Glycyrrhiza uralensis Fisch.), Renshen (Panax ginseng C. A. Mey.), Danggui (Angelica sinensis (Oliv.) Diels), Shengma (Cimicifuga foetida L.), and Zicao (Lithospermum erythrorhizon Siebold & Zucc.) were selected as the core CM-pair for further investigation. Network pharmacology analysis yielded 131 active components and 348 candidate targets for the core CM-pair. Quercetin and celabenzine were chosen as ligands for molecular docking. GO and KEGG enrichment analyses revealed that the core CM-pair could interact with targets involved in immune, inflammatory, and infectious diseases. Moreover, key mpox virus targets, F8-A22-E4 DNA polymerase holoenzyme and profilin-like protein A42R, were docked well with the selected core components. And molecular dynamic simulation indicated that the component (quercetin) could stably bind to the target (profilin-like protein A42R). Our findings identified potential prescriptions, herbs, and components that can offer potential therapies or drugs for addressing the MPX epidemic.Mpox (MPX) has escalated into a public health emergency of international concern, necessitating urgent prophylactic and therapeutic measures. The primary goal of this investigation was to systematically extract Wan Quan’s expertise in treating smallpox, as documented in Exclusive Methods for Treating Pox (Dou Zhen Xin Fa in Chinese), with the aim of identifying potential prescriptions, herbs, and components for alternative MPX therapies or drugs. This research utilized data mining to identify high-frequency Chinese Medicines (CMs), high-frequency CM-pairs, and CM compatibility rules. Network pharmacology, molecular docking, and molecular dynamic simulation were employed to reveal the potential molecular mechanisms of the core CM-pair. 119 prescriptions were extracted from Exclusive Methods for Treating Pox. We identified 25 high-frequency CMs and 23 high-frequency CM pairs among these prescriptions. Combined association rule mining analysis, Gancao ( This has escalated into a public health emergency of international concern, necessitating urgent prophylactic and therapeutic measures. Since there are no clinically proven specific treatments for MPXV infection, the use of computational chemical biology techniques to identify potential blockers of MPXV target proteins is critical in special situations, such as MPX outbreaks. For example, molecular docking, virtual screening, and artificial intelligence,5 (referred to as “ dry method” research) have been widely used in TCM to prevent and treat infectious diseases. These computational methodologies are pertinent for minimizing the use of animal models in pharmacological research, assisting in the rational design of innovative and safe drug candidates, repositioning marketed drugs, and supporting medicinal chemists and pharmacologists throughout the drug discovery trajectory. Two primary proteins, MPXV F8-A22-E4 DNA polymerase holoenzyme and profilin-like protein A42R, have been recommended as viable targets for screening drugs that inhibit MPXV replication and proliferation, taking advantage of ongoing efforts to solve protein structures that guide structure-based drug discovery.Mpox (MPX) is a rare viral zoonotic infectious disease, predominantly occurring in Central and West Africa, and is caused by the mpox virus (MPXV). However, From 1 January 2022 through 5 June 2023, a cumulative total of 87,929 laboratory-confirmed cases of MPX, including 146 deaths, have been reported to World Health Organization (WHO) from 111 countries/territories/areas in all 6 WHO Regions. The symptoms are similar to smallpox, but the disease is milder, primarily manifesting as high fever, headache, lymphadenopathy, and systemic blisters and pustules, with a case fatality rate of approximately 1% to 10%. Smallpox vaccination has been reported to offer 85% protection against MPXV, and anti-smallpox virus drugs can have anti-MPXV effects.Both MPXV and smallpox viruses belong to the family Poxviridae and the genus Orthopoxvirus. TCM has a long history of foreign exchange and has now spread to 196 countries and regions, receiving increasing attention. In recent years, with the promotion of the WHO and some professional groups and people, the acceptance and recognition of Traditional medicine by the international community has gradually increased. As the representative of Traditional medicine, TCM should enhance cooperation with other Traditional medicine, and jointly promote the application and promotion of international Traditional medicine. When dealing with the epidemic situation of such infectious diseases, TCM should also work with western medicine peers to jointly promote the international application and dissemination of Chinese medicine and make contributions to human health. Both MPXV and smallpox viruses fall under the category of “pox” in epidemic diseases in TCM. TCM had extensive clinical experience and numerous effective prescriptions for treating smallpox, which holds referential value for MPX treatment. In June 2022, the Diagnosis and Treatment Guideline of MPX was released by the National Health Commission and the National Administration of TCM of the People’s Republics of China, recommending TCM for MPX treatment. Consequently, it is of great importance to explore and extract TCM experience in treating smallpox based on historical TCM classics combined with contemporary medical research methods. The present study employed data mining and an association network to identify high-frequency Chinese medicines (CMs), high-frequency CM pairs, and CM compatibility rules from ancient prescriptions. Subsequently, network pharmacology was applied to reveal the components, targets, and related signaling pathways of action of the core CMs-pair. Moreover, molecular docking and molecular dynamics simulation were utilized to investigate the molecular mechanism between core components in the core CM pair and key MPXV targets. The overall framework based on an integrated screening system strategy was shown in Figure Traditional Chinese Medicine (TCM), a medical system with a lengthy history, exhibits exceptional clinical efficacy against pestilence. Pharmacopoeia of the People’s Republic of China, Chinese Pharmacy, and Chinese Materia Medica were utilized to screen prescriptions for treating smallpox. Exclusive Methods for Treating Pox (Dou Zhen Xin Fa) was authored by Wan Quan, a renowned pediatrician from the Ming Dynasty. He extensively incorporated the experience of previous generations of doctors in treating smallpox and combined it with his own clinical experience to write this book, which recorded prescriptions for treating smallpox at various stages.In this study, Exclusive Methods for Treating Pox (Dou Zhen Xin Fa in Chinese),http://www.worldfloraonline.org (last accessed on 20 Apr 2023) for subsequent data mining and association network analysis.The data preparation process comprised 2 steps: first, relevant prescription information in the book, including number, name, indication, and formula, was input into an Excel file. Second, prescription information was standardized based on the Pharmacopoeia of the People’s Republic of China and the full botanical plant names have been checked with https://www.ibm.com/cn-zh/products/spss-modeler) was used to construct a CMs association network, which was further visualized using Cytoscape  and analyze the compatibility rule of prescription with the apriori algorithm according to “the support ≥ 10.0%, the confidence ≥ 80.0%, and the maximum number of antecedents was 2.”In this study, frequency analysis, association rule mining, and association knowledge network construction methods were employed to analyze the standardized data. Excel was used to analyze the high-frequency CMs. SPSS Modeler 18.0 software , Renshen (Panax ginseng C. A. Mey.), Danggui (Angelica sinensis (Oliv.) Diels), Shengma (Cimicifuga foetida L.), and Zicao (Lithospermum erythrorhizon Siebold & Zucc.), were selected to explore their potential mechanism. The chemical components of these CMs were obtained from TCMSP . Then, active components and protein targets were screened according to ADME principles of “oral bioavailability ≥ 30% and drug-likeness ≥ 0.18.” Furthermore, Swiss Target Prediction (http://www.swisstargetprediction.ch/) was used to supplement target information. Next, the UniProt website (https://www.uniprot.org/) was utilized to match protein targets and gene names. Subsequently, the CM-component-target network was constructed and visualized using Cytoscape (Version 3.9.1). Finally, the protein–protein interaction (PPI) network for the targets was established using the String Database (https://cn.string-db.org/) and was further visualized using Cytoscape (Version 3.9.1).Based on previous prescription information analysis results and clinical experience, 5 core CMs: Gancao ( This resource was employed to conduct Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for potential targets, using a P value < .05 as the cutoff criterion. The resulting data was further visualized through the use of https://www.bioinformatics.com.cn (last accessed on 20 Feb 2023), an online platform dedicated to data analysis and visualization.DAVID, a database for annotation, visualization, and integrated discovery, assists investigators in comprehending the biological significance behind extensive gene lists.https://pubchem.ncbi.nlm.nih.gov/).The chemical components of the core CMs-pair were acquired as “2.2. Potential Active Mechanism of the Core CM-Pair.” Upon analyzing the CM-component-target network, 2 core components—Quercetin and Celabenzine—were chosen as ligands for molecular docking. Subsequently, these core components’ 3D sdf format structures were downloaded from PubChem  Fig. A. The hoThe high-resolution crystal structure of the MPXV profilin-like protein A42R was obtained from the PDB (PDB-ID: 4QWO)  were completed to achieve the initial equilibrium structure, followed by 50ps of heating (temperature from 50K to 300K), and then 500ps of equilibrium at 300K. The time steps were set to 2fs. When all the thermodynamic parameters are stable, the molecular dynamics simulation is carried out for 10ns with a time step of 2fs and a total number of steps of 5 × 10This study didn’t require an ethical review, so ethical approval wasn’t necessary.Chinese medicine prescriptions (fang ji in Chinese) served as the primary form of clinical practice in TCM. In our study, we selected 119 prescriptions encompassing 109 types of Chinese Medicines (CMs) from Exclusive Methods for Treating Pox (Dou Zhen Xin Fa) for smallpox treatment. Our findings revealed that in these 119 prescriptions, 42.23% of CMs possessed cool/cold properties, 41.18% had warm/heat properties, and 16.59% exhibited neutral properties. Sweet, bitter, or pungent CMs accounted for 90.63% of the total, with sweet CMs being the most frequent, followed by bitter and pungent CMs. Meridian affinity analysis results showed that 19.89% of CMs belonged to the lung meridian, while 18.74%, 14.86%, and 13.52% were associated with the spleen, heart, and stomach meridians, respectively. The results are presented in Table The Ming Dynasty marked the zenith of TCM development, featuring numerous renowned physicians. Wan Quan, a representative pediatrician of the era, devised these prescriptions, reflecting his approach to smallpox treatment: a combination of tonification and purgation, cold and warm treatments, adaptability based on the disease’s progression, and a focus on achieving therapeutic balance. This methodology could be traced back to the thoughts and experiences of previous generations of doctors. Ge Hong, during the Eastern Jin Dynasty, first documented smallpox in his work, Emergency Prescriptions Reserved Behind the Elbow (Zhou Hou Bei Ji Fang in Chinese). In the Song Dynasty, a relatively comprehensive diagnosis and treatment system for smallpox was established amid the academic contention between the Cold and Warm Schools. Qian Yi, representing the Cold School, excelled in treating smallpox with cold and cool CMs, while Chen Wenzhong, a representative of the Warm School, was adept at using warm and tonic CMs for smallpox treatment. Wan Quan integrated their thoughts and formed his academic system.In order to identify high-frequency CMs, we employed a frequency analysis method to count the occurrence of CMs in the selected prescriptions. The results revealed 25 types of CMs with the highest frequency in these prescriptions , Rheum officinale Baill. (Dahuang), and Zicao. These results highlighted the principle of TCM in strengthening the body’s resistance to eliminate pathogenic factors, which could alleviate symptoms and address the root cause of the MPX disease.Emerging evidence suggested that the host immune response to MPXV played a crucial role in disease pathogenesis and clinical manifestations. The MPXV-mediated immune injury lead to poor clinical outcomes in patients with MPX. a unique combination of 2 relatively fixed herbs in the clinic. To identify high-frequency CM pairs in the selected prescriptions, we used SPSS Modeler 18.0 software to construct a CM association network, which was subsequently visualized using Cytoscape 3.9.1. In this network, CMs with degree values greater than twice the median (degree = 24) were depicted in Figure CM pairs were a critical aspect of CM compatibility,Furthermore, the results identified 23 CM pairs with a frequency of more than 10% in the selected prescriptions  and the profilin-like protein A42R(PDB-ID:4QWO). The molecular docking results showed good docking activity and the molecular docking poses with the highest Libdock score are illustrated in Figure The DNA polymerase holoenzyme had been confirmed to play a crucial role in the DNA replication processivity of MPXV, and it might guide the development of anti-poxvirus drugs. Among the 2 selected compounds, celabenzine exhibits a higher Libdock score with the target. In celabenzine, conventional hydrogen bonds were formed with GLY531, TYR554, and TEU553, and mixed Pi/Alkyl hydrophobic interactions were formed with LYS661 and TYR554. In quercetin, conventional hydrogen bonds were formed with SER552 and ASN665, and mixed Pi/Alkyl hydrophobic interactions were formed with ASN551 and LYS661.,40 Studies had shown that A42R may engage in distinct interactions with phosphatidylinositol lipids. Although the role of A42R in MPXV virus replication was not yet fully understood, it could also be selected as a therapeutic target for structure-based drug design. Among the 2 selected components, quercetin exhibited a higher Libdock score with A42R. In quercetin, conventional hydrogen bonds were formed with ASN78, THR126, ARG127, and ARG129, and mixed Pi/Alkyl hydrophobic interactions were formed with ARG129 and ARG119. In celabenzine, conventional hydrogen bonds were formed with ARG129, ARG119, and ASN78. These interaction sites were important factors to consider in studying molecular structure-activity relationships and designing new drugs at the molecular level.The profilin-like protein A42R was a short protein encoded by the MPXV virus gp153 locus, displaying sequence similarity to eukaryotic profilins, which were actin-binding proteins involved in the regulation of cytoskeletal structure and function.,42 Molecular dynamic simulation could be used to study the stability of complexes. In this study, we selected quercetin and 4QWO, with better binding properties, for further molecular dynamic simulation. We calculated RMSD and RMSF in the simulation process to evaluate the stability of the complex. RMSD could reveal the position changes between the protein conformation and the initial conformation in the simulation process. RMSD fluctuated from frame to frame with an average value (0–0.8 Å), indicating that the system had reached equilibrium, and although there were structural differences between conformations, the differences were small and the trajectory was stable. RMSF showed the fluctuation of amino acid residues relative to the average structure of the trajectory, which could characterize the flexibility and motion intensity of amino acids in the whole simulation process. The smaller RMSF indicated the more convergent and stable the system. The RMSD and RMSF changes during the binding of quercetin with 4QWO were shown in Figures Molecular dynamic simulation was a computer experimental method to calculate the macroscopic properties of molecules by simulating the motion of microscopic particles on the basis of a molecular model.alue 0–0. Å, indic For thousands of years, under the guidance of the “holistic concept and syndrome differentiation” in TCM, Chinese medicines and their prescriptions have demonstrated multi-effect synergistic effects on infectious diseases such as smallpox. In modern society, their indispensable roles have been confirmed in SARS, H1N1 influenza epidemics, and COVID-19.–48 In the current MPX epidemic, Sheng-Ma-Ge-Gen decoction, Sheng-Jiang-San, and Zi-Xue-San were recommended for patients with fever, while Qing-Ying decoction, Sheng-Ma-Bie-Jia decoction, and Xuan-Bai-Cheng-Qi decoction were recommended for patients with high fever, acne, sore throat, and multiple swollen and painful lymph nodes by the National Health Commission and the National Administration of TCM of the People’s Republic of China. These are all ancient, classic, and famous TCM prescriptions. Thus, it is crucial to have a more in-depth analysis of these prescriptions and their mechanisms of action.The clinical manifestations of monkeypox are similar to smallpox. The most prevalent clinical manifestations include rash, fever, pruritus, pruritus, and lymphadenopathy. Other manifestations include fatigue, sore throat, headache, cough, myalgias, photophobia, arthralgia, difficult breathing, conjunctivitis, nausea/vomiting, and diarrhea. indicated for fatigue, cough and phlegm, myalgia, and difficult breathing caused by MPXV. Renshen was the most revered of the herbs in ancient times in China, Korea, Japan, and America, with the following actions: reinforcing vital energy, tonifying visceral qi, calming mind and benefiting intelligence, and helping produce saliva and slaking thirst, indicated for qi deficiency syndrome caused by MPXV. Danggui was usually used to invigorate blood circulation in the treatment of menstrual disorders, with the following actions: tonifying blood, promoting blood circulation, regulating menstruation, relieving pain, and moistening intestines, indicated for blood stasis and blood deficiency syndrome caused by MPXV. The combination of the Gancao, Renshen, and Danggui can play a synergistic effect of tonifying qi and blood and expressing toxin outward, which is suitable for deficiency syndrome caused by MPXV without the typical clinical manifestations and difficult to cure. What’s more, modern pharmacological studies had shown that they had the functions of antiviral, anti-inflammatory, anti-fatigue, and promoting immune-modulating,–54 which had certain effects in treating deficiency syndrome of MPX. Shengma was the main herb of Sheng-Ma-Ge-Gen Docotion, with the following actions: evacuating wind-heat, lifting yang qi, clearing heat and detoxifying, and promoting eruption, indicated for wind-heat syndrome or heat-toxin syndrome caused by MPXV. Zicao was a key herb for treating smallpox, with the following actions: clearing heat and cooling blood, promoting blood circulation, detoxifying, and promoting eruption, indicated for blood-heat syndrome or heat-toxin syndrome caused by MPXV. The Shengma and Zicao pair is suitable for excess heat syndrome caused by MPXV with typical clinical manifestations. And modern pharmacological studies had also shown that they had the functions of antiviral, anti-inflammatory, immune regulation, and easing pain,,56 which had certain effects in treating the excess heat syndrome of MPX. However, CMs had strict discipline on dosage and duration, otherwise, they will produce side effects. Studies had found that Gancao had salt corticosteroid components. Excessive Gancao consumption could lead to hyper mineralocorticoidism, a condition marked by salt retention, hypokalemia, hypertension, metabolic alkalosis, hypoaldosteronism, and low renin activity, leading to significant life-threatening consequences, particularly in individuals with cardiovascular disease.,58 Due to its action of decreasing blood glucose, Renshen should be used with caution for patients with low coagulation function. The most common side effects of Renshen resulting from its overdose were nervousness and excitability-- the inability to sleep and high blood pressure. Although Danggui was usually well tolerated in general use, it might increase the risk of bleeding in patients taking warfarin and antiplatelet drugs and cause photosensitivity reactions. Shengma and Zicao are relatively safe CMs, but long-term use will lead to liver and kidney damage. Due to their bitter taste and cold property, they easily affected digestive function, so they are generally used in the acute phase and are rarely used after symptom relief.To analyze the rules of Chinese medicines in treating smallpox and gain insights into the treatment of MPX, we selected 119 prescriptions from a medical book written by a renowned Ming dynasty doctor. We identified 25 high-frequency Chinese medicines and 23 high-frequency Chinese medicine pairs among these prescriptions. Combined with the results of the association rule analysis, Gancao, Renshen, Danggui, Shengma, and Zicao were selected as the core Chinese medicine pair for further study. Gancao is one of the most widely used CMs in the world, with the following actions: supplementing spleen qi, clearing heat and relieving toxicity, dispelling phlegm and relieving cough, relaxing tension and relieving pain, and harmonizing the nature of other herbal medicines,Humulus lupulus L. This extract was found to inhibit replication of various viral strains, at a different time from infection. And, quercetin was reported to inhibit viral neuraminidase activities in vitro and influenza infection in animal models. Celabenzine was a natural product found in Gymnosporia mossambicensis and Tripterygium wilfordii. Tripterygium wilfordii had significant anti-inflammatory and immune-modulating and is widely used in various autoimmune-mediated inflammatory diseases, including rheumatoid arthritis, nephrotic syndrome, systemic lupus erythematosus, and Behcet’s disease. The GO and KEGG pathway enrichment analysis revealed that the core Chinese medicine pairs exerted effects on the immune system, endocrine system, inflammatory response, and viral infections. MPX induces a robust immune response, internal environment disorder, and an inflammatory storm, during which numerous cytokines are activated. These results showed that the core Chinese medicine pair may regulate immune-related and inflammation-related pathways to alleviate and eliminate symptoms. Furthermore, the molecular docking and molecular dynamics simulation results demonstrated the binding mechanisms between the selected components and the known MPXV targets, indicating potential anti-MPXV virus activity.In the systematic network pharmacology analysis, we obtained 131 active components and 348 candidate targets of the core Chinese medicine pairs. We then constructed the herb-component-target and PPI networks. By analyzing the network topology, 2 core components, quercetin and celabenzine, were selected for molecular docking analysis. Quercetin was isolated from the hydroalcoholic extract of In conclusion, by combining the knowledge of ancient prescriptions with modern pharmacological research methods, we analyzed the compatibility of Chinese medicines for treating smallpox and explored the potential molecular mechanisms of the key herbs for addressing MPX. These findings underscored the characteristic multi-component and multi-target nature of Chinese medicines. However, it is important to note that virtual screening results have inherent limitations. Therefore, further in vitro and in vivo experiments are needed to validate the findings of this study in later stages. This research aims to provide a solid experimental foundation for the development of natural antiviral drugs.Conceptualization: Haozhong Wang.Data curation: Chengchen Xu.Formal analysis: Linyang LI.Funding acquisition: Yinling Guo.Investigation: Linyang LI.Methodology: Linyang LI, Haozhong Wang.Project administration: Yinling Guo, Haozhong Wang.Resources: Linyang LI.Software: Linyang LI, Chengchen Xu.Supervision: Linyang LI, Haozhong Wang.Validation: Linyang LI, Chengchen Xu.Visualization: Linyang LI.Writing – original draft: Linyang LI.Writing – review & editing: Linyang LI, Chengchen Xu, Haozhong Wang.


Exosomes, microvesicles, carry and release several vital molecules across cells, tissues, and organs. Epicardial adipose tissue exosomes are critical in the development and progression of coronary artery disease (CAD). It is hypothesized that exosomes may transport causative molecules from inflamed tissue and deliver to the target tissue and progress CAD. Thus, identifying and inhibiting the CAD-associated proteins that are being transported to other cells via exosomes will help slow the progression of CAD.Panax ginseng-derived compounds was conducted and then docked against the protein target to identify potential inhibitors and then subjected to molecular dynamics simulation (MDS) to understand the behavior of the protein-ligand complex till 100 nanoseconds. Finally, density functional theory (DFT) calculation was performed on the ligand with the highest affinity with the target.This study uses a systems biological approach that integrates differential gene expression in the CAD, exosomal cargo assessment, protein network construction, and functional enrichment to identify the crucial exosomal cargo protein target. Meanwhile, absorption, distribution, metabolism, and excretion (ADME) screening of Panax ginseng compounds, 27 showed drug-likeness properties. Inermin, a phytochemical showed good docking with −5.02 kcal/mol and achieved stability confirmation with SMAD2 based on MDS when compared to the known CAD drugs. Additionally, DFT analysis of inermin showed high chemical activity that significantly contributes to effective target binding. Overall, our computational study suggests that inermin could act against SMAD2 and may aid in the management of CAD.Through the systems biological approach, Mothers against decapentaplegic homolog 2 protein (SMAD2) was determined as a potential target that linked with PI3K-Akt signaling, Ubiquitin mediated proteolysis, and the focal adhesion pathway. Further, screening of 190  Coronary artery disease (CAD) is one of the most prevalent diseases that contributes to high mortality worldwide  with a svia exosomes will help to slow the progression of CAD.Exosomes are microvesicles sized between ∼20–140 nm in diameter . ExosomePanax ginseng C.A. Meyer). The beneficial effects of Panax ginseng include inhibiting oxidation, improving blood circulation, and protecting the cerebrovascular system  properties. Further, the affinity of ADME-screened compounds to the CAD target protein was assessed based on molecular docking. Furthermore, the molecular dynamics simulation (MDS) was used to investigate the structural conformation of the target protein with the docked compound. Additionally, the quantum property of the selected compound was studied through density functional theory (DFT) analysis. Overall, our study establishes the potential of a natural compound that could be useful in the CAD treatment.Herein, using a systems biological approach, we identified the crucial CAD protein that can be transported Panax ginseng were identified and its structures were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://tcmsp-e.com/tcmsp.php). Then, the retrieved compound structures were optimized with a OPLS3force-field using the LigPrep tool in the Schrödinger suite (Maestro 11.2). Likewise, the known CAD drugs  were downloaded as structure data files (SDF) format from PubChem (https://pubchem.ncbi.nlm.nih.gov/) for comparative analysis. Then the optimized natural compounds were screened with QikProp  (The bioactive compounds of r suite)  to predir suite) .https://www.ncbi.nlm.nih.gov/geo/) was selected with the following criteria that includes, (1) dataset containing expression profile of more than three samples in a group, (2) dataset containing CAD participants with the appropriate control executed in the CAD sample from the cardiac associated tissue, (3) the dataset providing human gene expression profile that uses microarray or RNAseq technique was considered, and (4) experiment carried out in animal or tissue culture were eliminated/excluded. To the selected dataset, limma R-program was implemented to identify the differentially expressed genes (DEGs) in CAD with log2fold-change (FC) and p-value < 0.05. Among DEGs, only the over expressed genes were selected for further assessment.A gene expression array dataset from the Gene Expression Omnibus (GEO) (http://www.exocarta.org/) which contains the exosomal cargo of DNA, mRNA, miRNA, lipids, and proteins from multiple species. All human exosomal cargo proteins were downloaded and duplicates were removed to obtain an unique protein list. These exosomal unique lists were mapped to the over expressed DEGs of CAD to discover a common protein list (SET-A). The proteins in SET-A were demonstrated to have qualities (1) over expressed in CAD and (2) carried via exosome. Then the SET-A was used to build an inter-protein network with the STRING database (https://string-db.org/). Thereby, the constructed network contains both SET-A and its interacting proteins, resembling that SET-A on exosomal delivery into a target cell can readily interact and activate the cellular proteins to perform functions.To gather the information on exosomal cargo proteins, the EXOCARTA database was used (http://bioinformatics.sdstate.edu/go/). The GO analysis was carried out to identify the biological process, molecular function, and cellular component of the network proteins with a false discovery rate (FDR) less than 0.05. Similarly, the molecular pathways analysis were explored using KEGG pathway module (FDR < 0.05).The function of these network proteins was assessed by gene ontology (GO) and KEGG pathway using ShinyG0 0.77 (https://prankweb.cz/) for grid generation around the pockets  database. Using a protein preparation module (Schrödinger suite), the target protein was optimized, which removes water molecules, heteroatoms, ions, and unwanted protein chains. Similarly, the LigPrep module (Schrödinger suite) was used for compound preparation such as adding hydrogen bonds. The binding pockets of protein were identified by P2Rank tool . Each simulation was carried out in three phases involving system construction, minimization, and the simulation run. Following the determination of the docked ligand–protein complex, the system was modeled using the SPC solvent system within the orthorhombic boundary and ions neutralized then subjected to energy minimization until it reached a gradient threshold of 25 kcal/mol at a pressure of 1 bar and a temperature of 300 K using the NPT ensemble class. The OPLS2005 force field optimization was used to minimize the system by carrying 2,000 iterations . A Nose–Hoover Chain thermostat was used to maintain the temperature, while a Martyna-Tobias-Klein barostat was utilized to sustain pressure with the equilibrium was set 200 ps. Finally, root-mean-square-deviation (RMSD), RMSF root-mean-square-fluctuation (RMSF) trajectories of the protein and ligand, protein–ligand interactions, and contacts with a variety of amino acids were used to determine the stability of the complex during the MD simulation.After gaining the understanding of the ligand–protein complex’s through docking, the molecular dynamics (MD) simulation was performed for the target protein with the compound from ++ basis set  were collected and optimized with LigPrep module. Then QikProp was used to screen 190 compounds that predict the ADME properties. On ADME screening, 27 compounds were noticed to fulfill most drug-likeness properties  from ten controls and nine CAD that experimented in microarray platform GPL6244 ((HuGene-1_0-st) Affymetrix Human Gene 1.0 ST Array (transcript (gene) version)). By employing limma programming, a total of 3,499 DEGs  with log2FC and p < 0.05 were identified. However, only the over-expressed 1,408 genes in CAD were selected for the subsequent assessment.Meanwhile, our search in NCBI, GEO datasets (Accessed on: 30 April 2023) allowed us to identify the suitable dataset n = 174) that represented as putative target for molecular docking.Using the data retrieved from EXOCARTA, 6,394 unique exosomal proteins were identified and mapped with the 1,408 over-expressed CAD genes. A total of 489 proteins identified to be common were termed as SET-A, as mentioned in the methodology section. Then, the protein network that was constructed with the 489 (SET-A) proteins was extended to have 1,487 (489+998) proteins to form a complex network , suggest5XOD) and optimized to perform a docking with 27 natural compounds along with five references CAD drugs . The active site for the SMAD2 was determined as GLU270, PRO271, PHE273, TRP274, TYR340, GLY342, GLY343, VAL345, PHE385, ASN387, LEU442, GLY444, and PRO445 using P2Rank tool. The compound inermin (MOL003648) has the highest binding score . Likewise, furosemide showed highest affinity of −4.472 kcal/mol with SMAD2 compared other analyzed CAD drugs. These results indicate that the compound from Panax ginsenghas a higher affinity towards SMAD2 than the CAD drugs. Top 10 molecular docking results were represented in The protein structure of SMAD2 was retrieved from PDB , electronegativity (χ), softness (S), hardness (η), and chemical potential (μ)). The electrophilicity scale shows that the atom has the ability to take up electrons. Likewise, the electronegativity scale describes the ability of the atom to attract electrons toward it. The hardness and softness values represent the electron charge transfer resistance and acceptance of an atom. The HOMO value was found to be −5.40 eV, while the LUMO was −0.45 eV, and has an energy gap (ΔE) of 4.95 eV (χ) was 2.93 eV, chemical hardness (η) was 4.95 eV, chemical potential (μ) was −2.93 eV, electrophilicity index (ω) was 1.46 eV, and chemical softness (σ) was 0.20 eV. Overall, the results suggest that inermin has high reactivity by quantum chemical analysis.The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are calculated with DFT-B3LYP6-311++ (dp). The frontier molecular orbital of inermin was illustrated in  4.95 eV . FurtherThe B3LYP/6-31G**++ in the Jaguar suite was utilized for ESP analysis. Inermin was analyzed to identify the reactivity, hydrogen bonding, and structural activity. The color variation denotes the electrostatic potential . ElectroThe molecules capacity of the binding was determined by the electronic charges. The Mulliken charges of the representative atoms of inermin were shown in Panax ginsengderived compounds have proven to have a multi-target potential, traditionally used to treat various diseases such as CAD, infertility, diabetes, and cancer , the molecular weight of the molecule should range between 130.0 and 725.0, donor HB should range from 0.0 to 6.0, HB acceptor should range from 2.0 to 20.0, the partition coefficient of predicted octanol/water has to be ranged between −2.0 and 6.5, solubility range should be −6.5 to 0.5, cell permeability (Caco-2) (range: <25 is poor and >500 is great), predicted central nervous system activity (−2 denotes inactive and +2 denotes active), brain/blood partition coefficient between −3.0 to 1.2, MDCK cell permeability (range: <25 is poor and >500 is great), oral absorption (range: <25 is poor and >80% is high), human serum albumin binding has to range between −1.5 to 1.5, Lipinski’s rule of five violations (0–4), and Jorgensen’s rule of three violations (0–3)  to find the active compounds from ns (0–3) . Particuns (0–3) . The comfunction , apoptosfunction , inflammfunction . Furtherfunction . Likewisfunction . Further pathway . Through pathway . It is i pathway . FurtherPanax ginseng along with five standard drugs currently used for the treatment of CAD. SMAD2 protein used for docking identifies that inermin  could be effective than furosemide  based on binding score. Henceforth, inermin will be effective in inhibiting the protein and might reduce the progression of CAD via exosome transport (We performed molecular docking between SMAD2 with active compounds of ransport . In thisMD simulation is used for analyzing the stability and conformation changes in the protein-ligand complex . The molA total of 35 ligand contacts  and 7 we2, Rg extended from 3.19 to 3.66 Å, SASA ranged from 153.77–474.58 Å2, molecular surface area ranged from 243.53–251.28 Å2. Similarly, the furosemide properties (2, 3.31−3.98 Å, 109.55–353.55 Å2 and 258.06–276.24 Å2, respectively. According to As represented in operties  such as Panax ginseng by ADME analysis. Molecular docking against SMAD2 determined that inermin has a higher binding score than the control drug. In addition, the MD simulation confirmed that the SMAD2 with inermin attained stability during the 100 ns MD run. Also, the quantum chemical properties of inermin showed high chemical reactivity. We conclude that the inermin will be a suitable alternative for the treatment of CAD. However, further validation was needed before proceeding with the clinical trial.The potential target SMAD2 was identified through systems biological analysis by integrating CAD-associated exosomal genes. Simultaneously, we identified 27 major lead compounds of 10.7717/peerj.16481/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.16481/supp-2Supplemental Information 2The drug-likeness properties of all 27 compoundsClick here for additional data file.10.7717/peerj.16481/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.16481/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.16481/supp-5Supplemental Information 5Panax ginsengDFT analysis of inermin compound derived from Click here for additional data file.


Several genome annotation tools standardize annotation outputs for comparability. During standardization, these tools do not allow user-friendly customization of annotation databases; limiting their flexibility and applicability in downstream analysis.StandEnA is a user-friendly command-line tool for Linux that facilitates the generation of custom databases by retrieving protein sequences from multiple databases. Directed by a user-defined list of standard names, StandEnA retrieves synonyms to search for corresponding sequences in a set of public databases. Custom databases are used in prokaryotic genome annotation to generate standardized presence–absence matrices and reference files containing standard database identifiers. To showcase StandEnA, we applied it to six metagenome-assembled genomes to analyze three different pathways.https://github.com/mdsufz/StandEnA.StandEnA is an open-source software available at Bioinformatics Advances online. NCBI ResourceProtein annotation can be performed using different query databases; for example, the Universal Protein Resource Knowledgebase (UniProtKB)  and NatiAnnotation tools, such as Bakta  and DFASWe developed the Standardized Enzyme Annotation pipeline (StandEnA) to overcome nomenclature and customizability problems in pathway annotation. StandEnA is a user-friendly command-line tool for Linux. It annotates pathways selected by users on prokaryotic genomes by generating a user-defined custom database of protein sequences from available protein synonyms and standard names . To alloEscherichia coli K12 genome (NC_000913.3) was annotated using StandEnA for the same three pathways and compared with annotations made using Prokka’s default database, the former revealing 3 times more standard enzyme annotations . Optionally, proteins can be downloaded either directly from KEGG via an OrtSuite command (In Step 1, StandEnA creates a custom database using a user-provided input file containing standard enzyme names, their EC numbers, and KEGG pathway names (Step 3 is divided into two parts. First, a reference file for each pathway is prepared. This file contains the KO identifier, EC number and standard name for each enzyme synonym used. After, the Prokka annotation output is searched for compiled enzyme synonym names within each pathway. Users can curate the synonyms from the Prokka annotation for further flexibility to remove any undesired protein name. Finally, Step 4 generates a standardized presence-absence matrix output for each pathway, including all of the genomes. Detailed information on input and output files is provided in We analyzed our pipeline using six MAGs (accession numbers of these MAGs can be found in We demonstrated that manual search using the UniProt database could expand StandEnA’s initial custom database and fine-tune its synonyms list providing access to desired protein sequences that are unavailable in StandEnA’s default search refer to . CompariFor six MAGs and three NCBI genomes, the entire pipeline was executed in under 8 h using a dual-core Intel Core i5 7th generation computer running Ubuntu 18.04.6 LTS on only 15 GB of free disk space. Compared to manual protein database construction, often reported to encompass a range of days to weeks , StandEnIn this application note, we presented StandEnA, a customizable and standardized Linux command-line tool for annotating desired pathways in prokaryotic genomes via user-defined custom databases. StandEnA creates outputs containing a standardized presence–absence matrix of pathway enzymes and a reference file of standard database identifiers for each enzyme synonym used during annotation. The workflow conveniently creates a custom database containing desired protein sequence files from multiple databases. StandEnA provides customizability to genome annotations, as users can monitor all intermediate files and manually curate them when necessary. Manual curation improved the annotation of the analyzed pathways. Moreover, our tool performs database cross-references standardizing the outputs for simple presence–absence matrix comparisons across genomes, facilitating downstream utilization such as genomic/functional potential comparisons through metabolic pathway predictions and feature extraction for machine learning applications across different studies.vbad069_Supplementary_DataClick here for additional data file.


New, artificial pathways promise higher yields and activity than natural ones like the Calvin-Benson-Bassham (CBB) cycle. The question remains how to best predict their in vivo performance and what actually makes one pathway “better” than another. In this context, we explore aerobic carbon fixation pathways by a computational approach and compare them based on their specific activity and yield on methanol, formate, and CO2/H2 considering the kinetics and thermodynamics of the reactions. Besides pathways found in nature or implemented in the laboratory, this included two completely new cycles with favorable features: the reductive citramalyl-CoA cycle and the 2-hydroxyglutarate-reverse tricarboxylic acid cycle. A comprehensive kinetic data set was collected for all enzymes of all pathways, and missing kinetic data were sampled with the Parameter Balancing algorithm. Kinetic and thermodynamic data were fed to the Enzyme Cost Minimization algorithm to check for respective inconsistencies and calculate pathway-specific activities. The specific activities of the reductive glycine pathway, the CETCH cycle, and the new reductive citramalyl-CoA cycle were predicted to match the best natural cycles with superior product-substrate yield. However, the CBB cycle performed better in terms of activity compared to the alternative pathways than previously thought. We make an argument that stoichiometric yield is likely not the most important design criterion of the CBB cycle. Still, alternative carbon fixation pathways were paretooptimal for specific activity and product-substrate yield in simulations with C1 substrates and CO2/H2 and therefore hold great potential for future applications in Industrial Biotechnology and Synthetic Biology.In the recent years, engineering new-to-nature CO Again, this is a conservative estimation as the enzyme has not yet been optimized for the substrates. From 2-ketoglutarate on, the pathway follows the traditional reverse TCA cycle.This reaction is catalyzed by a 2-hydroxyacyl-CoA lyase, an enzyme that has recently gained attention as an enzyme to assimilate C1 bodies , 56. In s enzyme , so fromThe detailed pathway maps, as well as the corresponding enzymes, the respective kinetic data, and their sources can be found in the Supplementary Information in Figure As a thought experiment to analyze and to understand the natural design of the CBB cycle, a hypothetically augmented version of the pathway was designed that uses 2 mol ATP less for the production of 1 mol C3 sugar. This is achieved by replacing the irreversible phosphatase reactions with phosphotransferase reactions in the following fashion:This net phosphate transfers are exergonic under standard conditions with rganisms , the phoEnzymes were found that are able to catalyze the described phosphate transfer reactions via pyrophosphate  in a pro(i)The 3-HP/4-HB cycle, the rCCC, the 2-HG-rTCA cycle, and the Serine cycle produce acetyl-CoA(ii)The CBB cycle and RuMP cycle produce glyceraldehyde-3-phosphate(iii)The 3-HP bicycle and rGlyP produce pyruvate(iv)The CETCH cycle produces glyoxylate as the primary productThe primary products of the pathways presented in the last sections are different for each pathway when looking at the illustrated stoichiometries Figures –4:(i)The2 is ubiquinol.This is a challenge for the comparison of the pathways as it is unclear how the “value” of a produced metabolite relates to another. To solve this, pathways were added to the SBtab model file that connect the primary products with each other. These additional connecting pathways will be called “connecting modules” as they can modularly be combined with the original primary pathways. The connecting modules that were implemented are described in the following sections and summed up with their net stoichiometries in Table 2 are produced per molecule of formed C6 sugar using the glyoxylate cycle. This is why some bacteria use different pathways for the assimilation of acetyl-CoA that are reductive and even fix CO2, like the ethylmalonyl-CoA pathway [This pathway is oxidative  which is contrary to the reductive nature of carbon fixation. Besides, for gluconeogenesis, two equivalents of CO pathway . Archaea pathway . In case pathway  in their2 with tartronate-semialdehyde as an intermediate [β-hydroxyaspartate cycle . Both gt et al.  was alsoβ-hydroxyaspartate cycle for glyoxylate assimilation instead which results in a cycle that needs 1 mol of ATP less than the original pathway per 1 mol of acetyl-CoA. A detailed map of this MCG-like pathway can be found in the Supplementary Information in Figure Another interesting case is the conversion of pyruvate or C4 dicarboxylic acids to acetyl-CoA. Plants use the pyruvate dehydrogenase complex, an oxidative decarboxylation reaction, which is in contrast to the reductive carbon fixing CBB cycle. To capitalize on this potential, it was proposed to rather split a C4 body into two C2 bodies, e.g., in the synthetic malyl-CoA-glycerate carbon fixation pathway (MCG pathway) (H. ). In thiMany pathway designs implemented in this work bear the potential to be superior to the pathways that are widespread in nature—at least in certain scenarios, like biotechnological applications. But how can we actually predict whether or not these pathways are feasible or even better apart from their stoichiometry? To rule out that pathways are thermodynamically and kinetically limited or even infeasible, the ECM algorithm was used which is able to factor in the thermodynamics and kinetics of the reactions based on all parameters that were collected. After assembly of all pathways and connecting modules, the SBtab model file comprised 113 reactions with 104 metabolites and 615 enzymatic parameters . The data file was then processed by Parameter Balancing to yield a complete and consistent parameter set for all reactions. Next, we evaluated the pathway-specific activities of all pathways with ECM towards the production of a certain metabolite.2-fixing pathways were compared for the production of glyceraldehyde-3-phosphate at atmospheric CO2 partial pressure as shown in Figure 2 source like hydrothermal vents. The flux distributions that were assumed for each pathway are defined by the pathway stoichiometries combined with the connecting modules, as listed on the left side of Figure μmol min-1 mg-1 which is in line with former estimates [μmol min-1 mg-1 which makes sense given the low CO2 partial pressure. Most pathways were predicted to have pathway activities in the same order of magnitude; some, however, were considerably slower: the MOG cycle was predicted to have only marginal activity which is in sharp contrast to the former predictions [2-fixing pathways, the rCCC, the CETCH cycle, and the crenarchaeal 3-HB/4-HB cycle showed pathway-specific activities equal to the CBB cycle with 10% of photorespiration.First, only the COstimates . The phodictions . Even th2 concentration has a dramatic impact on its pathway-specific activity which is less of a concern in alternative CO2-fixing pathways.Glyceraldehyde-3-phosphate is the main product of the CBB cycle which is why this pathway was likely favored when choosing it for comparison. Therefore, the same analysis was repeated using acetyl-CoA as the product of choice which is shown in Figure 2, and thus, respective limitations and also photorespiration can be avoided to a certain degree which will likely have a big influence on the resulting activities. Therefore, three different electron and carbon sources that are regarded as possible platform substrates for the future bioeconomy were evaluated as feedstock for the pathways: methanol, formate, and a mixture of CO2 and H2. The concentrations of CO2 and carbonate were increased by a factor of 100 to simulate active gassing with CO2.In most biotechnological scenarios, bioreactors can be gasified with COBacillus methanolicus use this enzyme at a high specific rate, but the elevated temperature requirement might play a role here. Alternatively, the enzyme kinetics and maybe even the thermodynamics could be fundamentally different in vivo since, for engineered E. coli with a functional RuMP cycle, it was speculated that formaldehyde was accumulating, and thus, the MDH was not the limiting factor . The PQF. Y. H. , the kinAs this is one of the key reactions and we also did not find a value for the forward kinetic constant, this standard deviation has a crucial impact on the total estimation of pathway-specific activity. All in all, the estimation for the rGlyP are consequently not completely trustworthy which stresses how urgently kinetic data for this system are needed to make accurate predictions. Disregarding the rGlyP, the rCCC, the 2-HG-rTCA cycle, and the CETCH cycle were paretooptimal for both criteria. The 2-HG-rTCA cycle in conjunction with the acetyl-CoA conversion module from the rCCC has a very low ATP-requirement per product. Although it has a lower pathway activity, it might still be a promising route, considering that conservative assumptions were made about the kinetic parameters of the enzymes.+ is almost irreversible. Likewise, reduction of formyl-CoA to formaldehyde that has been proposed by others [2/HCO3- which can be seen in Figure When formate was used as the substrate of choice, NADH was regenerated via formate dehydrogenase, a highly active enzyme in this reaction direction. Therefore, the pathway activity is in general less limited by the supply of reducing equivalents compared to methanol. The RuMP cycle needs formaldehyde which is difficult to produce from formate because formaldehyde oxidation with NADy others , 21 was y others . This va2 . In comparison to the CBB cycle, the new rCCC and the 2-HG-rTCA cycle  was assumed to regenerate NADH. In order to also include formate-dependent pathways, the formate dehydrogenase was run in the reductive direction as described or proposed by others . This iAll three scenarios presented in this section have also been performed with acetyl-CoA as the product of choice  compartments are necessary. Additionally, carbonate concentrations at a pH greater than 7—as found in most aquatic habitats—are generally higher than the CO2 concentrations at saturation, and therefore, the Michaelis constants of carbonate-fixing enzymes are not as restricted to low values.In the simulations described in the last sections, it could be seen that the orders of magnitude of both specific activity and product-substrate yield are similar for most pathways. Given the uncertainties of the enzymatic parameters that were used and the fact that enzyme kinetics were measured in vitro, there is no clear “optimal” pathway. It is rather likely that most pathways could have a niche in nature in which they are competitive which is in agreement with the sheer existence of many of them. The Serine cycle was very efficient and highly active for production of acetyl-CoA from formate, and the RuMP pathway was very efficient with methanol as a substrate which fits their natural roles. In the biotechnological scenarios described in the last section, the 3-HP/4-HP cycle and the 3-HP bicycle were predicted to be as active as the CBB cycle variants. Since these pathways occur in aquatic microorganisms and all carboxylation reactions are dependent on HCO2 and C1 compounds in Industrial Biotechnology. The GED pathway and the phosphatase-less CBB cycle were shown to not fall short off the CBB cycle in specific activity and might be interesting alternatives if better enzymes can be found for the critical reactions. However, the aminomutase variant of the MOG cycle seemed to be infeasible judging from our calculations. All model substrates—formate, methanol, and H2/CO2—showed good specific activities and hence are promising options for sustainable bioprocesses.The alternative and engineered carbon-fixing pathways match the activity of the CBB cycle but yield more product per substrate: the rCCC and the CETCH cycle outperform the CBB cycle in this regard. With formate as a substrate, the rGlyP was superior to the Serine cycle. Taken together, the rCCC, CETCH cycle, and rGlyP pathways have a high potential for the implementation in living organisms to create chassis organisms for the fixation of CO2-fixing pathways might also be implemented in crop plants which can be regarded as the Holy Grail of plant biotechnology and might boost crop productivity. To achieve this, the plant and chloroplast genome would have to be radically changed on a metabolic and regulatory level which, as of today, seems out of range of the methods of Synthetic Biology. First applications will consequently rather be found be in microorganisms that can be manipulated more easily and cultivated under controlled conditions.For future applications, the CO2 concentration and low RuBisCO activity :A second example that ATP efficiency is not the main driver in the evolution of photosynthesis can be found in C4-plant metabolism. C4 plants use a COclimates . CO2 con2 is released by the malic enzyme and refixed by the CBB cycle. Although a total minimum of 15 ATP instead of 9 ATP is necessary for the formation of one C3 sugar, C4 plants easily grow faster than C3 plants in most situations. Additionally, thinking of optimal design, the first steps in a CO2 concentration mechanism could also be catalyzed by pyruvate carboxylase:This set of reactions is quite exothermic in total with These reactions are also practically irreversible 2-fixing pathways except for the CBB cycle and the rGlyP. Being the most structurally complex of all vitamins, B12 biosynthesis is a complex pathway that does not occur in plants and animals. In addition, it also contains cobalt. The costs associated to biosynthesis of B12 and other important cofactors were not taken into account in this work. The other vitamins are structurally more compact, less demanding in their biosynthesis, and independent of metal ions and are needed for many other vital reactions in the metabolism as well. In the future, this requirement could be addressed by adding B12 as a metabolite and its biosynthesis to the SBtab model file if adequate data are available. This will help to evaluate whether or not B12 requirement has a critical impact.Specific activity and yields of different metabolic pathways were the main criteria for comparison in this work. While those are important factors, they are not necessarily decisive. One unconsidered factor in this context are the vitamins: interestingly, cobalamin (vitamin B12) is necessary for all COOther unconsidered factors in the analysis presented in this work are the controllability and robustness of the pathways. Previous works have shown that these factors are an essential part of the design principles in nature –77. AlloSo far, metabolite toxicity has also not been addressed in this work. Toxic metabolites in the pathways include, for example, aldehydes like formaldehyde, glyoxylate, succinate semialdehyde, malonate and semialdehyde, but also propionyl-CoA. To include this in the future, tighter constraints on these toxic compounds may be implemented, possibly based on concentrations that are actually measured within cells with a respective metabolism.2-fixing pathways was thoroughly investigated in a former study [Although the design space for artificial COer study , we couler study , 80 willin vivo data which might be very different from in vitro enzyme kinetics as has been pointed out for the ethylmalonyl-CoA pathway in this study. In this context, modelling approaches that are able to quantify uncertainties and identify missing data are ideal. This way, models and simulations can be iteratively improved to get closer and closer to the biological reality and a deeper understanding of metabolism and design principles of natural pathways.Using ECM and other advanced tools for pathway analysis can predict how to best implement a metabolic pathway based on measured data and which ones are most promising. This relies heavily on the quality of the kinetic and thermodynamic data that are used. For some sensitive parameters of reactions, there were only single studies available in which the enzymes were characterized. The ECM algorithm can predict, however, which reactions will be likely limiting the pathways and thereby gives good suggestions for enzymes that need to be better characterized and/or engineered. This stresses how important enzymology is for the understanding and the design of biochemical pathways as has also been noted by Erb recently . In fact


Bushenhuoxue (BSHX) formula, a ten-compound herbal decoction, is widely used to treat postmenopausal osteoporosis (PMOP) in China. However, the mechanism is not clear yet.The underlying biological processes and signaling pathways were predicted by network pharmacology. In vivo experimental study, 24 female C57BL/6 J mice were randomly divided into sham, ovariectomized (OVX) and BSHX formula groups. Mice in the latter two groups were subjected to bilateral ovariectomy, and mice in the BSHX formula group were extra treated by BSHX formula at an oral dosage of 0.2 mL/10 g for 8 weeks. The femur samples were harvested for tissue analyses including μCT assay, histology and immunohistochemical (IHC) staining of VEGF signaling.A total of 218 active ingredients and 274 related targets were identified in BSHX formula. After matching with 292 targets of PMOP, 64 overlapping genes were obtained. GO and KEGG enrichment analyses on these 64 genes revealed that angiogenesis and VEGF signaling were considered as the potential therapeutic mechanism of BSHX formula against PMOP. Animal experiments showed that mice in the BSHX formula-treated group presented increased bone mass, microstructural parameters, blood vessel numbers and an activation of VEGF signaling  compared to the OVX mice.This study revealed that BSHX formula exerts anti-PMOP effects possibly through activating VEGF signaling-mediated angiogenesis. Postmenopausal osteoporosis (PMOP) is a common bone disorder characterized by low bone mineral density and microstructure deterioration . It is eBushenhuoxue (BSHX) formula is a traditional herbal decoction composed of ten herbs  network. Furthermore, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on these overlapping genes to analyze the potential biological processes and singling pathways. According to the results of network pharmacology, an OVX mouse model was built to validate the above pharmacological mechanism.Eucommia ulmoides Oliv Eucommiaceae., Cornus officinalis Sieb Cornaceae., Glycyrrhiza uralensis Fisch Leguminosae., Aconitum carmichaeli Debx Ranunculaceae., Lycium barbarum L Solanaceae., Carthamus tinctorius L Asteraceae., Rehmannia glutinous Liboscb Scrophulariaceae. and Dioscoreae opposite Thunb Dioscoreaceae. were mixed and extracted with water at a ratio of 2:1:1:2:1:1:3:2. Other two herbs, Cinnamomum cassia Presl Lauraceae. and Prunus persica Batsch Rosaceae. were mixed at a ratio of 2:1 for ethanol extraction. These two parts were mixed and concentrated to 2 g crude drug/mL for intragastric administration.According to TCM theories, BSHX formula has functions of replenishing kidney, enriching essence and invigorating the circulation of blood. In this prescription, Shu Di Huang (SDH), Gou Qi Zi (GQZ), Shan Zhu Yu (SZY), Du Zhong (DZ), Fu Zi (FZ) and Rou Gui (RG) replenish kidney and enrich essence. Tao Ren (TR) and Hong Hua (HH) play the role in promoting blood circulation and removing blood stasis. All raw herbs in BSHX formula were provided by the First Affiliate Hospital of Zhejiang Chinese Medical University . The plant materials were authenticated by Professor Ge, and the voucher specimens were preserved in Zhejiang Chinese Medical University. The extraction process of these herbs was performed as previously described . EucommiIn our previous study, high-performance liquid chromatography (HPLC) analysis was performed to preliminarily detect chemical ingredients of BSHX formula, and six major ingredients were identified, including loganin, amygdalin, pinoresinol diglucoside, liquiritin, cinnamaldehyde and hydroxysafor yellow A .http://lsp.nwu.edu.cn/tcmsp.php) and Traditional Chinese Medicines Integrated Database  were used to search the chemical ingredients of BSHX formula. Oral bioavailability (OB ≥ 30%) and drug-like (DL ≥ 0.18) were set as the screening threshold [https://www.uniprot.org/) was used to predict the target genes for these ingredients.Traditional Chinese Medicine System Pharmacology Database  and DisGeNET Database (https://www.disgenet.org/) were used to collect PMOP-associated target genes, with the screening filter of score > 10 for GeneCards Database and score > 0.1 for DisGeNET Database. Then, a Venn diagram was constructed to determine the overlapping genes between PMOP and BSHX formula, which were termed as the potential therapeutic molecular targets.GeneCards Database (https://david.ncifcrf.gov/home.jsp). KEGG database (https://www.kegg.jp/) was used to identify the potential signaling pathways.The gene ontology (GO) enrichment analysis including biological process (BP), cellular component (CC) and molecular function (MF) terms was performed on the overlapping genes using DAVID database  to establish a Herb-Ingredient network; (2) the overlapping targets were analyzed using the String database (https://string-db.org/) to build a PPI network. The PPI network topological feature was evaluated by three topological characteristics: degree centrality (DC), betweenness centrality (BC) and closeness centrality (CC); and (3) the overlapping genes and signaling pathway obtained from KEGG database were imported into Cytoscape3.8.0 software to establish a Target-Pathway network.The network was constructed as follows: (1) The herbs of BSHX formula and its active ingredients obtained from TCMSP database and TCMID database were imported into Cytoscape3.8.0 software (n = 8 in each group): the sham group, the OVX group and the BSHX formula group. Mice in the latter two groups were subjected to bilateral ovariectomy, whereas a sham surgery only extracting the equal surrounding fatty tissues of ovaries were performed in the sham ones. At the next day post-surgery, BSHX formula was orally administered to the mice in BSHX formula group at a dosage of 0.2 mL/10 g body weight every two days [Ten-week-old female C57BL/6 J mice provided by Shanghai Laboratory Animal Center of Chinese Academy of Science  were randomly divided into three groups (two days . The micThe femur samples were obtained from the mice in each group for micro-computed tomography (μCT) analysis. Three-dimensional (3D) images of femoral metaphysis were reconstructed using NRecon software. The parameters of bone microstructure including bone volume fraction , average trabecular number , average trabecular thickness , average trabecular separation  and bone mineral density (BMD) were calculated as previously described .After μCT scanning, the femur samples were processed for paraffin sections at the thick of 3 μm as previously described . Alcian t-test (two groups) was performed with the software of SPSS 22.0. P* < 0.05 was considered as statistical significance, and P** < 0.01 was considered as highly statistical significance.All data were presented as mean ± standard deviation. Statistical analysis of unpaired Student's After screening the TCMID database and TCMSP database with the ADME thresholds of OB ≥ 30% and DL ≤ 0.18, a total of 218 active ingredients were identified in BSHX formula, including 87 ingredients (40.0%) in GC, 36 (16.5%) in GQZ, 26 (11.9%) in DZ, 18 (8.2%) in TR, 17 (7.8%) in HH, 14 (6.4%) in SZY, 12 (5.5%) in SY, 5 (2.3%) in FZ, 2 (0.9%) in SDH and 0 (0%) in RG. As shown in Fig. Target fishing on the 218 active ingredients was conducted in Uniprot databases, and we obtained 274 related targets of BSHX formula among which there were 220 in GC, 206 in DZ, 201 in HH, 190 in GQZ, 67 in SY, 46 in TR, 51 in SZY, 10 in, FZ 29 in SDH. Then, 292 related targets of PMOP were obtained from GeneCards and DisGeNet databases. After establishment of Venn diagram, we identified 64 overlapping genes between BSHX formula and PMOP  network was built on these 64 overlapping genes using the String database, which contained 64 nodes and 1110 edges Fig. B. Then, P-value . Thirdly, GO and KEGG enrichment analyses revealed numbers of underlying biological processes and signaling pathways when BSHX formula treats PMOP, while only angiogenesis and VEGF signaling were verified in animal experiments. Finally, cellular experiments or a rescue design in animal study with a signal inhibitor would further demonstrate the important role of VEGF-mediated angiogenesis for BSHX formula against PMOP.Overall, network pharmacology comprehensively analyzed the potential therapeutic molecular targets, biological processes and signaling pathways of BSHX formula against PMOP. The subsequent animal validation experiments revealed that BSHX formula exerts the anti-PMOP effects mainly via VEGF-mediated angiogenesis. Network pharmacology followed by experimental validation is an effective and reliable research pattern for pharmacological mechanism of Chinese medicine.


Active ingredients were screened by TCMSP and swissADME, meanwhile, PharmMapper combined with UniProt database was used to predict the active ingredient target information, GeneCard database was employed to obtain Alzheimer's disease (AD)-related genes, Cytoscapes 3.7.2 software was utilized to map the active ingredient-target effect. Besides, Cytoscapes 3.7.2 software Bisogenet and Cyto NCA plug-in combined with STRING platform were utilized to map the protein-protein interaction (PPI) network, DAVID was employed for GO annotation, while KEGG plug-in was used for KEGG pathway enrichment. Mice were tested for inflammatory damage induced by intracerebral injection of lipopolysaccharide (LPS), as well as learning memory and anxiety by water maze and open field tests. In addition, the expression of Caspase-3 and Caspase-9, together with inflammatory factors TNF-α, IL-6, and IL-1β was analyzed in serum. The expression levels of proteins related to PI3K-Akt signaling pathway in the brain were detected by Western blot (WB) assay. According to the results of network pharmacology, there were 35 active ingredients of licorice stem and leaf flavonoids screened, which exerted the anti-Alzheimer's disease (AD) effects via 67 targets and activated 41 signaling pathways including the PI3K-Akt pathway. Furthermore, Behavioural results revealed that Licorice stem and leaf flavonoids improved the learning and memory abilities of model mice and significantly improved the anxiety caused by inflammatory brain damage. Moreover, as suggested by HE staining and TUNEL staining of brain sections, Glycyrrhiza glabra stem and leaf flavonoids alleviated morphological lesions and cell nuclear damage in brain tissue. Results: of brain homogenate supernatant assay demonstrated that Glycyrrhiza glabra stem and leaf flavonoids had a significant effect on the levels of oxidative indicators superoxide dismutase (SOD), catalase (CAT), malonaldehyde (MDA), acetylcholine (Ach), acetylcholinesterase (AchE), Caspase-3, Caspase-9 and serum inflammatory factors TNF-α, IL-6 and IL-1β. Additionally, WB assay results indicated that the PI3K-Akt signaling pathway was activated. Licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat, or Glycyrrhiza glabra L., a leguminous plant that has a wide and long history of clinical use , 2. LicoNetwork pharmacology can analyze the mechanism of action of ingredients in diseases through network patterns, and serve as a purposeful guide for the mechanistic study of complex ingredients. When analyzing the therapeutic effects of Glycyrrhiza glabra stem and leaf flavonoids on Alzheimer’s disease (AD), a network pharmacology-based approach can clarify the molecular targets and mechanisms of action of Glycyrrhiza glabra stem and leaf flavonoids against AD. The intracerebral injection of lipopolysaccharide (LPS) in mice can lead to neuroinflammation and anxiety-like behavior, while inflammation can in turn trigger acetylcholine abnormalities and oxidative stress, satisfying the cholinergic hypothesis, the inflammatory response theory and oxidative damage as a model of AD, while its anxiety-like behavior can also be used as an indicator of model improvement –9. In thThe results are shown in Thirty-seven flavonoid active ingredients from the above-ground parts of Glycyrrhiza glabra were predicted as targets in PharmMapper, then their names were converted and de-duplicated in the Uniprot database to obtain 67 active ingredients as targets.The GeneCard database was searched to yield 10975 AD-related targets, among which, 1422 AD-related targets were screened and de-weighted with a Relevance score ≥10. These targets were later compared with those of active ingredients, and 36 potential targets were acquired from the flavonoids of Glycyrrhiza glabra against AD. The Venn diagram of the intersecting targets is presented in The 35 active ingredients and 36 intersecting targets were imported into Cytoscapes 3.7.2 software, and thereafter the active ingredient-core target network was plotted, including 71 nodes and 583 edges. As observed from Subsequently, 67 active ingredient targets and 1422 screened AD-related targets were imported into Cytoscapes 3.7.2 software Bisogenet plug-in to obtain a PPI network graph. Moreover, the NCA plug-in was utilized to analyze and filter the resulting intersection. The minimum interaction threshold was set at >0.7, and the TSV file was imported into Cytoscapes 3.7.2 software to construct a PPI network regarding the intersection of the PPI network for active ingredient targets of the licorice ground part and the PPI network for AD targets. There were 97 nodes and 604 edges in the graph. The node size and the edge thickness were set by the Degree value and the combined score value, with the larger Degree value indicating the larger node and the larger combined score value representing the thicker edge. The PPI network is displayed in DAVID was adopted to perform GO functional annotation and KEGG enrichment analysis on the key nodes obtained from the PPI network. To be specific, the GO functional annotation analysis was divided into biological process (GB), cellular component (GC), and molecular function (GM) categories. According to our results, GB yielded 239 entries, including negative regulation of RNA polymerase II promoter transcription, viral process, positive regulation of RNA polymerase II promoter transcription, negative regulation of apoptotic process, transcription, DNA template, intercellular adhesion, negative regulation of transcription, DNA template, positive regulation of transcription, DNA template, negative regulation of gene expression, and so on. Meanwhile, GC yielded 59 entries, such as protein complexes, intercellular adhesion junctions, nuclear chromosomes, nucleosomes, nucleoli, extracellular matrix, mitochondria, and intracellular ribonucleoprotein complexes. While GM yielded 77 entries, including protein binding, double-stranded RNA binding, enzyme binding, protein phosphatase binding, and estrogen receptor binding. The top 20 entries, ranked according to P-value from the smallest to the largest, were selected for visual analysis –2C.As for KEGG pathway enrichment analysis, there were 41 signaling pathways enriched , includiPositioning cruise test: the results are shown in Spatial exploration test: the results are presented in Normal mice tend to move around the edges of the field in an unfamiliar environment due to their avoidance, while anxiety will cause the mice to explore irregularly against their avoidance. The results of open field test are shown in The results of HE staining can be observed from The TUNEL staining results are shown in Serum inflammatory factor levels are presented in The brain oxidation indexes are displayed in Brain AchE and Ach levels are demonstrated in As shown in Compared with blank group, the p-PI3K/PI3K and p-Akt/Akt ratios in model group decreased markedly (P < 0.0001), and the administration of positive drugs and various concentrations of Glycyrrhiza glabra stem and leaf flavonoids significantly elevated the ratios to varying degrees (P < 0.05). The results are shown in Network pharmacology can be used to analyze the mechanism of action of ingredients in diseases through network patterns, and it plays a purposeful role in guiding mechanism research of complex ingredients. In this study, 75 major flavonoid components were collected from relevant database, 35 active ingredients were screened and 67 active ingredient targets were identified. Besides, 41 signaling pathways were obtained by constructing the PPI networks and enrichment analysis.PI3K-Akt signaling pathway is a neuroprotective signaling pathway that is widely present in all types of cells , its inhPatients with AD develop progressive memory and cognitive dysfunction, behavioral and personality changes, accompanied by psychiatric symptoms such as apathy, hallucinations and depression. In this study, the effects of licorice stem flavonoids on the pathological behavior of mice with an inflammatory model of AD were investigated by water maze and open field tests. In the water maze test, the model mice lost the spatial memory learning ability that the normal mice had, and licorice stem flavonoids inhibited the loss of memory learning ability in mice. Intracerebral injection of LPS and production of inflammatory factors caused anxiety-like behavior in model mice. In the open field test, model mice were engaged in frequent central area exploration against avoidance, which might reflect their cognitive dysfunction and personality changes , 15. It AD is characterized by cortical atrophy, widening of the sulcal gyrus and loss of neurons , 18. In Oxidative stress is one of the components of the inflammatory response . StudiesActivation of caspases is one of the typical characteristics of apoptosis, but in the late stages of apoptosis and in dead cells, caspases appear to significantly decrease by cleavage activation levels , 26. In Moreover, in this study, the effect of licorice stem and leaf flavonoids on the expression of PI3K-Akt signaling pathway-related proteins P-PI3K/PI3K and P-Akt/Akt was examined by WB assay. Consistent with the results of network pharmacological analysis, licorice stem and leaf flavonoids had a therapeutic effect on AD by activating the PI3K-Akt signaling pathway.The results of network pharmacology and animal experiments indicated that Glycyrrhiza glabra stem flavonoids inhibited neuronal apoptosis through the activation of PI3K-Akt signaling pathway, which was achieved possibly by regulating acetylcholine, inhibiting oxidative stress, inflammatory response, and modulating Caspase-3 and Caspase-9, thus improving the learning and memory abilities of mice.https://pubchem.ncbi.nlm.nih.gov/), TCMSP (http://tcmspw.com/tcmsp.php/), swissADME, (http://www.swissadme.ch/), PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), Uniprot (https://www.uniprot.org/), GeneCard (http://www.genecards.org/), STRING (https://cn.string-db.org/), DAVID (https://david.ncifcrf.gov/) were used.Databases including Pubchem , LPS, SOD assay kit, CAT assay kit, MDA assay kit, Caspase3 assay kit, Caspase9 assay kit , 0.9% saline , titanium dioxide , 10% formaldehyde fixative (laboratory preparation), TNF-α assay kit, IL-6 assay kit, IL1-β assay kit, Ach assay kit, AchE assay kit , electronic balance FA21041 , frozen centrifuge 1-16K , CNC ultrasonic cleaner KH-300DB type , Morris water maze , Absence field , Microplate spectrophotometer , and Embedding machine  were utilized in this study.The SMILES and two-dimensional (2D) chemical structures of the constituents were obtained from Pubchem by searching relevant literature for the above-ground flavonoid constituents of Glycyrrhiza glabra. Thereafter, the active ingredients were screened on the TCMSP platform for oral bioavailability (OB) and drug likeness (DL) values using the ingredient names and CAS numbers, and those not found on the TCMSP platform were screened on swissADME using the 2D structure diagrams.The 2D molecular structures of active ingredients were uploaded to PharmMapper as an SDF file, with the targets restricted to “Human Protein Targets Only”. The UniProt IDs of the obtained targets were then converted into Gene Symbols in the Uniprot database.AD-related targets were searched in the GeneCard database, then the search file was downloaded, filtered and de-duplicated using the Relevance score attribute. Later, the resulting disease targets were compared with the active ingredient targets, and a Venn diagram of the intersecting targets was created.Thereafter, the active ingredients and intersecting targets were imported into Cytoscapes 3.7.2 software to map the active ingredient-core target network.The PPI network was constructed by importing the active ingredient targets and the AD-related targets into the Cytoscapes 3.7.2 software Bisogenet plug-in. Later, the PPI intersection network was obtained by taking the intersection of the two plots, and then the PPI intersection network of licorice stem flavonoids and AD was obtained. Four attribute values of Degree, betweenness, Closeness, and LAC were later utilized to perform a second screening. The obtained key nodes were thereafter imported into STRING, and the minimum interaction threshold was limited to > 0.7. Afterwards, the PPI network maps were obtained and imported into Cytoscapes 3.7.2 software as TSV files, so as to construct the PPI maps regarding the intersection of PPI networks for active ingredient targets of the above-ground parts of Glycyrrhiza glabra and the PPI networks for AD targets.The key nodes obtained from the PPI network were imported into DAVID for Gene Ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and the species was restricted to “Homo sapiens” with a threshold of P-value < 0.05. Graph Pad Prism was employed to visualize the analysis.After one week of acclimatization, the mice were randomly divided into six groups, including blank group, model group, low dose group, medium dose group, high dose group and positive drug group.Mice in each group were injected with 5 g/L LPS solution (3 μL/mouse) in the lateral ventricle using a stereotaxic instrument , while those in control group were given injection of saline. Meanwhile, licorice stem and leaf flavonoids were administered by gavage at 200 mg/kg, 100 mg/kg and 50 mg/kg in the high, medium and low groups, respectively. Donepezil hydrochloride was administered at 3 mg/kg by gavage in the positive group, and saline was administered by gavage in the blank and model groups. The drug was administered for 21 days in total.On day 14 of administration, the Morris water maze was used to assess the spatial memory and learning ability of mice. To be specific, the water maze system consisted of a cylindrical pool , an escape platform, a camera apparatus and a system for analyzing the behavioral trajectories of mice, which was surrounded by shade. The water maze was divided into four quadrants, and mice were randomly selected to be placed in one of these four quadrants, and the time for each mouse to find the platform within 120 s was considered as the escape latency period. If the platform was not found within the specified time, the mice were guided to stay on the platform for 30 s. Subsequently, the mice were trained for 4 days and different quadrants were selected each day. At the end of the training period, mice were placed in any quadrant for a positioning cruise experiment, and the time required to find the platform within the time limit of 60 s was recorded. Thereafter, the escape platform was removed and a spatial exploration test was performed. The number of times that the mice entered the escape platform, the number of times that they entered the effective area, the time they stayed in the effective area and the time they stayed in the target quadrant within 120 s were recorded, respectively.The open field test set-up consisted of a black isolation box  and a camera system. Notably, the open field apparatus was cleaned before each experiment to avoid odor affecting the mice. The mice were placed into the box at a fixed point, then their trajectory and the time spent in the central area were monitored for 3 min.At the end of the behavioral experiments, blood was collected from eyes, placed in the refrigerator overnight at 4° C, and centrifuged at 4° C and 4000 r/min to extract the serum. Meanwhile, brain tissues were taken, ground on ice with 200 μL RIPA lysate per 100 mg, and centrifuged to extract the supernatant. Thereafter, the two brain tissues from each group were placed in 10% formaldehyde fixative and set aside.Brain tissues were fixed in 10% formaldehyde fixative, dehydrated in gradient ethanol solution , then transparentized in xylene, paraffin-embedded and sectioned for analysis. Subsequently, HE staining was performed on the paraffin sections in line with specific instructions to observe the histopathological changes in brain tissue of each group. Moreover, TUNEL staining was performed on the paraffin sections according to specific instructions to detect the apoptosis of brain tissue in each group.The ELISA kits were designed to detect the levels of TNF-α, IL-6 and IL-1β; SOD, CAT and MDA; acetylcholine and acetylcholinesterase; caspase-3 and caspase-9 in the supernatants of mouse brain tissue homogenates.The BCA Protein Concentration Kit was designed to detect the protein concentrations in the supernatants of brain tissue homogenates. To be specific, one sample with a final concentration of 1 mg/mL of protein was prepared using PBS and 5X sampling buffer, which was mixed well and boiled in boiling water for 3-5 min to denature the proteins in chains. After separation by polypropylene amide gel electrophoresis (PAGE), the samples were transferred to polyvinylidene difluoride (PVDF) membranes, and the excess was cut off according to the marker and protein molecular weight. Later, the membranes were incubated with 5% skimmed milk powder, primary antibodies and secondary antibodies in turn.All data were expressed as mean ± SEM and analyzed by one-way or two-way ANOVA with Tukey’s test for comparison between multiple groups. A p-value < 0.05 was considered significant.