http://www.rcsb.org/pdb). The main pharmacodynamic components of Ginkgo biloba were docked with the core target gene by AutoDock. PyMOL was used to display the interaction diagram between receptor proteins and ligand small molecules.In the analysis of the component-target action network, it is generally believed that a node with greater degree is the pivot node in the whole network. Therefore, the components of the core network were molecularly docked with the target. The 3D structure of the core components was obtained using PubChem .The We performed KEGG pathway enrichment analysis to examine the pathways for 27 putative therapeutic targets of EGb for antiaging. The top 20 pathways were determined , mainly Y-axis represents significant pathways of the target genes, and the X-axis shows the rich factor. The rich factor represents the ratio of the number of target genes belonging to a pathway to the number of all annotated genes located in the pathway. A higher rich factor represents a higher enrichment level. The color of the dot corresponds to different P values, and the size of the dot reflects the number of target genes expressed in the pathway.The Eight proteins were docked with four components using AutoDock, and 20 conformations were obtained. The binding energy scoring and docking parameters are shown in The binding pattern between receptor protein PPARG and beta-sitosterol ligand small molecules is presented in The binding pattern between the receptor protein PTGS2 and kaempferol ligand small molecules is shown in The binding pattern between the receptor protein PTGS2 and quercetin ligand small molecules is shown in Previous studies have shown that EGb is effective in the prevention and treatment of aging-related diseases –14. HoweThese eight targets, namely, DPP4, GSK3B, CCNA2, AR, ESR1, PTGS2, PPARG, and MAPK14, may play a crucial role in the antiaging network of EGb. DPP4 and GLP-1 had been found to play important roles in oxidative stress, lipid metabolism, insulin resistance, and inflammation. Recently, a study found that the balance between DPP4 and GLP-1 might be a therapeutic target for the management of vascular aging and atherosclerosis in animals . StudiesIn the GO enrichment analysis, the targets were closely related to positive regulation of transcription from RNA polymerase II promoter, nitric oxide biosynthetic process, smooth muscle cell proliferation, and negative regulation of the apoptotic process. CCs involve the nucleoplasm nucleus. MFs involve the binding of enzymes, proteins, and transcription factors. According to a study by Nakazawa et al., RNA polymerase II ubiquitination provides a two-tier protection mechanism by activating transcription-coupled nucleotide excision repair and, in parallel, processing of DNA damage-stalled RNA polymerase II, which together prevent prolonged transcription arrest and protect against neurodegeneration . Nitric α) contribute to decreased activation of signaling molecules, including PI3K and AKT, leading to insulin resistance [T cell receptor signaling pathway, Toll-like receptor (TLR) signaling pathway, and NOD-like receptor signaling pathway are responsible for generating innate immune responses and play a critical role in inflammation. Toll-like receptors play a significant role in promoting aging adipose tissue inflammation, and the study showed that old TLR4-deficient mice have improved glucose tolerance compared to age-matched wild-type mice [β-catenin signaling pathway is closely related to stem cell aging [KEGG pathway enrichment analyses demonstrated that EGb is involved in the regulation of multiple pathways. Increased oxidative stress and inflammation, including the HIF-1 and TNF signaling pathways, play significant roles in aging, especially vascular aging . The neusistance –55. The sistance . Focal asistance –59. Focasistance . Studiessistance . The MAPsistance . Inactivype mice . A diffeype mice . The FOXype mice . Vasculaype mice . Wnt proll aging .In this study, the active components and mechanism of EGb for aging were analyzed based on network pharmacology. Four compounds, eight targets, and 20 significant pathways in EGb were identified by network analysis, which explained the mechanism of EGb for aging by multiple components, targets, and pathways. Our study found that flavonoids  and beta-sitosterol, the main active components of EGb, might slow aging by inhibiting oxidative stress, inhibiting inflammation, ameliorating insulin resistance, and regulating BPs . Molecular docking results showed that PPARG had better binding capacity with beta-sitosterol, and PTGS2 had better binding capacity with kaempferol and quercetin. However, additional experiments must be conducted to verify these results for more evidence in the future.


Root canals were obturated with 35/0.04 gutta-percha (single-cone technique). The samples were obtained from apical, middle, and coronal thirds. Dentinal tubule penetration depth and percentage of penetration around the canal perimeter were measured. The statistical analysis was performed using Mann Whitney U test and Wilcoxon t-test . ES exhibited a significantly higher penetration than AHP in apical and middle thirds (p < 0.05), and in middle and coronal thirds relative to BR (p < 0.05). The percentage of penetration around the canal perimeter was significantly higher for ES compared to BR in all thirds, but only in the apical third for AHP (p < 0.05). The pre-mixed silicate-based sealer exhibited better penetration than the powder/liquid one.Dentinal tubule penetration influences root canal treatment sealing. The aim of this study was to compare dentinal penetration of two clinical presentations of silicate-based sealers using confocal laser. Sixty single-rooted human teeth from 50–70 year-old patients extracted for orthodontic/periodontal reasons were used. Canals were prepared using Mtwo system up to 35/0.04, with 5.25% NaOCl irrigation and final irrigation using 17% EDTA. Teeth were randomly assigned into study groups (n The objective of root canal obturation is to hermetically seal the canal system so that it is isolated, and thus prevent any leakage into or out of the root. The sealer applied in the process of obturation is responsible for dentin sealing, regardless of the technique used ,2. DiffeUnder the present criteria, endodontic sealers are expected not only to seal the root canal system, but also promote the healing of injured periapical tissues to produce a biological apical seal.Currently, new silicate-based sealers with a composition inspired by mineral trioxide aggregate (MTA) are available. These materials are formed, mainly, by calcium silicates and phosphates, and calcium hydroxide . They arEndoSequence BC Sealer  is a premixed silicate-based material presented in a ready-to-use format . BioRootSeveral studies have verified the excellent biocompatibility ,9 and biDentine tubular penetration represents a physical barrier for microorganisms and increases the retention of the sealer ,18, hencConfocal Laser Scanning Microscopy (CLSM) is a widely used technique for the study of tubular penetration of various materials. Several published studies in the field use this method to evaluate the penetration of sealing cements ,17,18,19Due to the increasing interest and clinical use of these endodontic biomaterials, there is a need to expand the existent information in this regard. The identification of significant differences in tubular penetration between the different presentation forms of the sealers could potentially lead to an increased clinical use of one form or another.Accordingly, the objective of the present in vitro study was to evaluate the dentinal tubular penetration of different endodontic sealers by comparing two silicate-based sealers, one premixed and the other in powder/liquid form with each other, and with a resin-based sealer; by means of an analysis with confocal laser scanning microscopy. The null hypothesis was that there is no difference between the tested materials in their tubular penetration.Universitat de València (ref. H1460624886119). Teeth with root resorptions, carious lesions , immature apices, fractures, or previous root canal treatments were excluded.Maxillary and mandibular single-rooted teeth extracted from 50–70 year-old patients due to periodontal or orthodontic reasons were selected (N = 60), all of which presented a type I canal configuration according to Weine’s classification , and an The selected teeth were decoronated at 15 mm from the apex to standardize sample lengths and they were prepared at a working length of 14 mm with the Mtwo rotary system  up to a 35/0.04 file, having previously achieved patency using K10 files . Irrigation during instrumentation was performed using 3 mL of 5.25% sodium hypochlorite , and final irrigation was carried out with 17% EDTA  during 1 min, followed by 5 mL saline solution.i = 20), according to the silicate-based sealers: group 1, EndoSequence BC Sealer (ES); group 2, BioRoot RCS (BR), and a control group (nc = 20) AH Plus (AHP). The sample size was established by comparison with similar studies in the field [Samples were randomly assigned to 2 study groups  to make them visible under confocal laser scanning microscopy (CLSM). One g of endodontic sealer was weighed on an analytic scale  with a precision of 0.0001 g. Next, 0.002 g of the Rhodamine B was weighed.Endodontic sealers were prepared following their manufacturers’ instructions, adding a fluorochrome  by means of the single-cone technique.During the storage period, before and after the instrumentation and obturation, the roots were kept in a 100% humidity environment. After a minimum of 72 h of storage, to ensure the complete setting of the materials, samples were obtained from the apical, middle, and coronal thirds at 3, 5, and 8 mm from the root apex, respectively, using a diamond disc to obtain a 1 mm section of each third. The obtained samples were fixed onto microscope slides and were polished using fine and superfine grain SoftLex discs . The samples were then stored in a light-free environment to avoid a previous exposure of the fluorochrome to light before it was viewed under CLSM.The equipment used for the microscopic study was CLSM Leica TCS SP2  with 543 nm and 578 nm excitation and emission wavelengths, respectively. Sample analysis was performed using 10× magnification. Images were registered and posteriorly analyzed via ImageJ v1.51 software . Dentinal tubule penetration depth (in μm) and the percentage of penetration around the canal perimeter was measured from the images obtained by CLSM .® SPSS V25.00 . Distribution was evaluated by the Kolmogorov Smirnov test, and it was determined that the variables did not present a normal distribution. Accordingly, values of penetration into dentinal tubules of the two silicate-based sealers were compared between them and with AH Plus sealer (control) in each root third  by U Mann Whitney test. Comparisons between thirds in each group was performed two by two by means of the Wilcoxon T-test. The confidence interval was established at 95%.Data obtained were statistically analyzed using IBMp < 0.05), and in the middle and coronal thirds relative to BR. The percentage of penetration around the canal perimeter was significantly higher for ES compared to BR in all thirds, but only in the apical third for AHP (p < 0.05).The quantitative results of the CLSM image analysis in terms of the tubular penetration depth and percentage of dentinal tubule perimeter penetration are presented in T-Test. Both in the silicate-based sealers and AHP groups, the penetration depth was significantly decreased from the coronal to the apical thirds (p < 0.05). In relation to the percentage of dentinal tubule perimeter penetrated by cement, ES group showed significant differences between the coronal and apical thirds (p < 0.01); BR group between the coronal and apical thirds, and between the coronal and middle thirds (p < 0.005); while AHP exhibited significant differences between all thirds (p < 0.05).Comparisons within each group, between thirds were performed two by two by means of the Wilcoxon p < 0.05).To the authors’ knowledge, the novelty of this paper resides in the fact that this is the first study to explore the differences in dentinal tubule penetration between silicate-based endodontic sealers in terms of their form of presentation (pre-mixed or powder/liquid).ES and BR are two pure silicate-based sealers. Although they present a similar composition, they differ in their presentation. While ES comes premixed, in a ready-to-use format, BR is available in powder/liquid form. This difference means that, despite the fact that both materials require water to start and complete their setting reaction, ES will only depend on the remaining humidity in the dentinal tubules, while BR starts its reaction when mixing it .Currently, no endodontic sealer meets all the ideal properties expected of them, such as dimensional stability, biocompatibility, and adhesion to root canal walls . HoweverCLSM is a widely used procedure for the study of tubular penetration ,26,27,28Techniques like scanning electron microscopy (SEM) have also been used for the study of tubular penetration ,31,32,33In relation to the results of the present study, ES showed a higher penetration compared to the other sealers although not significantly in all cases. This may be due, partially, to the size of the sealer particles (<1 μm) , which iEl Hachem et al.  and EymiThe median values showed by AHP are similar to those from various studies ,40,41. AThe assessment of the percentage of penetration around the canal perimeter has an important clinical relevance, acting as a representation of the sealing ability against microorganisms in the dentinal tubules, regardless of the depth of penetration of the sealer ,42. TherWang et al.  reportedDespite the fact that the tubular penetration of BR was not significantly different from that of AHP, the values obtained in our study were lower than ES. The difference in penetration between both silicate-based sealers may be due to their different forms of presentation. Even having followed the manufacturers’ instructions for the preparation of BR, small variations in the dosage during the mixing process may have been produced. Consequently, this may have acted as a limitation, potentially having an effect on the final results by varying the fluidity of the sealer, and thereby, influencing its tubular penetration. Since ES is presented in a premixed, ready-to-use format, its fluidity and consistency are not altered, so they are always optimal.Despite using human teeth extracted for different reasons , the tubular penetration pattern followed the same trend in all teeth. Both for the depth of penetration inside the dentinal tubule and for the percentage of tubular penetration, the apical third showed the lowest results, followed by the middle and coronal thirds; coinciding with other authors ,36,41,42The ready-to-use attribute from the premixed silicate-based sealers, together with their potentially higher dentinal tubule sealing exhibited in the present study, could act as supporting evidence for their clinical use versus powder/liquid sealers. However, the lack of similar studies regarding this comparison can act as a limitation for the applicability of the results of the present study. In order to extrapolate the results into the clinical setting, future studies comparing similar biomaterials under different conditions should be performed.ES exhibited the highest tubular penetration, while no significant differences were found between BR and AHP. Both the maximum depth of penetration inside the dentinal tubules and the percentage of penetration presented higher values in the coronal thirds for all the studied materials. Within the limitations of the present study, it can be concluded that tubular penetration was higher in the premixed endodontic sealer.


One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC.56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples  from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes , and RNA Transcription/Expression.We performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n A large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC. In order to scale network inputs and outputs, we normalized our input matrix so that they had zero mean and unity standard deviation. We then processed the input matrix using principal component analysis (PCA) to reduce dimensionality. The SOM algorithm was then used to cluster the data based on similarity and topology using 100,000 training epochs. The SOM translates the differentially expressed transcriptome profile into a two-dimensional quadratic 7 × 7 pixel map and a color code for similarity values.Self-Organizing Map (SOM) analysis was performed to identify clusters of transcripts with similar expression patterns and was conducted for every time point analyzing pairwise comparisons of p-values and are based upon the match results from up/down-regulation. Given the observed differential regulation of a transcript in the dataset, the activation state is determined for each specific functional pathway, and the directionality effect is then assigned. If an activation z-score can’t be predicted for a significant pathway based on the available data, and after bias correction, NA (white color) is assigned for that specific pathway [Next, we performed functional pathway analysis using IPA  , on sele pathway .Additional file 1.


Depressive disorder is a common mental disorder characterized by depressed mood and loss of interest or pleasure. As the Herbal medicines are mainly used as complementary and alternative therapy for depression. This study aimed at exploring antidepressant activity of Huang-lian Jie-du Decoction (HLJDD), and evaluating active components and potential depression-associated targets. HLJDD was administered on chronic unpredictable mild stress-induced (CUMS) depressive mice. Behavior evaluation was performed through force swimming test (FST), novelty-suppressed feeding test (NSF), and open field test (OFT). Active components of HLJDD, potential targets, and metabolic pathways involved in depression were explored through systemic biology-based network pharmacology assay, molecular docking and metabonomics. FST assay showed that CUMS mice administered with HLJDD had significantly shorter immobility time compared with control mice. Further, HLJDD alleviated feeding latency of CUMS mice in NSFand increased moving distance and duration in OFT. In the following network pharmacology assay, thirty-eight active compounds in HLJDD were identified based on drug-like characteristics, and pharmacokinetics and pharmacodynamics profiles. Moreover, forty-eight molecular targets and ten biochemical pathways were uncovered through molecular docking and metabonomics. GRIN2B, DRD, PRKCA, HTR, MAOA, SLC6A4, GRIN2A, and CACNA1A are implicated in inhibition of depressive symptoms through modulating tryptophan metabolism, serotonergic and dopaminergic synaptic activities, cAMP signaling pathway, and calcium signaling pathway. Further network pharmacology-based analysis showed a correlation between HLJDD and tryptophan metabolism. A total of thirty-seven active compounds, seventy-six targets, and sixteen biochemical pathways were involved in tryptophan metabolism. These findings show that HLJDD acts on potential targets such as SLC6A4, HTR, INS, MAO, CAT, and FoxO, PI3K/Akt, calcium, HIF-1, and mTOR signaling pathways, and modulates serotoninergic and dopaminergic synaptic functions. In addition, metabonomics showed that tryptophan metabolism is the primary target for HLJDD in CUMS mice. The findings of the study show that HLJDD exhibited antidepressant effects. SLC6A4 and MAOA in tryptophan metabolism were modulated by berberine, baicalein, tetrahydroberberine, candicine and may be the main antidepressant targets for HLJDD. MDD patients present with depressed mood, pessimistic thinking, loss of enjoyment in usual activities and lack of energy. Worldwide, more than 350 million people are suffering from depression . World HHypertension patients treated with reserpine often present with depression , therefoAlthough more than a dozen antidepressants are available, most individuals with depression do not respond to these treatments. Most antidepressants are associated with unfavorable long-term outcomes and paradoxical effects, such as induction of depression, worsening of symptoms and withdrawal symptoms . Multi-tIn addition to use of complementary and alternative medicine as prophylactic interventions or treatments as, there is increased interest in use of Chinese herbal medicine for depression treatment. Huang-lian Jie-du decoction (HLJDD), is a well-known Chinese herbal initially recorded in Prescriptions for Emergent Reference over 1500 years ago . HLJDD has heat dissipation and detoxification activities . It is aNetwork pharmacology is an emerging approach for evaluating systematic mechanism of action for multi-target drugs based on biological network associating with a disease. The approach is used to explore biological basis of herbal formulae and bioactive ingredients present. Several herbal formulae for treatment of neurological or mental disorders have been developed through network pharmacology . HLJDD mIn this study, experience-based approach and evidence-based assay were used to explore antidepressant activities of HLJDD, a traditional Chinese medicine formula. We explored the symptoms for depression in CUMS-induced depressive animal model after treatment with HLJDD. Further, network pharmacology assay using bioinformatics tools, systems biology, and polypharmacology was performed to determine genes, proteins, and signaling pathways implicated in activity of HLJDD against depression . CurrentRhizome Coptis , RadixScutellariae , CortexPhellodendri amurensis  and Fructus Gardeniae  were purchased from Fu Chun Tong Chinese Herbal Pieces Co., Ltd. . Herbal materials were authenticated by Dr Qinan Wu . A voucher number was assigned to each specimen and voucher specimens were deposited in Nanjing University of Chinese Medicine. Fluoxetine HCL (FXT) was purchased from Beijing Yinuokai Technology Co., Ltd. .HLJDD extract was prepared according to a protocol described in our previous study . Coptis SPF male C57BL/6 mice, weighing 18–22 g , were housed in a animal laboratory with regulated temperature and humidity of 25 ± 1°C and 60 ± 5%, respectively. Animal were acclimatized for a week and randomly divided into 4 groups: control group, model group (CUMS), fluoxetine hydrochloride group  and HLJDD group . HLJDD and FXT were dissolved in 0.5% CMC-Na. A total of 20 ml/kg body weight for both HLJDD and FXT was administered orally every week. Mice in the control and model group were given 0.5% CMC-Na. HLJDD dosage was calculated using dosage conversion relationship between mice and human and clinical dosage of HLJDD extract. Behavioral experiment was performed 1 h after the last administration and data were analyzed using Video Analysis System for Animal Behavior . The study was carried out following recommendations provided by Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . The Animal Care and Use Committee of Nanjing University of Chinese Medicine approved the protocol and total number of mice used in this study.Chronic unpredictable mild stress (CUMS) mice model is developed by exposing animals to a set of unpredictable mild stressors for several weeks to induce depression-like phenotypes . In thisMice exhibits behavioral despair under unavoidable stressors, which is similar to MDD phenotypes in human . Forced Novelty-suppressed feeding test (NSF) is an effective behavioral paradigm commonly used to evaluate depression-like behaviors in experimental animals after fasting. The test uses the conflict between feeding and fear of a novel environment . After sOpen field test (OFT) is used to measure of exploratory behavior, locomotor activity and general depressive-like activity . Mice weγ-aminobutyric acid (GABA), glutamic acid (Glu), 5-hydroxytryptamine (5-HT), dopamine (DA) and acetylcholine (Ach) in the hippocampus, cortex, striatum and amygdala were determined using LC-MS/MS. Accurately weighed cerebral tissues were added to 10-fold volume of 0.1% formic acid and completely homogenized for 3 min. 100 μL of the homogenate were added to 200 μL 0.2% formic acid acetonitrile, vortexed for 3 min and centrifuged at 12,000 rpm at 4°C for 10 min. Supernatants were concentrated and reconstituted with 0.1% formic acid.Neurotransmitters including A. Ionization source conditions were capillary voltage, 3.0 kV; sampling cone voltage, 30.0 V; source temperature, 150°C and desolvation temperature, 500°C.A 2.1 mm × 100 mm, 2.7 μm chromatographic column  was used. Other chromatographic conditions were; mobile phase consisting of A, 0.1% formic acid in water, and B, acetonitrile; column temperature, 30°C; flow rate: 300 μL/min; injection volume, 2 μL; gradient elution conditions, 0–2 min, 98–95% A; 2–3.5 min, 95–40% A; 3.5–4 min, 40–98% A; 4–6.1 min, 98% p < 0.05 was considered statistically significant. Data analysis was carried out using SPSS 17.0 software.Data were presented as mean ± SD. Analysis of variance (ANOVA with a Fisher’s LSD test) was carried out to determine differences between groups. http://lsp.nwu.edu.cn/tcmsp.php), BATMAN-TCM database (http://bionet.ncpsb.org/batman-tcm/), PharmMapper database (http://lilab-ecust.cn/pharmmapper/index.html), OMIM database (http://www.omim.org), PharmGkb database (https://www.pharmgkb.org/), TTD (http://bidd.nus.edu.sg/BIDD- Databases/TTD/TTD.asp), System Dock database Site database (http://systemsdock.unit.oist.jp), String Version 10.5 server (https://string-db.org/), RCSB PDB database (http://www.rcsb.org/pdb/home/home. do), DAVID database (https://david.ncifcrf.gov/) and Pubchem database (https://pubchem.ncbi.nlm.nih. gov) were used for network pharmacology analysis. Cytoscape 3.8.0 software was used to construct an interaction network. AutoDock Vina software was used to predict the binding mode and binding energy scores between active compounds and molecular targets.TCMSP database  ≥ 30% and drug-likeness (DL) ≥ 0.18 parameters in TCMSP were used to screen BATMAN-TCM results. The final cluster of chemical ingredients of HLJDD was a combination of BATMAN-TCM and TCMSP results.Active compounds were screened using TCMSP and BATMAN-TCM databases. TCMSP was used to predict ADME/T and other biological, chemical and physical parameters of active compounds using mathematical and computational models. SysDT model is used to find relevant targets, map diseases, tissues and organs, and to predict drug efficacy and toxicity mechanism . BATMAN-Targets of the active compounds were predicted using BATMAN-TCM, TCMSP and PharmMapper databases. PharmMapper Server is an open-source web-server for identification of potential target for small molecules  using pharmacophore mapping approach . 3D struHomo sapiens targets were selected for study.OMIM database, TTD database, and PharmGkb database were used for identification of potential targets implicated in depression and tryptophan metabolism. Depressive, antidepressant, depressed, depression for depressive symptoms analysis, and tryptophan, and tryptophan metabolism for tryptophan metabolism assay were used as keywords in the three databases and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze signaling pathways and bioprocesses associated with depression that are modulated by HLJDD. Moreover, targets implicated in HLJDD activity and tryptophan metabolism were selected for enrichment assay. Enrichment assay was used to predict possible association between HLJDD and tryptophan metabolism. Further, predicted targets were submitted to DAVID database. OFFICIAL_GENE_SYMBOL item gene list were selected for analysis. GO enrichment analysis was performed together with KEGG pathways analysis.Data files and attribute files of compound-target interaction, target-pathway of depressive disorders and tryptophan metabolism were saved in Microsoft Excel. Data files were subsequently imported to Cytoscape 3.2.1 software and ingredient-target interaction networks for depression and tryptophan metabolism constructed.Interaction network for screened target proteins implicated in depression and tryptophan metabolism was constructed using String database. Top five important targets in the interaction network were selected for molecular docking. Molecular docking was used to predict interactions between protein receptors and HLJDD active compounds. Five important targets and related active compounds were selected using Dock website database for molecular docking. Molecular docking was performed by uploading the PDB ID of each target and 3D structure of active ingredients in SDF (.sdf) format. A cutoff value of 4.25 was set for the binding energy score to ensure good binding affinity of active compounds against selected targets. Binding energy scores greater than 5.0 implied stronger binding affinity. Moreover, molecular docking was carried out using AutoDock Vina . AutoDocHippocampus of each animal was isolated and frozen separately in liquid nitrogen. Hippocampus sample to be analyzed using LC-Q/TOF-MS was accurately weighed. ddH2O was added to the weighed hippocampus sample and homogenized for 3 min. 100 μL homogenate was added to ice-cold 80% methanol solution  and vortexed for 3 min. The mixture wascentrifuged at 13,000 rpm for 15 min at 4°C. The supernatants were transferred to a centrifuge concentrator for concentration and drying. The concentrated supernatant was reconstituted with 0.1% formic acid.Mice were sacrificed with carbon dioxide immediately after behavioral assay. B.A 2.1 mm × 100 mm, 2.7 μm chromatographic column was used . The mobile phase consisted of A and B, A, 0.1% formic acid in water, B, 0.1% formic acid in acetonitrile. The column temperature was set at 30°C; flow rate at 400 μL/min; injection volume at 5 μL and gradient elution conditions at 0–0.5 min, 1% B; 0.5–4 min, 1–20% B; 4–8 min, 20–100% B; 8–9 min, 100% B; 9–9.5 min, 100–1% B; 9.5–12 min, 1% Electrospray ionization source in positive mode and negative mode was used for the mass spectrometer (AB SCIEX Triple TOF™ 5600). Ionization source conditions were capillary voltage set at 3.0 kV; source temperature set at 120°C; desolvation temperature set at 350°C set at sampling cone voltage set at 30 V; ion energy at 1.0 V; cone gas flow set at 50 L/h; desolvent gas flow set at 600 L/h and mass scanning range was 100～1000 m/z.p(corr)| > 0.58, and those with no significant difference (p > 0.05) were excluded. Spectrum structure of each metabolite was obtained using HMDB database. Potential endogenous metabolites were determined using PeakView software. MetaboAnalyst 4.0 was used to predict metabolic pathways implicated in depression.Data were normalized using MarkerView software, and then imported into SIMCA 14.1 software. Principal component analysis (PCA) and orthogonal partial least-squares discrimination analysis (OPLS-DA) were performed after standardization. Potential different metabolites were screened using VIP >1.0, correlation coefficient |p < 0.01). One-week administration of HLJDD significantly increased weight in CUMS mice (p < 0.01) (p < 0.01). Mice in HLJDD and FXT groups showed no significant difference in immobility time  . Mice inity time . These fp < 0.01)  .p < 0.01) (p < 0.05)  . Movemen < 0.05) .Profile of neurotransmitters in different brain regions  were analyzed using LC-MS/MS. Mice in the CUMS group showed reduced levels of GABA in hippocampus, cortex, striatum and amygdala. HLJDD restored GABA levels in the four brain regions . On the Area total of 1059 promising targets of HLJDD were identified after deleting duplicate targets within TCMSP, BATMAN-TCM and PharmMapper databases . To explore the relationship between HLJDD and depression, 267 targets related to depressive disorders were retrieved from OMIM, TTD, and PharmGkb databases. area total of 48 common targets for HLJDD and depression were identified. Most common target proteins were D2 dopamine receptor (DRD2), Sodium-dependent serotonin transporter (SLC6A4), D1A dopamine receptor (DRD1), and Glutamate receptor ionotropic NMDA 2A (GRIN2A). A total of 67 active ingredients of HLJDD were correlated with the 48 proteins. A total of 38 active ingredients was shown to directly target the selected proteins. Common active compounds targeting the 48 proteins were Berberine, Jasmone, Skatole, Menisperine, Phellodendron, and Shihunidine. Top twenty depression-associated compounds and targets are listed in p < 0.05 were selected as potential pathways implicated in the activity of HLJDD against depression. Pathways identified under BP category included serotonin receptor signaling pathway, serotonin biosynthetic process, neurotransmitter biosynthetic process and aromatic amino acid family metabolic process , molecular function (MF) and cellular components (CC). Pathways that showed  process . Two metA compound-target-pathway network for active compounds in HLJDD implicated in treatment of depression was constructed using Cytoscape software . The netIn this study, interactions between anti-depressant targets and HLJDD active compounds were analyzed using String database. Interaction network of anti-depressant targets and HLJDD active compounds was constructed based on the degree value greater than medium of ranking. The network consisted of 24 nodes and 134 edges. Results were saved in TSV format and imported into Cytoscape for identification of key targets . Higher p < 0.05 were selected. Under the BP category pathways involved in drug response, regulation of insulin secretion and inflammatory response were identified. A total of 16 tryptophan metabolism-related pathways (p < 0.05) were screened in five categories using KEGG pathway analysis. Three pathways were metabolic-related pathways, nine were signaling-related pathways, two were synaptic-related pathways, one was a disease-related pathway and one inflammation pathway.We further explored interaction network between HLJDD and tryptophan metabolism as tryptophan metabolism plays a key role in pathological process of depression. GO enrichment analysis and KEGG pathway analysis of the 76 targets involved in tryptophan metabolism was carried using DAVUD database. Pathways in which targets were implicated in were classified into three levels: BP, MF, and CC and those pathways with Compound-target network for tryptophan metabolism was constructed using Cytoscape based on active compounds and predicted target. The network had a total of 113 nodes. Out of these nodes, 37 were active compounds expressed in blue and 76 were targets expressed in orange. Active compounds and targets were connected by 372 edges. Main targets identified were SLC6A4, HTR2A, HTR2B, and INS which were mainly targeted by Jasmone, Skatole, berberine. SLC6A4 had the highest degree value among the selected targets . In addiInteraction network of tryptophan metabolism targets and HLJDD active compounds was constructed using a degree value greater than the medium target. The network consisted of 38 nodes and 326 edges. Results were saved in TSV format and imported into Cytoscape software for selection of key targets . Nodes wFive important depression targets identified using the network map  and twenty-one related active compounds were selected for molecular docking. The PDB-ID for each target was retrieved from RCSB PDB database. The largest search term match score was selected as the optimal PDB-ID value. 3D structural formula of the active compounds were retrieved from Pubchem database. Four pair ingredient-target docking scores were greater than 7.0 and accounted for 3.81%. Seventy-one docking scores were between 7.0 and 5.0 and accounted for 67.62%. Further, sixteen docking scores were between 5.0 and 4.25 and accounted for 15.24% whereas fourteen docking scores were less than 4.25 and accounted for 13.33%. Five tryptophan metabolism targets including CAT, INS, MAOA, MAOB, ALDH2, and SLC6A4 and eight related active compounds were selected for molecular docking. Docking results showed five docking scores greater than 7.0 which accounted for 12.50%; twenty-five docking scores between 7.0 and 5.0 which accounted for 62.50%; four docking scores between 5.0 and 4.25 which accounted for 10.00% and six docking scores less than 4.25 which accounted for 15.00%.−1, respectively for Binding energy scores of berberine, baicalein, tetrahydroberberine, and candicine against MAOA were −8.4, −8.4, −7.6, −6.2 kcal mol−1 | > 0.58 in S-Plot mode was used as the screening criteria for exploring different metabolites involved in depression using ANOVA (p < 0.05). A total of 18 different metabolites were identified based on secondary spectrum information and HMDB database in which 8 metabolites were up-regulated and 10 metabolites were down-regulated. Variation of the 18 metabolites was reversed by administration of HLJDD  showed a positive correlation with metabolites involved in kynurenine metabolism . Serotonin metabolism showed a negative correlation with kynurenine metabolism was observed . This imMood disorders, including major depressive disorder (MDD) and bipolar disorder (BD), are the most prevalent psychiatric conditions worldwide. World Health Organization predicted that MDD will be the leading cause of disease burden by 2030 . AlthougHypericum  showed significantly higher activity compared with placebo in the treatment of depression using Hamilton Depression Rating Scale. Hypericum mediates antidepressant effect by inhibiting reuptake of 5-HT, NA, and DA into the presynaptic neurons  a si® . DopaminCurrently, monoamine hypothesis of depression is the leading theory guiding drug discovery and clinical practice. The theory suggests assay of serotonin level in plasma and cerebrospinal fluid for diagnosis of depression . First gin vitro and in vivo experiments. Regulation of tryptophan metabolism by HLJDD is mediated through serotonin metabolism and kynurenine metabolism. Although, active phytochemicals were selected based on drug-like rules, novel available delivery system should be explored to improve bioavailability of compounds to specific brain regions. More studies on development of a multi-component therapy from HLJDD and explore different key targets for treatment of depression.An interaction network for depression-associated targets and corresponding compounds in HLJDD showed that several pathways are implicated in activity of HLJDD against MDD. In addition to tryptophan metabolism process and receptor modulation, dopamine receptors DRD2 and DRD4 are involved in therapeutic effects of HLJDD. Insulin signaling associated with glucose metabolism in neurons was identified, which is consistent with findings that intravenous insulin improves mood and cognition, and depression subjects have glucose disruption . AlthougHLJDD, a TCM formula showed significant therapeutic effects on depressive disorder. HLJDD ameliorated depression-like behavior of CUMS mice using FST, NSF and OFT assays. Multiple pharmacological actions of HLJDD on depression are mainly achieved by modulation of tryptophan metabolism, monoamine nervous system function and energy metabolism. HLJDD provides scaffolds for development of a novel alternative antidepressant therapies.


This article comments on:Chen J, Zhu M, Liu R, Zhang M, Lv Y, Liu Y, Xiao X, Yuan J, Cai H. 2020. BIOMASS YIELD 1 regulates Sorghum biomass and grain yield via the shikimate pathway. Journal of Experimental Botany 71, 5506–5520. The shikimate pathway is key to the synthesis of aromatic amino acids and many secondary metabolites in plants.et al. (2020)Chen biomass yield 1 (by1) mutant in Sorghum using an ethylmethane sulfonate (EMS) mutagenesis screen. The mutation mapped to an amino acid substitution in the sequence ofidentified the dby1 mutants resulted in severe defects in growth and development, such as reduced height, narrow leaves and stem, and abnormal floral organs, which ultimately led to less biomass and yield production. The by1 mutant reveals novel information regarding the integration of primary and secondary metabolism and how this influences growth and development.-arabino-heptulosonate-7-phosphate synthase (DAHPS)—the enzyme catalysing the first committed step of the shikimate pathway. Reduced DAHPS activity in enolpyruvate (PEP) and erythrose-4-phosphate (E4P), produced by the glycolytic and pentose phosphate pathways, respectively. The pathway results in the formation of chorismate, which serves as a precursor molecule for the synthesis of the aromatic amino acids (AAAs); Phe, Tyr, and Trp  and erythrose-4-phosphate (E4P) into chorismate in seven enzymatic steps. Chorismate is the precursor for aromatic amino acids and many secondary metabolites. The by1 mutation in Sorghum was mapped to an amino acid substitution in the enzyme catalysing the first committed step in the shikimate pathway, the DAHP synthase (highlighted in red).The shikimate pathway converts the substrates phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroquinate dehydratase/shikimate:NADP oxidoreductase (DHQase/SORase), 5-enolpyruvylshikimate-3-phosphate (EPSP).Abbreviations: PhosphoThe shikimate pathway is required for growth and development. The well-known herbicide, glyphosate, inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase—an enzyme involved in the shikimate pathway. Also, mutations of various shikimate enzymes were identified in the screen for embryo-lethal mutants in Arabidopsis .Sorghum bicolor to isolate candidate genes regulating biomass production and grain yield. The mutant by1 showed a drastic reduction in both biomass and yield. The characteristic growth features associated with decreased biomass of by1 were reduced plant height, thinner stems, and smaller, narrower leaves. In by1, internode, leaf blade, and vascular cells were developed normally except that the final cell volume was significantly reduced. The authors conclude that reduced cell expansion was associated with abnormal growth phenotypes of by1, which ultimately resulted in decreased plant biomass. Developmental defects including poorly developed panicles, anthers, and pollen contributed to the final reduction of grain yield in by1. In by1, anthers were thin and small and, moreover, pollen viability was reduced by ~50% and pollen grains appeared shrunken.As Sorghum is an important staple food and biofuel crop, by1 phenotype, by1 was outcrossed to Shangzhuang broomcorn. Using a map-based cloning approach, an EMS-induced single single nucleotide polymorphism  was mapped to the Sobic.002G379600 (BY1) gene, which resulted in a Pro to Leu amino acid substitution (Pro192Leu) in the protein product. The gene BY1 encodes a DAHPS, which catalyses the first reaction in the shikimate pathway. The authors report that the Pro192Leu substitution reduced the BY1 enzyme activity by ~33%. BY1 function was validated in rice by targeting Loc_Os07g42960 (a homologue of Sorghum BY1) by CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein]. Growth and developmental abnormalities were similar to Sorghum by1, and both biomass and yield were also drastically decreased.To map the causative gene responsible for the by1 to dissect the developmental pathways that were affected in the mutant line. Most of the genes involved in pathways operating upstream of the shikimate pathway  and the phenylpropanoid pathway that operates downstream of the shikimate pathway were significantly up-regulated. In contrast, the levels of metabolites in those pathways including PEP, shikimate, Phe, and chalcone were reduced. Interestingly, most of the metabolites with significantly lowered levels were phenylpropanoids, and 19 of 22 flavonoids were less abundant. The authors propose that a positive feedback signal in response to reduced metabolites enhances the expression of genes involved in the shikimate pathway and its upstream pathways to restore the carbon flux into the shikimate pathway. This is in agreement with previous studies that showed that shikimate pathway genes in response to AAAs and metabolite levels were regulated at the gene expression level rather than at the post-translational level  and tryptophan synthase mutants were smaller and free auxin levels were reduced in IGS mutants (transparent testa4 (tt4), plant height and inflorescence stem thickness were decreased due to elevated basipetal auxin transport  and rice dmd1 (defective microspore development1) knockout mutants that showed a delay in the timely degradation of the callose wall /ferredoxin (Fd) system (The amino acid substitution Pro192Leu could affect DAHPS activity in many different ways. The authors propose that the In summary,