Acorus tatarinowii (AT) is widely used in the treatment of Alzheimer disease, epilepsy, depression, and stroke, which leads to disorders of consciousness disease. However, the systemic mechanism of AT treating IS is unexplicit. This article is supposed to explain why AT has an effect on the treatment of IS in a comprehensive and systematic way by network pharmacology.  Stroke is the second significant cause for death, with ischemic stroke (IS) being the main type threatening human being's health.  ADME  is an important property for screening-related compounds in AT, which were screening out of TCMSP, TCMID, Chemistry Database, and literature from CNKI. Then, these targets related to screened compounds were predicted via Swiss Targets, when AT-related targets database was established. The gene targets related to IS were collected from DisGeNET and GeneCards. IS-AT is a common protein interactive network established by STRING Database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analysed by IS-AT common target genes. Cytoscape software was used to establish a visualized network for active compounds-core targets and core target proteins-proteins interactive network. Furthermore, we drew a signal pathway picture about its effect to reveal the basic mechanism of AT against IS systematically. α,α,4-trimethyl,acetate, cis,cis,cis-7,10,13-hexadecatrienal, hydroxyacoronene, nerolidol, galgravin, veraguensin, 2′-o-methyl isoliquiritigenin, gamma-asarone, and alpha-asarone. We obtained 398 related targets, 63 of which were the same as the IS-related genes from targets prediction. Except for GRM2, remaining 62 target genes have an interactive relation, respectively. The top 10 degree core target genes were IL6, TNF, IL1B, TLR4, NOS3, MAPK1, PTGS2, VEGFA, JUN, and MMP9. There were more than 20 terms of biological process, 7 terms of cellular components, and 14 terms of molecular function through GO enrichment analysis and 13 terms of signal pathway from KEGG enrichment analysis based on P < 0.05.  There were 53 active compounds screened from AT, inferring the main therapeutic substances as follows: bisasaricin, 3-cyclohexene-1-methanol- AT had a therapeutic effect for ischemic via multicomponent, multitarget, and multisignal pathway, which provided a novel research aspect for AT against IS. It not Sheng Nong's herbal classic thousands years ago, and its dried rhizomes attributed to Araceae Juss, with the effect of reducing phlegm by resuscitation, improving intelligence, dehumidifying, and appetizing [Traditional Chinese medicine is always used without side effect to reduce disability rates, improve the quality of life, and decrease the risk of recrudesce for cerebral ischemic patients. The major mechanism involved anti-inflammatory reaction, antioxidant, inhibiting neuron apoptosis, promoting vascular regeneration, and maintaining the BBB permeability. AT was firstly recorded in petizing , which wpetizing . Many stpetizing , 13, whipetizing , and regpetizing .It is unclear whether AT and AT-contained ingredients have other mechanisms against IS. Due to lack of systematic and comprehensive explanation about it, network pharmacology was used to elucidate the interactive relation among drug, targets, and disease. Potential target prediction helps us to understand more mechanisms for multiple pharmacological functions of AT and its role in biological network, which will be beneficial for the curative effect improvement , 17. ManThe flowchart of this study is shown in http://tcmspw.com/tcmsp.php), TCMID Database , Chemistry Database [http://chemdb.sgst.cn/scdb/default.asp), and literature from CNKI to provide more useful and important compounds. Based on pharmacokinetics , we set up a series of parameters, such as BBB absorption>−0.3 and DL ≥ 0.18 [https://pubchem.ncbi.nlm.nih.gov/) to finish Swiss ADME prediction [We searched active compounds AT contained via TCMSP Database   and DisGeNET [https://www.disgenet.org/) with the keywords “ischemic stroke,” “cerebral ischemic,” “cerebral infarction.” These gene targets were put into IS-related targets database, respectively. Genes from Genecards in searching different keywords will be screened by its scores, which more than its median are constituted in IS-related targets database from Genecards.We obtained IS-related gene targets by searching in Genecards , where diversity of shape nodes was distinguished from AT, active compounds in AT, and targets, where edges represented the interactive relationship between AT and compounds and compounds and targets.http://www.bioinformatics.com.cn/).By the aid of Microsoft Excel, we intersected potential target database related to compounds in AT- and IS-related target database to get the core target related to AT-IS, which was used to draw a Venn diagram online (https://string-db.org/), selecting species “Homo sapiens”. Potential protein targets with a medium confidence score of 0.400 were designed as an interaction network, with hiding disconnect core targets to get PPI analysis results. All these results could be used in constructing AT-IS PPI visualized network, based on its degree value by analysing.Core targets related to AT-IS were imported into STRING , choosing species “Homo sapiens” and then clicking “custom analyse.” GO enrichment analysis is composed of biological process, molecular function, and cellular components. KEGG enrichment analysis is used to uncover the signal pathway showing potential therapeutic effect of some diseases. Before GO and KEGG enrichment for above core genes, “Min Overlap = 3,” “P value cutoff = 0.01,” and “Min enrichment = 1.5” should be set for passway and process enrichment and “Min Network Size = 3” and “Max Network Size = 500” for protein-protein interaction enrichment.GO and KEGG enrichment analysis of core targets related to AT-IS was accomplished by an online tool, Metascape   and AT-22  were lower than this median; no target was related to AT-28 blumenol A; thus, these three compounds were out of the network). 1409 targets were predicted, 398 targets remained after deleting repeat ones, and specific information is shown in Supplementary We screened 2391 gene targets in Genecards Database, after filtering out based on median, when 560 gene targets were obtained from DisGeNET  and contained fluid shear stress and atherosclerosis, HIF-1 signaling pathway, IL-17 signaling pathway, arachidonic acid metabolism, platelet activation, bladder cancer, inflammatory mediator regulation of TRP channels, transcriptional misregulation in cancer, PPAR signaling pathway, complement and coagulation cascades, GnRH signaling pathway, regulation of lipolysis in adipocytes, and serotonergic synapse , uncovered its core role in AT.Absorbed, distributed, metabolized, and excreted are significant parameters to determine the transformation and delivery of one drug in the body. A CNS-active drug having a high BBB permeability is crucial for its antistoke effect for entering into the brain. It was evidenced than if BBB permeability was lower than −0.30, and it had no effect on entering the brain through BBB, so we set it for more than that . Only 6 There were 62 core targets for AT against IS, IL6, TNF, IL1B, TLR4, NOS3, MAPK1, PTGS2, VEGFA, JUN, and MMP9 involved in the top 10 degree targets. IL6, TNF, IL1B, TLR4, and NOS3 are related to the HIF-1 signal pathway, fluid shear stress and atherosclerosis, IL-17 signal pathway, and inflammatory mediator regulation of TRP channels, which are called inflammatory factors as effect for enhancing the level of inflammatory reaction in cerebral ischemic –33. GO eα, IL-1β, and IL-6, increasing anti-inflammatory factor IL-10, and suppressing oxidative stress level [α and IL-1β proinflammatory factors expression to antiatherosclerosis [Atherosclerosis is the main pathological mechanism leading to cerebral ischemic, half of which has a connection with it. It was commonly known that it was beneficial to decrease the incidence or palindromia of cerebral ischemic by intervening atherosclerosis as secondary prevention . In factss level . JNK andss level . In addiclerosis .γ and PPARδ expression through overactivated p38MAPK, AMPK, and PKA to promote white adipose cell browning and increased lipid degeneration [Disorder of fatty metabolism not only induces atherosclerosis but also is a high risk of stroke. It is necessary to regulate adipose lipolysis to avoid metabolism syndrome occurrence as possible to decrease the incidence of stroke indirectly. However, it required to further prove the role of adipose lipolysis in ischemic stroke for fat mice. It was confirmed that stroke rats with a high level of inflammatory factor in plasma and adipose tissue induced adipose lipolytic enzymes and free fatty acids expression increased . It is aneration .α and PPARγ, nuclear transcription from nuclear receptor family, and PPARα/γ agonist decreased inflammatory reaction to induce the neuroprotective effect after cerebral ischemic [α-Asarone conjugate structure activated by PPARα, increasing CPT1A gene expression related to degeneration and metabolism of fatty acid [α ligand directly [PPAR signal pathway has a close relationship with fatty metabolism, which also exerts a protective effect on cerebral ischemic. PPARischemic . α-Asarotty acid . Linaloodirectly , which idirectly .2 is a positive feedback protein, platelet agonist, a second wave mediater, which is not unnecessary for its accumulation. It can trigger platelet activated by G protein-coupled receptor [2 produce. SQ29548, TXA2R antagonist, was found to inhibit microglia/macrophages activation and enrichment to reduce injury of cerebral ischemic [β-asarone could be against coagulation via APP, PTGS2, and TBXAS1 [2 expression and decreased ET-1 and TXA2 expression to inhibit platelet activation and accumulation to suppress thrombus formation [α-asarone [Antiplatelet aggregative activity and anticoagulation are common and major therapy to prevent emboli and thrombus formation for ischemic stroke. Previous studies confirmed AT suppressing platelet activation and thrombus formation , 53. Plareceptor . In our ischemic . It has d TBXAS1 . Eugenold TBXAS1 . Paeonolormation . 3,11-Euormation  and α-as2 formation leading to platelet accumulation. A series of evidence has proved that a high level of COX-2 and PLA2 was filled with MCAO animal models and stroke patients [α-Iso-cubebene inhibited iNOS, COX2, and MMP9 expression and the phosphorylation of JNK and p38 to exert a neuroprotective effect, which was released by microglia amyloid beta induced [α-iso-cubebene has a neuroprotective effect for IS via inhibiting AA metabolism need to be further proved.In addition, arachidonic acid (AA) metabolism promotes platelet accumulation. COX-2(PTGS2) makes AA degeneration, whereas PLA2 promotes AA synthesis, which takes participation in TXApatients –63. Eugepatients . In vitrpatients . α-Iso-c induced . HoweverAT antithrombus was involved in an intrinsic way of coagulation cascades. FXa activated JNK pathway through PAR-1, which triggered F2 to induce neuron death . In vitrα in the early stage of clinic, indicating a serious expectation for 90 days [α weakened the increase in TLR4 in BV-2 cells in hypoxia condition with downregulation of TNF-α expression [α [β-Asarone had the ability of antioxidation, which decreased HIF-1α in cortex [Acute cerebral ischemic patients had a high level of HIF-1 90 days . TLR4 ispression . In factssion [α . β-Asaron cortex .Both HRPA1 and TPR1 are members of TRP superfamily, which are structurally dependent nonselective cation channels and mediators of several signaling pathways. Methyl eugenol was proved to be hTPR1 agonist, selectively activating hTPRA1 . MicroglTaken together, these signal pathways, related to AT preventing and treating IS, could be approximately classified into three categories as follows: atherosclerosis, regulation of lipolysis in adipocytes, and PPAR signal pathway were associated with lipid metabolism; coagulation cascades, arachidonic acid (AA) metabolism, and platelet activation had a connection with the therapy of antiplatelet aggregative activity and anticoagulation; and HIF-1 signal pathway, inflammatory mediator regulation of TRP channels, and Th17 differentiation might be the potential therapeutic signal pathway. In addition, we inferred that an intrinsic way of coagulation cascades and Th17 differentiation was probably new therapeutic or preventive direction for the role of AT against IS.In summary, we explored and explained multiple compounds, multiple pathways, and multiple targets of AT-regulated ischemic stroke treatment based on a network pharmacology method. Our data indicated that amounts of compounds contained in AT might prevent and treat ischemic stroke through some signal pathways, most of which need an elaborated detailed mechanism in the related signal pathway. Two novel research directions  of AT therapeutic for preventing IS were proposed. In the future, more studies should focus on providing experimental evidence and enhancing the effect of AT in the cerebral ischemic on a comprehensive level and improving ability of AT targeting to the brain and crossing the BBB.


Shennong Bencao Jing are effective and widely used as an herb pair for the treatment of TA, while the active ingredients and the functional mechanism of Tusizi-Sangjisheng herb pair treating TA are still unknown. In order to exploit the relationship between those two herbs and TA, systems pharmacology analysis was carried out in this study. A total of 75 ingredients of Tusizi-Sangjisheng were collected from Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP). 12 bioactive compounds were screened, and 153 directly related targets were predicted by systematic models. Besides, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to systematically explore the potential mechanisms of Tusizi-Sangjisheng treating TA. Meanwhile, Compound-Target (C-T), Target-Disease (T-D), and Target-Pathway (T-P) networks were constructed to further quest the underlying functional mechanisms of Tusizi-Sangjisheng. As a result, 31 targets and 3 key pathways were found to be directly related to TA that includes mitogen-activated protein kinases (MAPKs), phosphatidylinositol-3-kinase/protein kinase B (PI3K-Akt), and transforming growth factor-β (TGF-β) signaling pathways. The results in this study may provide some valuable clues about the molecular mechanisms of the efficient Chinese herb pair Tusizi-Sangjisheng in the treatment of TA.Threatened abortion (TA) is a common complication with high incidence in the first trimester of pregnancy, which will end in miscarriage if not treated properly. The Chinese herbs Cuscutae Semen (Tusizi in Chinese) and Herba Taxilli (Sangjisheng in Chinese) first recorded in the ancient classic medical book  Threatened abortion (TA), defined as vaginal bleeding with or without lower abdominal pain or backache with a closed cervix and an intrauterine viable fetus, is the commonest complication that occurs in early pregnancy , especiaShen Jing in Chinese) and nourishing the liver [In clinical practice, targeted and directed treatment should be used to prevent miscarriage when specific causes are identified. For example, combined aspirin and heparin are effective in TA induced by antiphospholipid syndrome while antibiotics are useful for TA induced by bacterial vaginosis  and surghe liver , 18. Zenhe liver  and Li'she liver  researchhe liver . It has he liver  and antihe liver , 24 and he liver . Herba The liver  and has he liver  and antihe liver . GeneralThe objective of this study is to systematically explore the functional mechanism of the herb pair Tusizi-Sangjisheng in the molecular level and also to understand the relationship among those small chemicals, their related targets, and diseases. The results of this study may facilitate clarifying the mechanism of the Chinese herb pair Tusizi-Sangjisheng and offer some valuable methods for studying other akin herbs.http://lsp.nwsuaf.edu.cn/tcmsp.php) which comprises more than 510 herbal entries registered in Chinese pharmacopoeia with more than 33,000 ingredients. The rational assessment of absorption, distribution, metabolism, and excretion properties (ADME) of compounds is essential to decide drug candidates. In the present study, two ADME-related models, PreOB  and PreDL (predict drug likeness) of the drugs, were employed to prescreen the bioactive compounds. Oral bioavailability (%F) is considered a key parameter in drug development. Oral drug absorption is mainly about two basic parameters: solubility and gastrointestinal permeability of the drug. In detail, the PreOB model, developed on the basis of a robust in-house system OBioavail 1.1 [http://www.drugbank.ca/), compounds with DL ≥ 0.18 were selected as the candidate bioactive chemicals in this study. Finally, a total of 75 compounds from Tusizi-Sangjisheng were obtained and 12 bioactive compounds among them were screened.All compounds of Tusizi-Sangjisheng in this study were collected by using the TCMSP database  was used to search the genes of the human species and the corresponding UniProtKB related to the predicted targets of the bioactive molecules. Information on the physiological functions of all targets was obtained from TTD (http://bidd.nus.edu.sg/group/ttd/) and UniProt (http://www.uniprot.org/) databases. Additionally, to systematically study the effects of herbal medicines and to characterize the therapeutic effects on a pathway level, three visualized networks including Compound-Target (C-T), Target-Disease (T-D), and Target-Pathway (T-P) were constructed. And the obtained target profiles were organized into several pathways by mapping to KEGG (Kyoto Encyclopedia of Genes and Genomes). All bipartite graphs were drawn by Cytoscape 3.5.1 software [Target identification for bioactive compounds is an essential step for drug discovery. Predicting drug-target and drug-pathway interactions could help understand the biological mechanisms from the perspective of network pharmacology. To obtain the related targets of these active compounds, a SysDT  model basoftware .In this study, a total of 75 compounds of the Tusizi-Sangjisheng (29 for Tusizi and 46 for Sangjisheng) were included. After screening all compounds at the criteria that OB ≥ 30% and DL ≥ 0.18, 12 bioactive compounds with excellent ADME properties were obtained and the specific information is shown in Traditionally, herbal medicines contain numerous pharmacological compounds, which offer bright prospects for the treatment of complex diseases in a synergistic manner. Network pharmacology has undergone a rapid development in recent years and emerged as an invaluable tool for describing and analyzing complex systems in pharmacology studies. Considering that the multicomponent herbs exert effects on diseases by acting to specific protein targets, drug targeting may shed light on the mechanism of herbs treating TA from the perspective of network pharmacology. In this study, after target fishing by the SysDT  model, aIn order to uncover the synergistic effects of multiple components and targets in Tusizi-Sangjisheng, a C-T network analysis was carried out. After excluding 2 bioactive compounds which have no directly related targets, a graph of C-T interactions was drawn  using 10Disease-related genes interact in their products and expression patterns. Disease and disease genes are linked together to form a network through known correlations. A variety of disease network diagrams can be generated from the interdependence of cell networks based on human diseases. In these figures, if the phenotype of a disease is related to a molecule, different phenotypes will be linked . In addiExisting data and theories make it possible to use network methods to study diseases from different scales. Complex network systems can visually reveal the complex relationships among diseases, genes, and pathways to a certain extent. Since the application of network models to biology, many researchers have constructed networks of different functions and properties. These network models have shown us their value in biological researches. At present, the research on diseases mainly includes protein-protein relationship networks, metabolic networks, and regulatory networks. We established a Target-Disease network to study protein-protein interactions and to understand the complex mechanism of action of TCM. This network showed that different diseases have common pathological changes and can be cured by the same compound; that is, one compound can correspond to multiple diseases.http://www.omim.org/) was used to search target genes related to TA, and the search term was “threatened miscarriage” or “threatened abortion”. The target genes corresponding to the disease is compared with those corresponding to the bioactive compounds, and the common ones are the target genes of the drug in treating TA. A total of 455 targets related to TA were collected through the OMIM database. Comparing it with the 153 targets corresponding to the bioactive compounds of Tusizi-Sangjisheng, 31 common targets preventing miscarriage were obtained. As shown in The OMIM database  and perform GO enrichment analysis. We found that these targets are closely related to various biological processes, such as the RNA polymerase II promoter positive regulation of transcription, positive regulation of reactive oxygen metabolic processes, and inflammatory responses to multicellular biological processes.Upload the predicted target genes to the DAVID database  signal transduction pathway. As shown in β signal pathway acts on TGFB1 and Myc targets.The top three pathways with the highest degrees in β-sitosterol, and isorhamnetin. Quercetin, kaempferol, and isorhamnetin are all flavonoids that are abundant in amount and widely distributed in nature. Quercetin is one of the phytochemicals with anticancer and antioxidant activities that widely exists in nuts, teas, vegetables, herbs, and generally diet of people [γ, and TNF-α and increasing the decreased ratio of Bcl-2/Bax apoptotic proteins induced by lipopolysaccharide (LPS) revealing the putative value in preventing miscarriage caused by bacterial infection [κB signaling activity, and its antioxidative effect is associated with ROS blocking [β-Sitosterol, one of the several phytosterols, is a natural micronutrient in higher plants possessing numerous physiological effects including antioxidative, anticancer, anti-inflammatory, antidiabetic, and immune modulation activities [β-sitosterol have wide pharmacological functions, the detailed correlation with TA is still unclear and needs to be further studied.In this study, we used systematic pharmacology to study Tusizi-Sangjisheng and found that this herb pair has a total of 12 compounds with good OB and DL properties, which are related to 153 target proteins. The most potent compounds in Tusizi-Sangjisheng is quercetin which is followed by kaempferol, f people , 35. In nfection –38. Kaemnfection , 40. It nfection , and thenfection –43. Isorblocking . Besidesblocking . β-Sitostivities . AlthougPTGS, also known as cyclooxygenase (COX) , is a crNOS is the only rate-limiting enzyme that synthesizes NO. It belongs to isozymes and has three subtypes: neuronal (nNOS/NOS1), inducible (iNOS/NOS2), and endothelial NOS (eNOS/NOS3) , among wα and TNF-β are the two known types of TNF that can be expressed in immune cells such as T and B cells. IL-6 (interleukin 6), which belongs to the Th2-type cytokine, is a type of cytokine involved in a variety of immune inflammatory responses that could inhibit Th1-mediated immune responses. Th1 immunity causes loss of pregnancy while Th2 immunity helps to maintain pregnancy, so the ratio of Th1/Th2 is crucial for pregnancy. If the balance of Th1/Th2 is broken, TA or miscarriage may occur. TNF-α is mainly expressed by Th1 and NK cells that can kill tumor cells and also attack trophoblast cells and embryonic tissues. A high serum level of TNF-α and inadequate expression of IL-6 in endometrial tissue have been found in patients with recurrent abortion, revealing that the overproduction of TNF or underactivation of IL-6 may cause fetus damage and loss [α to promote the expression of IL-6, increase its level in plasma and decidual tissue, and then inhibit the expression of TNF-α so as to reduce the maternal immune response against the fetus and maintain the normal development of the pregnancy.TNF is a cytokine secreted by macrophages that can directly cause death of tumor cells. TNF-and loss , 58. In Caspase is a type of protease and an important mediator of programmed cell death (apoptosis) that includes caspase-1 , caspase-2 (containing caspase-2 and 9), and caspase-3 . Among them, caspase-3 is mainly expressed in the cytoplasm of trophoblast cells and could regulate cell growth and apoptosis by activating the Fas pathway. Apoptosis is essential for the normal physiology of pregnancy during implantation since apoptosis of trophoblast cells is important for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo , 60. TheMAPK is a class of serine/threonine protein kinases and widely present in various cells. It has important regulatory effects on gene expression. After activation, the protein migrates into the phosphorylated nucleoproteins and membrane receptors, regulating gene transcription and other life events. It consists of four separate signaling cascades: the JNK/SAPK ; the ERKs ; the ERK5 or big MAPK1; and the p38MAPK group of protein kinases. When stimulated by extracellular signals, the MAPK cascade which is an evolutionarily conserved tertiary kinase cascade transduction signal will be activated by a series of complicated chemical reactions, resulting in the successive activation of MAPKKK, MAPKK, and MAPK through the phosphorylation of amino acid residues . EvidencThe PI3K-Akt signal pathway consists of phosphoinositide 3-kinase (PI3K) and protein kinase B  and is involved in transcription, protein synthesis, migration, apoptosis, and proliferation activities . Identicβ is a multifunctional cytokine with three isoforms  and acts mainly through Smad pathways [β plays pivotal and dual roles in the regulation of cell growth and apoptosis which is vividly described as a “switch” [β signal pathway is one of them that could help to maintain normal pregnancy by balancing the proliferation and apoptosis of trophoblast cells [β signal pathway is also involved in immune and inflammatory responses by regulating indoleamine 2,3-dioxygense (IDO) expression to promote immune tolerance of the maternal-fetal interface [β signal pathway in TA. Yet we can hypothesize based on the above information that the active ingredients in the herb pair Tusizi-Sangjisheng exert their TA-preventing function by regulating the immune responses in maternal-fetal interface and controlling the normal apoptosis and proliferation of uterine cells through the TGF-β signal pathway.TGF-pathways . Evidenc“switch” . During st cells . In addinterface , inhibitnterface . At presIn normal pregnancy, the balance of the immune microenvironment at the maternal-fetal interface can ensure immune tolerance and protect the embryo from maternal immune rejection. The ability of gestational trophoblasts to proliferate, differentiate, migrate, and invade is crucial for embryo implantation, placenta formation, and embryo growth and development. Trophoblasts are the main cell types of placental tissues that differentiate into extravillous trophoblasts, syncytiotrophoblasts and cell trophoblasts. Extravillous trophoblasts can be further differentiated into cells with high infiltration capacity. Interstitial trophoblast cells and intravascular trophoblast cells then invade the uterine decidua and reshape the spiral arteries of a pregnant uterus, thereby forming placental tissues which provide nutrients for the development of the embryo. Therefore, infiltration of trophoblasts into the uterine decidua is the key step to the success of early pregnancy. If the ability of trophoblasts to proliferate, migrate, and invade is weakened, it will affect the remodeling of decidual tissue blood vessels and the formation of placenta and further develop into pregnancy-dependent complications such as abortion, preeclampsia, and intrauterine growth restriction. These processes during pregnancy are jointly regulated by many signal transduction pathways. However, the exact regulatory mechanisms are still not very clear. Three key signaling pathways mentioned above were screened by systematic methods in this study revealing that Tusizi-Sangjisheng may effectively reduce miscarriage rates via regulating the normal expression and function of those three signal pathways, correcting under- or overexpression and maintaining normal pregnancy.β signaling pathways. The mechanism of Tusizi-Sangjisheng on the treatment of TA may be through regulating the normal expression of the above three signal pathways, correcting the state of under- or overexpression, and maintaining normal pregnancy.The putative mechanisms in the molecular level of the commonly used herb pair Tusizi-Sangjisheng treating TA were analyzed by systems pharmacology. 12 bioactive molecules with OB ≥ 30% and DL ≥ 0.18 of Tusizi-Sangjisheng were obtained by calculating the ADME properties. Besides, a total of 153 direct targets of 12 bioactive molecules were predicted by systematic models. By constructing a T-D network for analysis, 31 targets mainly containing PTGS1, NOS3, TNF, and caspase were found to be directly related to TA among the corresponding targets of Tusizi-Sangjisheng and TA was considered a complex disease with multifaceted causes that need to be treated from multiple angles by GO analysis. Through the T-P network enrichment analysis, Tusizi-Sangjisheng was found to act on multiple pathways by several active molecules that include MAPK, PI3K-Akt, and TGF-


Atractylodes macrocephala Koidz (AMK) is employed in traditional Chinese medicine (TCM) to treat various disorders. AMK can be efficacious against CG, but the active ingredients, drug targets, and its exact molecular mechanism are not known. We employed network pharmacology to analyze the active ingredients, drug targets, and key pathways of AMK in CG treatment. Seventy-seven AMK candidate ingredients were selected from four databases, and 27 active ingredients were selected for CG treatment. Twenty-five overlapping gene symbols related to CG and drugs were obtained from GeneCards and OMIM databases. A protein–protein interaction (PPI) network and TCM comprehensive network (Drug–Ingredients–Gene symbols–Disease network) were constructed, and 528 Gene Ontology (GO) terms and 26 pathways were obtained by analyses of enrichment of GO pathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We suggest that the interleukin-17 signaling pathway, C-type lectin receptor signaling pathway, tumor necrosis factor signaling pathway, and AGE-RAGE signaling pathway in diabetic complications might serve as the key points and principal pathways for CG treatment. We also evaluated the reliability of some important active ingredients and targets by in vitro experiments. We showed that AMK probably influences the inflammatory response, amino acid synthesis, and energy metabolism when treating CG. This study provides novel insights for researchers to explore the mechanism of action of TCM systematically.Chronic gastritis (CG) is an inflammatory disease.  Chronic gastritis (CG) is an inflammatory disease in which the epithelium of the gastric mucosa is invaded by various pathogenic factors, which results in persistent and chronic inflammatory changes. CG can be divided into three categories: chronic non-atrophic, chronic atrophic, and “special.” If not treated in a timely manner, CG can transform into gastric cancer . Most paTraditional Chinese medicine (TCM) has been used to treat various diseases for thousands of years. In TCM theory, CG is divided into six types according to the pattern of: accumulation of damp heat in the spleen–stomach; dampness obstructing the spleen–stomach; spleen–stomach Qi deficiency; spleen–stomach deficiency cold; liver–Qi stagnation; stagnant heat in the liver–stomach .Atractylodes macrocephala Koidz. (AMK) can invigorate the spleen and Qi, reduce dampness and moisture, act as an antiperspirant, and stop fever developing. In TCM, it is often used to treat spleen deficiency, malnutrition, abdominal distension, diarrhea, dizziness, palpitations, edema, spontaneous sweating, and fetal restlessness , Chinese Academy of Sciences Chemistry Database (www.organchem.csdb.cn), TCM Database@taiwan (http://tcm.cmu.edu.tw), and Traditional Chinese Medicine Integrated Database (https://omictools.com/tcmid-too). TCMSP is a special platform based on systems pharmacology for Chinese herbal medicines that includes the relationships between drugs, targets, and diseases. TCMSP has information on 500 drugs and >12,000 ingredients  refers to the percentage of unmodified drugs that enters the circulatory system after oral administration . OB is awww.talete.mi.it/products/dragon_description.htm) and β denotes the average molecular property for all drugs in the DrugBank database (www.drugbank.ca/) (where α is the molecular property of the AMK ingredient on the basis of Dragon software (ank.ca/) .Most ingredients in Chinese formulations have poor pharmacologic properties, so they cannot bind to specific protein targets on cells efficaciously. Therefore, several researchers have recommended that molecules with OB ≥ 30% or DL ≥ 0.18 are considered to have better pharmacologic effects, and are selected as candidate ingredients for further analyses . We usedTarget forecasts and target-prediction methods can be divided into four categories based on: the predictions of a ligand ; the predictions of a receptor (molecular docking); machine learning ; a combined forecast. Considering the limitations of the experimental conditions of molecular docking and machine learning and our previous experience, we chose a prediction method based on ligands for subsequent research .http://lsp.nwu.edu.cn/tcmsp.php). We removed redundant information and retained only those targets that could interact directly with each of these ingredients in AMK. Then, the target was transformed using the UniProt knowledge database (www.uniprot.org) with the selected species as Homo sapiens. After deletion of redundant items, data were merged to obtain gene symbols.All the protein targets of the ingredients in AMK were retrieved from TCMSP (www.genecards.org/). GeneCards is a searchable, comprehensive database that offers all comments and predicts human genetic information comprehensively in a user-friendly manner  (We collected gene targets for CG from two sources. The first source was the GeneCards v4.9.0 (y manner . We usedry 2019) .www.cytoscape.org/) , ingredients, gene symbols, and disease (CG). Next, we used Cytoscape v3.7.1 (pe.org/)  to underhttps://string-db.org/, updated on 19 January 2019)  ry 2019) . Then, thttp://bioconductor.org/, released on 31 October 2018) for analyses.GO is an international standard classification system for gene function . We usedhttp://bioconductor.org/, released on 31 October 2018) for analyses.The KEGG database analyzes the metabolic pathways of gene products in cells and the functions of these gene products . We usedwww.rcsb.org); the PDB entry code for these proteins is 1ALU, 2NVH, and 4DAJ, respectively. During molecular docking, we adopted the GOLD Score fitness function. The GOLD fitness function comprised three main terms: a hydrogen energy; a pairwise van der Waals steric energy between the protein and the ligand; and an internal energy term for the ligand.We wished to ascertain the interaction between active ingredients and their protein targets and explore their binding modes. Hence, we selected three active ingredients and three targets, a total of four ingredients–targets interactions for verification of molecular docking. We used GOLD v5.1 . We obtained the X-ray crystal structures of interleukin (IL)-6, IL-1β, and muscarinic acetylcholine receptor M3 (CHRM3) from the RCSB Protein Data Bank (PDB)  was purchased from Must Biotechnology . A stock solution of 100 mM ATL-I in dimethyl sulfoxide was prepared and stored at 4°C. Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and IL-1β were purchased from Elabscience Biotechnology .2 for all experiments.RAW264.7 cells were obtained from the cell bank of the Chinese Academy of Sciences . RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum (Gibco). Cells were cultured at 37°C in an atmosphere of 5% CO3 cells/well in 96-well culture plates. After incubation for 24 h, Raw264.7 cells were exposed to ATL-I . After treatment for 24 h, 20 μL of Cell Counting Kit (CCK-8) assay solution  was added to each well, and cells were incubated for 4 h at 37°C in an atmosphere of 5% CO2. The absorbance at 450 nm was measured by a microplate reader. Cell survival was calculated as: absorbance/absorbance of control ×100%.RAW264.7 cells in the logarithmic phase were seeded at 8 × 103 cells/well) were incubated with lipopolysaccharide  for 24 h and then treated with ATL-I  for 24 h. Supernatants were harvested and the level of IL-6 and IL-1β determined by ELISA kits (Biosharp).RAW264.7 cells , and reverse-transcribed with oligo-DT using HiScript™ Reverse Transcriptase  according to manufacturer instructions. The primers used were synthesized by Tsingke . The sequences were  5′-TCAGGCAGGCAGTATCACTC-3′ and 5′-AGCTCATATGGGTCCGACAG-3′ for IL-1β; 5′-CACAGAGGATACCACTCCCAACAGA-3′ and 5′-ACAATCAGAATTGCCATTGCACAAC-3′ for IL-6; 5′-ATGGGTGTGAACCACGAGA-3′ and 5′-CAGGGATGATGTTCTGGGCA-3′ for the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Total RNA was extracted with TRIzol−ΔΔCT method was the basis for relative expression of genes.qRT-PCR was done using SYBR™ Green Master Mix (Vazyme) in the QuantStudio 6 Flex system . The PCR cycling profile was: one cycle at 50°C for 2 min and 95°C for 10 min, 40 cycles at 95°C and 60°C for 30s. Fluorescence signals were detected using the QuantStudio 6 Flex system. Gene-expression data were normalized to that of the endogenous control GAPDH. The 2p < 0.05 was considered significant. All experiments were repeated at least three times.Data are the mean ± SD. The significance of results was determined based on one-way analysis of variance using Prism 8.0.1 . p < 0.05). ATL-I inhibited expression of Notch-1, Hey-1, Jagged-1, and Hes-1 in the Notch signaling pathway. ATL-I inhibited expression of the mRNA of Notch-1, Hey-1, Jagged-1, and Hes-1 (p < 0.05). ATL-I has been shown to inhibit proliferation of mgc-803 cells in human gastric cancer by inhibiting the Notch signaling pathway , (L)-Alanine , -3,6,10-trimethyl-4,7,8,11-tetrahydrocyclodeca[b]furan (5), 3β-acetoxyatractylone (4), ATL-I (4), and atractylone (2). Moreover, it meant that AMK could act on multiple targets through the same active ingredient. For example, ATL-I could have an effect on vascular endothelial growth factor A (VEGFA), IL-6, and IL-1β if used for CG treatment. Wang and colleagues showed that ATL-I can achieve anti-inflammatory effects by inhibiting expression of IL-6 and IL-1β . DetaileWe constructed a PPI network consisting of 25 nodes and 77 edges    -stimulated RAW264.7 cells. We undertook ELISAs and qRT-PCR for IL-6 and IL-1β to confirm the predicted anti-inflammatory effects of the ingredients. IL-6 production showed a significant decreasing trend with increasing ATL-I concentration  curative effects than those of western medicine . A detaiWe showed that 27 active ingredients in AMK influence 25 overlapping gene symbols that have important roles in CG treatment. Thirty GO terms and 26 pathways were obtained by analyses of enrichment of GO pathways and KEGG pathways. We postulate that the IL-17 signaling pathway (hsa04657), C-type lectin receptor signaling pathway (hsa04625), TNF signaling pathway (hsa04668), and AGE-RAGE signaling pathway in diabetic complications (hsa04933) might serve as the key points and principal signaling pathways in CG treatment.ATL-I and atractylone have been identified as bioactive ingredients by researchers . In addiIn addition, the same target can be linked to multiple active ingredients, for example, PTGS2 can be associated with D-camphene, α-longipinene, akridin, and 14-acetyl-12-senecioyl -2E,8Z, 10E-atractylentriol. These data suggest that AMK can act on the same target through multiple active ingredients. ATL-I can be associated with IL-6, IL-1β, and VEGFA, which also suggests that AMK can act on multiple targets through the same active ingredient.Analyses of a PPI network showed that CAT, IL-6, IL-1β, and VEGFA were the most correlated proteins, followed by PTGS2. In combination with analyses of enrichment of GO pathways and KEGG pathways, we believe that the mechanism of action of AMK in CG treatment is closely related to inflammation regulation . For exaTo some extent, the data mentioned above also suggest that AMK has multiple ingredients, multiple targets, and multiple approaches. Such data provide the basis for multi-ingredient synergies in future research.To summary, network pharmacology has been used widely in TCM research .In China, AMK can be used to treat constipation, irritable bowel syndrome (IBS) and diarrhea . In TCM We explored, systematically, how AMK may operate in terms of CG treatment. Our data may offer insights into how TCM can be employed in CG treatment.The data used to support the findings of our study are included within the article, or within the SY and HY conceived and designed the study. YY conceived and designed the experimental validation in vitro. All authors participated in drafting of the manuscript and revising it before final submission. These authors: SY, JZ and YY have contributed equally to this work and share first authorship.This work was supported by the National Key Research and Development Project of China (2018YFC1707206).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.


Tripterygium wilfordii Hook.f and Tripterygium hypoglaucum (H.Lév.) Hutch is effective herbs to prevent aggravation of Rheumatoid arthritis (RA). However, both of them show severe side effects in the reproductive system and other systems. Kunxian Capsule (KX), a Traditional Chinese Medicine (TCM) patent prescription, comprised of 4 herbs, including H.Lév. Hutch, is reported to be an available prescription in treating RA with fewer side effects as compares to Tripterygium tablets. To reveal the pharmacological mechanism of KX in RA treatment and side effect alleviation, we collected related information of KX from open-access databases and performed various analyses. 1354 targets were identified in KX. These targets were enriched in the calcium signaling pathway, cAMP signaling pathway, cGMP-PKG signaling pathway and PI3K-AKT signaling pathway, forming biological functions, such as cofactor binding, coenzyme binding, etc. These pathways or functions mostly affect cell cycle, differentiation, and maturation of Th17 cells, macrophage, and synovial fibroblast. These targets also act on the IL-17 signaling pathway, Th17 cell differentiation signaling pathway and TNF signaling pathway, which is related to inflammation response inhibition. Next, a disease network was constructed, which indicated IMPDH2, MTHFD1 are the key genes answering for the side effects of H.Lév. Hutch. The side effect–related genes lead to the negative regulation of nucleic acid, which could be restored by the rest 3 herbs through some positive amino acid metabolism. In conclusion, KX is a relatively safe alternative approach in RA intervention. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by symmetry and peripheral polyarthritis. Epidemiology shows that the prevalence of RA is 0.5% to 1%, varying by geography and population . ChronicTripterygium hypoglaucum (H.Lév.) Hutch (Kun Ming Shan Hai Tang), Epimedium brevicornu Maxim (Yin Yang Huo), Cuscuta chinensis Lam (Tu Si Zi), and Lycium barbarum L (Gou Qi Zi). Previous research has demonstrated that KX can down-regulate chemokines, inhibit inflammation response and has analgesic and immunosuppressive effects of the whole-course intervention . KunXian Capsule (KX) is a Traditional Chinese Medicine (TCM) patent prescription which is widely used in RA treatment. There are four main herbs in KX, namely rvention . Howeverrvention , the advrvention . Thus, wCurcuma longa L (E’Zhu) in the treatment of breast cancer  network was carried out to identify the key targets of KX in RA treatment. The whole network contains 1348 nodes, 7164 edges. For better analysis in adverse reactions and presentation, we select the nodes whose degree is above 10 to construct a PPI network Figure 3H.Lév. Hutch, i.e., IMPDH2, MTHFD1, PKD2, PTGIS, SLC25A13, AGTR1, NR0B1, AR, and AGT  of KX, which pointed out that they may help in reducing the side effect of H.Lév. Hutch. The analysis of the GO enrichment of rest herbs shows that the primary function involved in animal organ development, positive regulation of the cellular biosynthetic process, positive regulation of protein metabolic process, positive regulation of gene expression, etc. , Celastrol (Cel), wilforgine, etc. We made a review of the Tripterygium wilfordii genus herbs or their active ingredients to validate our hypothesis. The KX capsule or its active ingredients could promote varieties of cell apoptosis, including T cells, macrophages, dendritic cells, and fibroblast-like synoviocytes (FLS). Experimental evidences of KX capsule or its active ingredients for apoptosis were summarized in The PI3K/AKT/mTOR pathway has a pivotal role in mediating many cellular responses, including protection from apoptosis. The KX capsule or its ingredients could target at NF-κB, AP-1, IKK, HIF-1α, resulting in varieties of phenotype changes, such as inhibition of inflammatory cytokines released by macrophages, differentiation of T cell, invasion of FLS. Experimental evidence of the KX capsule or its active ingredients for inhibiting inflammatory network were summarized in required in cell survival. Methotrexate (MTX), the main treatment in RA, inhibits the enzyme dihydrofolate reductase and then lead to tetrahydrofolate exhausted. The depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production (via disrupting the pathways (H.Lév. Hutch) increases the level of cysteinylglycine and cysteine conjugates derived from NADPH-independent metabolism and promotes hepatotoxicity, which could be ameliorated by the supplement of glutathione  stimulates the growth and metabolic activity via increasing amino acid and nucleotide metabolism, short-chain fatty acid-related metabolism . All these pathways are involved in cell signal transmission and could have crosstalk with PI3K/AKT/mTOR signaling pathway, which is used to achieve the proliferation, differentiation, and apoptosis of immune cells in RA . In physAt the same time, KX can alleviate the inflammatory response and reduce the disease activities by regulating multiple key inflammatory signaling pathways in RA, such as IL-17 signaling pathway, Th17 cell differentiation signaling pathway, and TNF signaling pathway . In physH.Lév. Hutch can act on IMPDH2 and MTHFD1.IMPDH2 encodes the rate-limiting enzyme (inosine 5′-monophosphate dehydrogenase) in the de novo guanine nucleotide biosynthesis. It is thus involved in maintaining cellular guanine deoxy- and ribonucleotide pools, which needed for DNA and RNA synthesis  could play a role of detoxication through positive regulation of the amino acid metabolism or supplement of some amino acids. In conclusion, KX is a relatively safe alternative approach in RA treatment.KX treats RA H.Lév. Hutch (Kun Ming Shan Hai Tang), Epimedium brevicornu Maxim (Yin Yang Huo), Cuscuta chinensis Lam (Tu Si Zi), and Lycium barbarum L (Gou Qi Zi). The component and targets of Epimedium brevicornu Maxim (Yin Yang Huo), Cuscuta chinensis Lam (Tu Si Zi), and Lycium barbarum L (Gou Qi Zi) were searched at BATMAN-TCM. The Cut-off value is set to 20. Since the BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm/) database does not record H.Lév. Hutch component data, a literature search is required. Finally, we used the data of a published master’s thesis in Chinese .There are four main components of KX,  Chinese . The theCTD is a publicly available database that contains robust disease genes. “Rheumatoid arthritis” was entered into the CTD database to obtain any possible disease target genes.via pathological disorder. The OMIM database was retrieved by PubMed, and the retrieved relevant disease genes were recorded.OMIM is a comprehensive, authoritative compendium of human genes and genetic phenotypes database, which can be used to establish disease networks https://string-db.org/) to attain protein interaction networks, with a minimum required interaction score 0.9. Cytoscape 3.7.0 (https://cytoscape.org/) software was applied for PPI visualization. The adverse reaction disease network was constructed using the gene retrieved in OMIM with a minimum required interaction score of 0.4.Targets of the Kunxian capsule and RA-related genes were input into STRING . Among the 11 methods, the MCC PPI network has better performance in predicting the accuracy of key targets . The MCCBioconductor’s “clusterProfiler,” “pathview,” and other functional packages  in the RThe original contributions presented in the study are included in the article/YT: data analyses, figure preparation, manuscript preparation, and study initiation. YZ: interpretation, data analyses, and manuscript preparation/submission. LL: data analyses, and manuscript preparation. ZX: data interpretation, figure preparation. CW: critically reviewed the manuscript, project funding. LH: critically reviewed the manuscript and study initiation. All authors approved the final version of the manuscript.Research was funded by National Key R&D Program of China (2018YFC1705500).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.


Avascular necrosis of femoral head (AVNFH) is a debilitating disease, which affects the middle aged population. Though the disease is managed using bisphosphonate, it eventually leads to total hip replacement due to collapse of femoral head. Studies regarding the association of single nucleotide polymorphisms with AVNFH, transcriptomics, proteomics, metabolomics, biophysical, ultrastructural and histopathology have been carried out. Functional validation of SNPs was carried out using literature. An integrated systems analysis using the available datasets might help to gain further insights into the disease process. We have carried out an analysis of transcriptomic data from GEO-database, SNPs associated with AVNFH, proteomic and metabolomic data collected from literature. Based on deficiency of vitamins in AVNFH, an enzyme-cofactor network was generated. The datasets are analyzed using ClueGO and the genes are binned into pathways. Metabolomic datasets are analyzed using MetaboAnalyst. Centrality analysis using CytoNCA on the data sets showed cystathionine beta synthase and methylmalonyl-CoA-mutase to be common to 3 out of 4 datasets. Further, the genes common to at least two data sets were analyzed using DisGeNET, which showed their involvement with various diseases, most of which were risk factors associated with AVNFH. Our analysis shows elevated homocysteine, hypoxia, coagulation, Osteoclast differentiation and endochondral ossification as the major pathways associated with disease which correlated with histopathology, IHC, MRI, Micro-Raman spectroscopy etc. The analysis shows AVNFH to be a multi-systemic disease and provides molecular signatures that are characteristic to the disease process. AVNFH has been categorized basically into idiopathic and secondary varieties. The secondary causes of AVNFH include haemoglobinopathies, storage disorders, SLE, decompression sickness etc.5 alcohol, Steroid, trauma, coagulopathies7 Numerous other factors like genetic factors, environmental factors, physiological process like pregnancy, lifestyle involving smoking, drug abuse, diseases like haemochromatosis have also been implicated as risk factors in AVNFH10.Avascular Necrosis of Femoral Head (AVNFH) is a debilitating disease, usually affecting younger individuals. Clinically AVNFH is associated with reduction in vascular supply to the subchondral bone of the femoral head which leads to necrosis and collapse of articular surface and eventual degeneration of hip joint11. Histopathology shows increased cement lines and lacunae while electron microscopy shows micro-cracks, dead bone and woven bone substitution1. Immuno histochemistry of AVNFH bone shows increased osteocalcein1, iNOS12, RANKL13, BMP-2 and HIF-1 staining15. AVNFH is also associated with coagulation disorders and coagulopathies possibly play an important role in the pathophysiology of the disease. Consistent with this, increased microparticles have been observed in the blood of AVNFH patients which might clog capillaries and contribute to avascularity17. High amounts of Von Willebrand Factor which is associated with AVNFH will also lead to thrombosis18. Coagulation factor disorders have been correlated with AVNFH19. Endochondral ossification, where cartilage is replaced by bone was also shown to be inhibited in AVNFH20. These studies point to a complex association of hypoxia, thrombosis, reduced osteoblast activity and increased osteoclast activity in the pathogenesis of AVNFH.Biophysical characterization of bone from AVNFH patients and piglet model of disease shows reduced mineral to matrix ratio and increased carbonate to phosphate ratio indicative of extensive remodelling due to osteoclast activity4. The genetic factors include mutations in Col2A2, MTHFR, NOS, PAI-1, PON-1, alpha-2-Microglobulin, SREBP-2, VEGFC, ADH-2, and sometimes associated with Increased Lipoprotein A and protein C deficiency21. However, MTHFR mutations do not correlate with the disease in Korean and Japanese populations22, pointing to a possible population specific differences.More so, AVNFH has now been considered as a Multi systemic disease rather than as a disease involving just the femoral head26. The transcriptomic and proteomic analysis have largely reiterated on the signalling pathways that are deregulated in AVNFH26. These pathways are largely binned into osteoclastogenesis, osteoblasts and their function etc. Microarray studies have been carried out using rat models of steroid induced AVNFH and cartilage from diseased area of human AVNFH patients27. The results show deregulation of key pathways involved in the pathogenesis of AVNFH. Microarray analysis of whole transcriptome of AVNFH articular cartilage was performed at different time points from the induction of ischemic injury and compared to control24. The study showed genes involved in ischemic injury and angiogenesis were significantly upregulated at 2 weeks post AVNFH onset and those belonging to the inflammatory pathway four weeks post onset of the disease. Proteomic studies have also been carried out on AVNFH patient bone samples28. These studies points to deregulation of multiple signalling and metabolic pathways which are implicated in cartilage and bone biology. Targeted metabolomic analysis shows elevated levels of metabolites belonging to Methionine–Homocysteine pathway, Polyamine and urea pathway concomitant with low levels of B12, B6 and Betaine in AVNFH patients1. Metabolomic analysis of bone tissue in Chinese population showed changes in amino-acid and fatty acid metabolism25. Serum metabolomic analysis of rabbit model of steroid induced AVNFH showed differentially regulated phospholipids29. These studies show perturbations in different signalling and metabolic pathways in AVNFH.Omic techniques like transcriptomic, proteomic or metabolomics analysis of serum or tissues from AVNFH patients or model systems have been carried out6, B12 and Folate and its implications for enzyme activity concomitant with deregulation of metabolic pathways has been analysed. The implications of the deficiency of these vitamins has been analysed in the light of transcriptomic and proteomic data obtained from cartilage or bone from patients and animal models. Further, the SNP data, transcriptomic data and proteomic data have been integrated with Vitamin-cofactor enzyme data. The role of SNPs, transcriptomic, proteomic and metabolomic data and its role in osteoblast, osteoclast and cartilage biology, their function as well as its implications for AVNFH has been discussed. The data analysis shows deregulation of multiple pathways consistent with the multi-systemic nature of AVNFH. In addition, the genes from integrative analysis common to at least two data sets were analysed using DisGeNET. The results show the involvement of these genes with various diseases. These diseases are risk factors associated with AVNFH. Further, we validated the role of elevated homocysteine on MSC differentiation to osteoblast and mineralization. We also show that RANKL is elevated in the AVNFH patients. Using literature mining we show elevated homocysteine, vitamin B6, B12, glutathione, N-acetylcysteine, CBS and MUT Knock out and knock down modulate RANKL induced osteoclastogenesis. Our analysis provides a theoretical basis to understand the disease and raises further questions which could shed light on the disease process. This could in turn provide potential biomarkers and newer therapeutic targets in the disease.However, a concerted effort to analyse the disease from a systems perspective by integrating the data sets from omic-studies as well as data from those reported in literature has largely been lacking. In the present study an integrative analysis of AVNFH associated SNPs, transcriptomic data sets of bone and cartilage, proteomic data sets and metabolomic data have been carried out. The data is analysed using cytoscape and binned into different pathways using its plugin ClueGO. The SNP data was validated by literature search for experimentally verified effects or phenotypes. The deficiency of vitamins/cofactors like B12 metabolism, Folate metabolism, TGFβ signalling, matrix metallo proteases, Clotting and coagulation, HIF-1 pathway, angiogenesis, Endochondral ossification etc. The Reactome pathway binned genes into vacuolar pathway, matrix metallo proteinases, VEGF and purine metabolism. The KEGG pathway binned genes into Coagulation cascade, Fluid shear stress and atherosclerosis, HIF-1 pathway, VEGF signalling and AGE/RAGE pathway , alcoholism, estrogen pathway etc.  transport and metabolism as well as . The genes with an adjusted P. value of ≤ 0.05 were selected for analysis using cytoscape. The genes are subsequently binned into pathways using the plugin ClueGO. The results of analysis using KEGG shows deregulation of signalling and metabolic pathways having significant term P. value like alanine aspartate and glutamate metabolism, glycolysis/gluconeogenesis, methionine and cysteine metabolism, B26. The dataset showed 128 significantly upregulated proteins and 52 significantly downregulated proteins. The proteins with significant changes in the levels of expression were used for pathway annotation analysis using ClueGO a plugin of Cytoscape. Analysis using Reactome pathways binned the proteins into pathways involving—defects in cobalamin (B12) metabolism, Platelet degranulation, scavenging of Heme from plasma, Defective AMN causes hereditary megaloblastic anemia, and Glycolysis pathways . An interaction network of the cofactors and the enzymes were generated and pathway annotation analysis was carried out using ClueGO. The results of this analysis for B6, B12, Folate shows enrichment in amino acid metabolism, Fatty acid and steroid metabolism, taurine and hypotaurine metabolism, one carbon metabolism, cysteine and homocysteine metabolism, selenocysteine metabolism, glycogen and glucose metabolism . Two of the nodes, CBS and MUT had degree 3. Defective MUT and CBS due to Vitamin Bs35 Fig. . These 1s35 Fig. . Reactoms35 Fig. . Pathways35 Fig. . FurtherIn addition, the data from AVNFH patients and animal model systems for MRI, CT, Histopathology, IHC and Micro-Raman Spectroscopy of bone was correlated with molecular signatures obtained from –Omic analysis. Micro-Raman spectroscopy of human AVNFH bone and piglet AVNFH model showed reduced mineral matrix ratio and increased carbonate to phosphate ratio indicative of increased osteoclastogenesis and decreased osteoblast function  lead to elevated levels of homocysteine and methyl malonic acid which modulate MSC differentiation to osteoblasts, macrophage differentiation to osteoclasts as well as their function with potential implications for the disease.Taken together our –Omic analysis results as well as integrative analysis using different data sets shows CBS and MUT to be associated with the disease. CBS and MUT, for their activity require B12 and coagulation which has implications for disease process. The cofactor-enzyme network conjured pathways that are deregulated in the disease, which has major implications for disease progression. The integrated analysis of SNP, Transcriptomic, proteomic and cofactor network shows common pathways that are deregulated in the disease that are very critical to the disease process. Analysis of the genes that are common to at least two data sets in the integrative analysis using DisGeNET showed association with multiple diseases. Interestingly cross validation using literature mining showed that these diseases are risk factors associated with AVNFH. In order to validate the prediction of a role for elevated homocysteine in vitro studies were carried out using MSC. Experimental validation of the role of homocysteine shows impaired MSC differentiation to osteoblasts and their function. In addition to this we also probed into the role of RANKL in AVNFH by measuring the levels of RANKL in our patient cohort. Subsequently the factors that modulate RANKL induced osteoclastogenesis was validated by literature mining. Our analysis showed elevated levels of RANKL in AVNFH patients. Using literature mining we show that RANKL induced osteoclastogenesis is augmented by homocysteine, depletion of B6 and B12, knock down/knock out of CBS or MUT. RANKL induced osteoclastogenesis could be attenuated by supplementation of glutathione or N-Acetylcysteine in CBS KO mice. Taken together the analysis recapitulates the observed processes associated with the disease which could be validated by experiments and literature mining.SNPs associated with AVNFH are collected from the literature. Network of SNPs associated with AVNFH was generated using Cytoscape and the SNPs in genes were binned into pathways. Furthermore, the functional implications of SNPs that were validated experimentally was collected from literature and its implication for AVNFH was discerned. The SNPs were associated with elevated homocysteine, hypoxia, osteoclastogenesis or coagulation and vasoconstriction. Transcriptomic analysis binned the genes into signaling pathways, metabolic pathways and other pathways that has implication for bone biology and disease manifestation. Proteomic data collected from the literature shows changes in pathways like B1. The analyzed data at the level of SNPs, Transcriptomic, proteomic, metabolomics or their integrative analysis shows involvement of cysteine and methionine pathway, one carbon metabolism, B12 metabolism, B6 and folate metabolism, megaloblastic anemia etc. In addition, alcoholism which is a risk factor for AVNFH also leads to reduced levels of B12 and increased levels of homocysteine37. Our previous studies have shown lower levels of hemoglobin in AVNFH patients compared to age and gender matched controls1. However, whether the reduced hemoglobin correlated with megaloblastic anemia was not confirmed. Homocysteine has implications for bone biology as it remodels osteoblastogenesis, osteoclastogenesis and their function39. Homocysteine is also an inflammatory molecule and induces inflammatory response in the macrophages. Homocysteine is known to inhibit the activity of NOS. NOS is an important enzyme which is essential for vascular function40. Inhibition of NOS also leads to increased osteoclastogenesis41. Homocysteine also modulates blood coagulation42. In addition, homocysteine is known to induce ROS43. B6 is an important vitamin which is a cofactor for cystathionine beta synthase involved in the synthesis of cysteine and thus glutathione44. Reduced levels of B6 not only contribute to increased homocysteine but also to decreased cysteine, which is essential for the synthesis of glutathione. Our integrative analysis shows CBS as a key enzyme in the transsulfuration pathway which might be involved in AVNFH. Thus SNPs or vitamins like B6, B12 and folate or alcoholism might modulate the levels of homocysteine in different populations which in turn influences multiple pathways in the progression of AVNFH.Previous studies have shown significant association of elevated levels of homocysteine with AVNFH45. Previous studies have shown an association of homocysteine with AVNFH1. Homocysteine potentiate ADP induced platelet degranulation and coagulation46. Increased Von Willebrand factor which is involved in coagulation process is also associated with AVNFH47. SNP in Collagen associated with AVNFH is shown to induce platelet activation49. In addition, microparticles in blood are associated with the disease16. The association of coagulation and vasoconstriction is also supported by histopathology and MRI-BOLD as well as blood velocity studies50. Hence multiple factors at the genetic, transcriptomic, metabolomic or at the level of cofactors might contribute to coagulation cascade which is associated with AVNFH.AVNFH has also been associated with coagulation disorders. Consistent with this SNP analysis Using the Plugin ClueGO binned genes into blood clotting cascade, complement and coagulation cascade. ClueGO analysis of network generated with the SNP containing genes along with first neighbors show that the genes are binned into Platelet degranulation pathway. ClueGO analysis of Network generated from Proteomic data sets shows genes binned into platelet degranulation, complement and coagulation cascade. Platelet degranulation is an important process in coagulation pathway11. CT scan shows reduced Houns field unit an indication of reduced bone density1. The data is suggestive of increased osteoclast and decreased osteoblast function. The increased resorption of bone is ascribed to increased osteoclastogenesis and its function51. In addition, AVNFH bone shows increased bone turnover as observed in the Immunohistochemistry (IHC) for osteocalcein1. Homocysteine is shown to modulate osteoclastogenesis. Studies have reiterated a role for homocysteine in osteoblast induced osteoclastogenesis by way of RANKL expression and secretion32. B12 and B6 depleted media are shown to augment the differentiation of macrophages to osteoclasts52. Our AVNFH patient cohort show elevated level of RANKL in plasma. IHC of patient bone also shows increased RANK, RANKL staining13. More so, Iron sequestration is an important event in osteoclastogenesis53. Consistent with these facts, RANKL induced osteoclastogenesis shows upregulation of genes involved in iron metabolism54. Knocking down genes involved in endosome acidification was shown to inhibit osteoclastogenesis55. SNP analysis shows HIF-1 signaling pathway and VEGF signaling pathway are associated with hypoxia. SNP and Transcriptomic analysis shows regulation of gene expression by HIF-1α. Traumatic AVNFH is associated with hypoxia as seen in animal model of AVNFH56. In addition, the microparticles in blood associated with ANVFH is proposed to clog capillaries inducing hypoxic conditions17. Consistent with this immuno-histochemistry shows increased HIF-1 staining in AVNFH bone15. MRI-BOLD technique also shows changes in the oxygenation of arterial and venous blood in AVNFH patients compared to controls  into osteoblasts60. However, contradictory results are obtained in case of homocysteine induced mineralization61. Increased iron also inhibit differentiation of MSC into osteoblasts62. Consistent with this a role for iron is conceived in osteoclastogenesis, osteoblastogenesis and their function. Iron overload diseases like sickle cell anemia, beta-thalassemia and hemochromatosis are risk factors for AVNFH. The histopathological changes also report empty lacunae in bone, indicative of absence of osteoblasts, reverse cement lines, marrow calcification etc., which shows deregulation and imbalance in osteoblast and osteoclast function1. The different pathways might act in concert to impede MSC differentiation into osteoblasts and promote differentiation of macrophages into osteoclast as well as modulate their function.Homocysteine is shown to inhibit the expression of LOX163. Network analysis of SNPs associated with AVNFH and transcriptomic analysis of AVNFH cartilage shows binning of genes involved in endochondral ossification pathway. Cartilage in AVNFH shows hypertrophy and death in AVNFH64.Endochondral ossification is a process where cartilage is replaced by bone65. Bisphosphonate acts at multiple levels by inhibiting osteoclastogenesis and preventing coagulation by inhibiting platelet degranulation66. Bisphosphonate also helps in increasing mineralization65. However, in the absence of osteoclastogenesis, this might lead to woven bone formation which might hamper bone strength67.Treatment with Bisphosphonate is the standard care treatment for AVNFHIn the present study, analysis of genes common to at least two data sets using DisGeNET showed association with various diseases. Validation using literature showed these diseases are risk factors associated with AVNFH  based on the platform GPL13497 (Agilent-026652 Whole Human Genome Microarray 4 × 44 K v2), deposited by Ruiyu L24. Gene expression profiling of AVNFH articular cartilage was carried out by collecting hip articular cartilage specimens from 12 AVNFH patients and 12 healthy controls. Microarray data of GEO accession GSE74847 was used which studied RANKL induced osteoclastogenesis using RAW 264.7 cells76. The Proteomic data from AVNFH bone tissue compared to healthy control bone tissue was used from a published dataset26. Data from three published metabolomic analyses were used for our study. Our previous study involving targeted metabolomics of plasma samples from 14 patients compared to 14 healthy control samples were used1, a global non-targeted metabolomics of plasma samples from 30 AVNFH patients compared to 30 healthy controls23, and a bone trabecular metabolomics study of 28 patients compared to 20 controls25.The gene expression microarray study involved a genome wide Gene expression profiling of hip articular cartilage with AVNFH. Gene expression microarray data of GEO accession GSE74089 was obtained from Gene Expression Omnibus77. The GEO2R tool uses the GEO-query and limma R packages from the Bioconductor project to compare the original processed data tables supplied by the submitter. The GEOquery R package parses GEO data into R data structures that can be used by other R packages77. The limma  R package has statistical tests for identifying differentially expressed genes78. The GEO2R tool was used to define groups such that the logFC gave fold change of NFH group with respect to Control group. i.e., a positive logFC implied upregulation of gene in NFH group and negative logFC implied downregulation in NFH group.The GEO2R web tool was used to define two groups of samples namely  ‘NFH’ and ‘Control’, and to perform Differential Gene Expression analysis79 was used for adjustment of the microarray data and provide a good balance between discovery of statistically significant genes and limitation of false positives. Significant differentially expressed genes of Adjusted P. Value ≤ 0.05 were used for this study. Significant changes in the proteomic and metabolomics analyses were obtained from the respective published datasets.The adjusted p. Value from the Limma package was used to identify genes that were significantly differentially expressed between the Control and NFH patient sample. The Value adjustments, also called multiple-testing corrections, attempt to correct for the occurrence of false positive results. The Benjamini & Hochberg false discovery rate method71. ClueGo can perform Enrichment (right-sided hypergeometric test), Depletion (left-sided hypergeometric test) or Enrichment/Depletion (two-sided hypergeometric test). We have used Enrichment/Depletion (two-sided hypergeometric test) for our analyses which are recommended80. The significant differentially expressed genes were used for querying Reactome, KEGG and WikiPathways74 databases for pathway functional analyses. The pathway terms showing term P. Value ≤ 0.05 were considered for further analysis. Significant pathways of interest were used for creating subnetworks containing specific daughter pathways with genes involved to understand differential gene expression and predict mechanism of derangement of metabolic pathways. Cytoscape tools were used to generate specific pathway networks from global pathways.The ClueGO plugin of Cytoscape was used for pathway annotation analysis81. Subnetworks of pathways showing pathway terms and their associated genes were created. logFC data of genes was then imported into cytoscape and was overlaid with the subnetwork. MetaboAnalyst tool was used to bin significant metabolites into KEGG Pathways30.Cluepedia plugin of Cytoscape was used to add all genes present in all the pathway terms of the network6, B12 and Folate cofactors are implicated in AVNFH82, an interaction network involving these cofactors and respective proteins was created and visualised in Cytoscape71. We have created this network based on the published dataset involving cofactors–protein interaction network used for understanding relation between human nutrition and diseases31. The created Vitamin B6, B12 and Folate cofactors – protein network was used for ClueGo analyses.Since Vitamin BNetworks for individual analyses involving AVNFH SNPs, Proteomic, Transcriptomics and Cofactor-protein networks were created using Cytoscape. Next these individual networks were merged in Cytoscape to obtain an integrated network. To understand common key nodes in AVNFH, network centrality analysis was carried out using CytoNCA plugin of Cytoscape 83. The hub nodes obtained from network centrality analysis was used for further functional analyses using ClueGO.The 125 hub nodes from integrated omics network with degree 2 and 3 were used to carry out Drug/Disease enrichment analyses. Enrichr web—based tool was used to carry out Drug / Disease enrichment analyses 84. We used gene—set libraries from DisGeNet and Rare Diseases GeneRIF Gene Lists. DisGeNet is a comprehensive repository of human gene—disease associations. Rare Diseases GeneRIF Gene Lists has gene—disease associations based on GeneRIF (Gene Reference into Function) to enrich the functional annotation of genes. The enriched terms were sorted using combined score ranking after carrying out the enrichment analyses using Enrichr tool.All the blood samples of AVNFH (n = 5) and controls (n = 7) were procured from patients visiting Sri Sathya Sai Institute of Higher Medical Sciences in a de-identified manner by a honest broker as per approval of the SSSIHMS institutional bioethics commission  Informed consent was obtained from all subjects and the methods were carried out in “accordance” with the approved guidelines and regulations. Plasma samples from healthy control, and AVNFH were frozen at − 80 °C until assay was done. Commercial ELISA kits (Peprotech) of human sRANK ligand (Cat. No. 900-M142), were used in the study as per manufacturer’s instructions. Dilutions of antibodies were carried out as per manufacturers instruction unless otherwise specified. Briefly, 96 well micro-titer plates (Corning Product #3590) were coated with capture antibody and incubated overnight at room temperature. Following this, the plate was first washed with wash buffer and blocked with block buffer for 1 h at room temperature. Standards and sample dilutions were prepared using the diluent and pipetted into designated wells. Detection antibody was added immediately into the standards and sample wells and incubated for 2 h at room temperature. After thorough washing, Avidin- HRP conjugate was added and incubated for 30 min at room temperature. Finally, ABTS Liquid substrate provided by the manufacturer in the respective kit was added into each well and incubated for 30 min at room temperature. Absorbance at 405 nm (reference absorbance 650 nm) was obtained within 30 min of adding the stop solution and the results were calculated using a log–log or 4- parameter curve fit. P. value ≤ 0.05 was considered significant.g for 10 min. The cell pellet was suspended in 1 ml MSC culture medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 2 mM glutamine . Cells were seeded in 35 mm cell culture dishes at a density of 1 × 106 cells/cm2 and incubated in a humidified incubator at 37 °C, 5% CO2. After 1 day, nonadherent cells were removed by washing twice with DPBS and fresh MSC culture medium was added. The medium was replaced with fresh MSC culture medium every 2 days.Animal experiments were approved by the Institutional Animal Ethics Committee, Indian Institute of Science, Bangalore, India. All animal protocols were performed in accordance with the guidelines for care and use of laboratory animals set by Indian National Science Academy. Six to eight -week-old CD-1 female mice, weighing 25–28 g, were used for the isolation of BM-MSCs BM-MSCs were isolated as previously described (PMID: 29760732). Briefly, the mice were sacrificed by cervical dislocation and the femurs and tibias were dissected out. The bone marrow was flushed with Dulbecco's phosphate-buffered solution . The cell suspension was filtered through a 70 μm cell strainer , and centrifuged at 300×60 was studied by using DL Homocysteine (Sigma-Aldrich). Medium was changed every 3 days.Mouse bone marrow derived Mesenchymal Stem Cells (MSCs) were cultured in basal medium (BM) consisting of DMEM (Gibco) supplemented with 10% FBS (Gibco), 1% Penicillin –Streptomycin solution (Gibco) and 1% Glutamax (Gibco). Cells were cultured in 4 well plates in basal medium till cells reached 70% confluence following which the basal medium was replaced with Osteogenic medium (OM) which consisted of basal medium supplemented with Ascorbate—100 µM (Sigma-Aldrich) and β-glycerophosphate—5 mM (Sigma-Aldrich). During osteogenic differentiation, effect of appropriate concentration of Homocysteine—30 µMEffect of Homocysteine on Alkaline Phosphatase activity during osteogenic differentiation was carried out after 7 days of treatment. ALP activity staining was carried out by fixing cells with 4% para formaldehyde. After fixation the cells were stained with staining solution containing Napthol–AS–MS Phosphate—0.4 mg/ml (Sigma-Aldrich), Fast Red TR salt—1 mg/ml (Sigma-Aldrich), dissolved in Tris-Maleate buffer—30 mM at pH 9, in presence of 0.08% MgCl2 solution. At pH 9, in presence of Mg2 + and Alkaline Phosphatase, Napthol–AS–MS Phosphate and Fast Red TR combine to form an insoluble Azo red end product.Effect of Homocysteine on matrix mineralization, was carried out using Alizarin Red S staining to stain calcium deposits. After 14 days of treatment, the cells were fixed with 4% para formaldehyde. Then the cells were stained with 2% Alizarin Red S solution (Sigma-Aldrich) whose pH was adjusted to 4.2 using ammonium hydroxide solution. Alizarin Red S stains calcium deposits to give red colouration.https://imagej.nih.gov/ij/), by calculating the intensity of the stained area. Data is presented as mean fold change of at least 5 sample images from each group. P. Value ≤ 0.05 was considered significant.ALP activity staining and Alizarin staining was quantified using ImageJ software (Version: 1.46r URL: Experiments on humans and the use of human blood samples: We confirm that all experiments were performed in accordance with relevant guidelines and regulations.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.


Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for “natural” 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by  CurrentSaccharomyces cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia fermentans, Pichia anomala, Schizosaccharomyces pombe, Yarrowia lipolytica, Zygosaccharomyces rouxii, and Hansenula anomala can synthesize 2-PE from L-phenylalanine (L-Phe) or glucose through the Ehrlich pathway and phenylpyruvate (PPA) pathway  sensor system] and intracellular amino acid-sensing system [target of rapamycin (TOR) pathway], which are crucial for sensing extracellular and intracellular amino acids, respectively  families with conserved sequences and architectural characteristics are critical for the transportation of amino acids . Amino aynthesis . To dateno acids . In addino acids . These tno acids .AGP1 gene is a general amino acid permease and has a broad substrate range and low substrate affinity. Earlier research found that asparagine and glutamine are the major substrates of Agp1p in S. cerevisiae YCC5, with Km < 1.0 mM. Moreover, Agp1p can transport L-Phe and other uncharged amino acids when these amino acids are present in millimolar concentrations  and can be inhibited in the presence of ammonium salts  and a UASAA motif, and transcription of the BAP2 gene is regulated by Leu, the SPS sensor system and the GAAC pathway , poor nitrogen sources (urea or proline) or the addition of rapamycin. When S. cerevisiae is cultured in the presence of preferred nitrogen sources, the transcription of ARO9 is low or negligible , poor nitrogen sources (urea or proline) or the addition of rapamycin  are upregulated in tetraconazole-enriched medium. Therefore, the activity of aminotransferase, decarboxylase, and dehydrogenase may be enhanced by tetraconazole . Therefore, α-ketoglutarate is the converging point of L-Phe catabolism and glucose metabolism. α-Ketoglutarate is modulated by the retrograde regulation (RTG) pathway, which mainly regulates the expression of and ACO1 . In the and ACO1 . TherefoSaccharomyces cerevisiae is generally recognized as safe (GRAS) and is typically used in food and industrial production. Saccharomyces cerevisiae can also serve as one of most promising microorganisms for the biosynthesis of natural 2-PE. Saccharomyces cerevisiae recognizes extracellular L-Phe through the SPS-sensing pathway, regulates the expression of genes that are critical for the transport of L-Phe, and then recognizes intracellular L-Phe through the TOR-sensing pathway. Finally, L-Phe is converted into 2-PE via the Ehrlich pathway. The currently known transporters involved in the transport of L-Phe are Agp1p, Bap2p, Bap3p, and Gap1p.S. cerevisiae Y294 is grown with ammonium salt as the sole nitrogen source, the harvested cells are transferred to the buffer system containing L-Phe and leucine, in which Bap2p is the principal L-Phe transporter. Agp1p is the major transporter of L-Phe when S. cerevisiae Y294 gap1∆ is cultured in synthetic medium. The transporters of L-Phe are regulated synergistically by different pathways. For example, Bap2p is regulated simultaneously by the SPS-sensing pathway and GAAC pathway, and Agp1p is regulated simultaneously by the SPS-sensing pathway and NCR. The details of the regulatory mechanism of L-Phe transport remain unclear, and further study is necessary to determine how the regulatory pathways synergistically regulate the transport of L-Phe.However, the four permeases contributing to L-Phe transmembrane transport could be finely tuned based on different nitrogen sources. When In situ product removal, two-phase extraction and in situ product adsorption are used to alleviate the toxicity of 2-PE, which has been shown to be effective but uneconomical. Many efforts have been made to screen robust strains  from glucose or glycerol , aromatic amino acids , and sulfur-containing amino acids (methionine) are assimilated by the Ehrlich pathway to produce other higher alcohols, such as 2-methylbutanol, propanol, and 3-methylbutanol. This research could play a guiding role in the production of other higher alcohols.XC and CY conceived the review. JD and HX wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that this study received funding from China Tobacco Corporation and Hubei tobacco company. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.


In this perspective article, we highlight the use of protein-protein interaction (PPI) networks as a multi-omics integration approach to unravel information flow and mechanisms during complex biological events, with a focus on the immune system. This involves a combination of tools, including: InnateDB, a database of curated interactions between genes and protein products involved in the innate immunity; NetworkAnalyst, a visualization and analysis platform for InnateDB interactions; and MetaBridge, a tool to integrate metabolite data into PPI networks. The application of these systems techniques is demonstrated for a variety of biological questions, including: the developmental trajectory of neonates during the first week of life, mechanisms in host-pathogen interaction, disease prognosis, biomarker discovery, and drug discovery and repurposing. Overall, systems biology analyses of omics data have been applied to a variety of immunology-related questions, and here we demonstrate the numerous ways in which PPI network analysis can be a powerful tool in contributing to our understanding of the immune system and the study of vaccines.Systems biology is an approach to interrogate complex biological systems through large-scale quantification of numerous biomolecules. The immune system involves >1,500 genes/proteins in many interconnected pathways and processes, and a systems-level approach is critical in broadening our understanding of the immune response to vaccination. Changes in molecular pathways can be detected using high-throughput omics datasets  by using methods such as pathway enrichment, network analysis, machine learning, etc. Importantly, integration of multiple  In the field of immunology, a systems biology approach is necessary to understanding the immune response to vaccination, infection and diseases, since these involve complex interactions between a large number of genetic, epigenetic, physiological and environmental factors. Systems-level strategies can ultimately be applied to better understand the molecular changes in humans upon exposure to a vaccine or an immunotherapeutic, to understand the mechanisms underlying disease or pathogenesis, and to characterize the effect(s) of specific challenges to the immune system –5. OmicsA key challenge in systems biology is building the appropriate bioinformatics tools to integrate omics datasets, ultimately enabling the correlation of global changes with the underlying biological events that drove those changes. Statistical and machine learning approaches have been applied to omics datasets [reviewed previously –11] to iDownstream characterization of dysregulated molecules can further our understanding of underlying biological mechanisms at play. This can be achieved by interrogating curated functional genomics information from databases of gene ontologies , pathways, known interactors, transcription factor binding sites (TFBS) upstream of dysregulated genes, etc. through various enrichment analyses. However, a large proportion of genes have not been assigned to canonical pathways in pathways databases (such as KEGG or Reactome), so pathway enrichment limits the ability of such approaches to reveal novel insights .per se, and must be eventually tested using downstream wet lab experiments  immune system development, (b) mechanisms of host-pathogen interactions, (c) discovery of mechanism-based biomarkers, and (d) strategies to define prospective new interventions based on drug repurposing. While the methods are somewhat biased toward the study of innate immune and inflammatory responses, it is worth mentioning that “innate immunity instructs adaptive immunity”  in that www.innatedb.com) focused on elucidating the genes, proteins, and molecular “interactome” of the innate immune response, with an emphasis on curation of experimentally-validated PPIs and signaling pathways in human, mouse and bovine. The interactome can be used to understand the interplay between multi-omics datasets that measure different parts of a larger system of physical, metabolic, and regulatory networks. For example, human TRAF6 and MyD88 are usually defined as having a role in the major TLR4 to NFκB signaling pathway of innate immunity. However, in InnateDB, they are experimentally documented to interact with 398 and 129 proteins, respectively, in humans. This means that there is a massive potential for these proteins to bridge and/or participate in multiple biological pathways when activated by innate immune stimuli.InnateDB , 44 and InnateDB is an important tool in immunology as evidenced by the >6,000,000 hits from more than 55,000 visitors annually. While all known pathways  and molecular interactions  are present, the emphasis on innate immunity is achieved through the contextual review, curation and annotation of molecular interactions and pathways involved in innate immunity. To date, the InnateDB curation team has reviewed more than 5,200 publications annotating >27,000 molecular interactions of >9,400 separate genes in rich detail including annotation of the cell, cell-line and tissue type; the molecules involved; the interaction detection method; etc. By including interaction and pathway data relevant to all biological processes, a much broader perspective of innate immunity can be achieved, especially since an effective innate immune response requires the coordinated efforts of many important processes including the endocrine, circulatory, and nervous systems . AdditioInnateDB facilitates systems-level analyses by enabling the integration, analysis and visualization of user-supplied quantitative data, such as gene expression data, in the context of molecular interaction networks and pathways. This includes the statistically robust analysis of overrepresented pathways, interactomes, ontologies, TFBS, and networks. One can, for example, refine the network to show only molecular interactions between a list of differentially expressed (DE) genes (and their encoded products) or view all potential interactors regardless of whether they are DE. This can aid in the identification of important nodes that may not be regulated transcriptionally or which are expressed at an earlier or later time. Networks derived from InnateDB can be interactively visualized using the Cerebral plug-in for Cytoscape  to generIn addition to these outlined methods, there are bioinformatics tools available for performing other types of network analyses specifically for studying the immune system. Examples include immuneExpresso , a data Most recently, as a part of the EPIC-HIPC consortium, we published a study that revealed a robust developmental trajectory of immune ontogeny during the first week of life in newborns using a multi-omics integration approach . TranscrImportantly, through this study, we were able to show that multi-omics integration using PPI networks  provided similar biological insights, but greater depth, when compared to data-driven supervised integration approaches , it The analyses outlined in this article merely scratch the surface of what is possible using systems biology and high-throughput omics techniques to study the immune system, e.g., the major tools described here , 50–52 hBD, MS, and AB all contributed to writing of the first draft of this article. AL performed data analysis of the ontogeny study and provided valuable feedback through the writing process. RH supervised all authors, edited the manuscript and provided critical insights and feedback. All authors contributed to the article and approved the submitted version.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.