Introduction: The increasing mortality in patients with sepsis-induced pulmonary fibrosis owes to a lack of effective treatment options. This study aims to explore the possibility and possible targets of Reduning in the prevention of sepsis-related pulmonary fibrosis.Methods: The active components and targets of Reduning were searched and screened from the database and analysis platform of traditional Chinese medicine (TCM) system pharmacology. GeneCards, human genome database, DisGeNET database, and the OMIM database were checked to determine the targets associated with sepsis-induced pulmonary fibrosis. DAVID Bioinformatics Resources 6.8 was used for GO and KEGG enrichment analysis to predict its possible signaling pathways and explore its molecular mechanism. The protein–protein interaction (PPI) network was used to identify key active components and core targets. Molecular docking technology was applied to screen the complexes with stable binding of key active components and core targets. Molecular dynamics simulations were used to verify the binding stability and molecular dynamics characteristics of the complexes. The protective effect of RDN on sepsis-induced pulmonary fibrosis was verified by in vitro and in vivo experiments.Results: There were 319 shared targets between sepsis-induced pulmonary fibrosis and RDN. GO enrichment analysis showed that they mainly regulated and participated in the positive regulation of kinase activity, mitogen-activated protein kinase (MAPK) cascade, and protein phosphorylation. KEGG enrichment analysis showed that they were mainly enriched in the mitogen-activated protein kinase cascade signaling pathway, the calcium signaling pathway, the apoptosis pathway, and other signaling pathways. The results of molecular docking and molecular dynamics simulations showed that the active components, stigmasterol, beta-sitosterol, and quercetin, had good binding activities with ERBB2, and they exhibited good stability. Molecular validation experiments confirmed RDN could alleviate lung fibrosis induced by cecum ligation and puncture (CLP), in parallel with the inhibition of the ERBB2-p38 MAPK pathway in mouse alveolar macrophages (AMs).Discussion: Reduning may prevent sepsis-induced pulmonary fibrosis by regulating the ERBB2-p38 MAPK signaling pathway, which provides a possibility for the prevention of sepsis-induced pulmonary fibrosis with traditional Chinese medicine. Sepsis-induced acute respiratory distress syndrome (ARDS), a syndrome characterized by acute respiratory failure and intractable hypoxemia, may trigger persistent fibrosis . PatientIn recent years, scholars have gradually deepened their exploration of the possible effects and related mechanisms of TCM in the prevention and treatment of acute and chronic lung injury caused by sepsis . EspeciaThe rise of network pharmacology has provided ideas for revealing the mechanism of TCM compounds in the prevention and treatment of diseases recently. Network pharmacology integrates the advantages of pharmacology, proteomics, and systems biology and has the characteristics of holistic and systematic research. These advantages and characteristics are consistent with the holistic view of TCM, which is conducive to reveal the scientific connotation of multi-component, multi-target, multi-pathway, and holistic regulation of diseases in TCM . This sthttps://tcmspw.com/tcmsp.php/) on artemisinin, honeysuckle, and gardenia correspond to active component retrieval. Taking oral bioavailability (OB) ≥ 30% and drug like (DL) ≥ 0.18 as screening conditions, 49 active compounds of TCM were screened. The PubChem database (https://pubchem.ncbi.nlm.nih.gov/) was used to retrieve 49 active compounds, which was be the primer number input to predict the 782 targets using the SwissTargetPrediction platform.The analysis platform and database system pharmacology of traditional Chinese medicine , the NCBI database (https://www.ncbi.nlm.nih.gov/), and the OMIM database (https://omim.org/) were used to obtain the target genes of disease. The target retrieval was performed using “pulmonary fibrosis in sepsis” as the search term, and the species was set as human. The disease targets retrieved from the database were merged and de-weighted to obtain 2,121 final disease targets.GeneCards, human genome database  to construct the PPI network, and the biological species was set as “Homo sapiens” to obtain the PPI network. There are 319 nodes and 5,487 edges in the network, with an average degree of 34.4 (The common targets of drugs and diseases were input into the STRING database ( of 34.4 .https://david.ncifcrf.gov/home.jsp) to analyze GO enrichment, including biological processes , cell component (CC) enrichment, and molecular function (MF). p-value cutoff = 0.05, q-value cutoff = 0.05, and the rest defaulted to original settings. The shared targets were imported into DAVID Bioinformatics Resources 6.8 to clarify KEGG pathway enrichment. We chose the top 10 pathways according to p-value and used microbioinformatics (http://www.bioinformatics.com.cn/) to visualize.Shared targets of RDN and sepsis-induced pulmonary fibrosis were imported into DAVID Bioinformatics Resources 6.8 (http://www.rcsb.org/). Then, AutoDock software was used to prepare the ligands and proteins required for molecular docking. The crystal structure of the target protein was created by removing water molecules, hydrogenation, modified amino acids, optimized energy and adjusted field parameters, and then the low-energy conformation of the ligand structure was satisfied.The compound name, molecular weight, and 3D structure of the active compounds were determined using the PubChem database, and the corresponding 3D structures of the active compounds were downloaded from the RCSB PDB database  was used for small molecules. The TIP3P water model was used to add the TIP3P water model to the complex system to establish the water box, and sodium ions were added to construct the equilibrium system . In the Anti-p-p38 MAPK (Thr180/Tyr182) antibody, anti-p38 MAPK antibody, and FITC-anti-F4/80 antibody were from Cell Signaling Technology. Anti-p-ERBB2/HER2 (phospho Y877) antibody, anti-ERBB2/HER2 antibody, anti-GAPDH antibody, anti-CD206 antibody, and Alexa Fluor 647-anti-ERBB2 (phospho Y877) antibody were from Abcam. A macrophage isolation kit (peritoneum) was purchased from Miltenyi Biotec.Male wild-type (WT) C57BL/6J mice aged 6–8 weeks were purchased from Beijing Huafukang Biotechnology Co., Ltd. All mice were subsequently bred and settled under specific pathogen-free (SPF) conditions at Tsinghua University. In our study, we anesthetized WT mice with an intraperitoneal injection of tribromoethanol (10 mg/kg). After midline laparotomy, for cecum ligation and puncture (CLP) groups, the cecum was exposed and ligated immediately below the ileocecal valve, causing intestinal obstruction. After two punctures with an 18G needle, the cecum was placed back and the abdominal wall was sealed. Reduning group and CLP+Reduning group were administered with Reduning (50 or 100 mg/kg) by intraperitoneal injection once per day for 7 days before the operation. For the sham group, the cecum was exposed but did nothing, and then the abdominal wall was closed. BAL fluid (BALF) was collected by rinsing the alveolar cavity with 1 ml precooled PBS, and it was stored at −20°C. The right lungs were embedded in 10% formalin prepared for morphology examination. For all these groups, normal saline was injected intraperitoneally just after the operation to simulate clinical fluid replenishment. At 21 days after the operation, mice were sacrificed, and the inferior lobe of the right lung was used as the study sample. All study designs were supervised and approved by the Ethics Committee of Beijing Tsinghua Changgung Hospital (protocol code NCT05095324).After fixation in 4% paraformaldehyde, the lung tissues were dehydrated, embedded in paraffin, and sliced for Masson staining. The morphological structure, inflammatory infiltration, and degree of fibrosis of the lung tissues were evaluated under a microscope after the sections were stained. The Ashcroft score for pulmonary fibrosis was performed on the lung histopathological sections of each mouse, and the scoring criteria were as follows : 0: normIHC was performed following the manufacturers’ protocols. Sections were backstained, dehydrated, removed, and mounted with modified methylethoxylin. All regiments included appropriate positive and negative controls. The percentage of F4/80 protein positive in the lung tissue was derived from three separate data.After the sections were dewaxed and hydrated, lx EDTA antigen repair solution (PH 9.0) was used for antigen repair. After PBS cleaning, endogenous peroxidase block-agent was added to the tissue sections to block the activity of endogenous peroxidase. Goat serum was added to the tissue sections for sealing and incubated in a wet box at room temperature for 30 min. After that, primary antibody diluted with sterile PBS (a-SMA antibody concentration 1:500) was added to the covered tissue sections and incubated at 4°C overnight. An appropriate amount of immunofluorescent secondary antibody was added and was inoculated for 1 h against light at room temperature. Finally, the nucleus was stained with DAPI dye solution, and the tablet was sealed with an anti-fluorescent quench agent.Cells from mice BALF were collected and harvested after centrifugation at 1,000 rpm for 10 min at 4°C and suspended in precooled sterile PBS containing 2% FBS. Mouse alveolar macrophages (AMs) were isolated using a macrophage isolation kit (peritoneum) following the manufacturer’s instructions. Then, Western blot was applied to detect the expression level of the indicated proteins.2) in the laboratory of Tsinghua Changgung Hospital. The experiments were exhibited after two passages. Cells were incubated with LPS (100 ng/ml). After the indicated length of time, cell culture supernatant and cells were collected. The supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to evaluate pro-inflammatory and anti-inflammatory factors. Then, Western blot were applied to detect the expression level of the indicated proteins.AMs were cultured with Dulbecco’s modified Eagle medium (DMEM) and high glucose  under standard conditions  and incubated with secondary antibodies at room temperature for 40 min. After washing with TBST again, the protein signal was observed, and the membrane signal was displayed by an enhanced chemiluminescence Western blot detection system. The staining intensity of the strip was measured using ImageJ software. The antibodies used in our study are described in the previous “reagents” section. Protein expression levels were defined as gray values normalized to the steward gene GAPDH and expressed as multiples of the control. All experiments were performed three times and three times independently.p < 0.05 was considered statistically significant. Moreover, Prism 8  was responsible for visualizing.Data were expressed as the mean ± SD. The differences between variables were compared using one-way ANOVA or a two-tailed Student’s t-test using SPSS software . In order to better understand the intricate relationship among Reduning, active components, and corresponding targets, a network diagram of TCM, compound, and target was constructed based on the included TCM, active components, and target sites. The network was imported into Cytoscape 3.7.2 software to draw the network diagram, and the results are shown in p-value were selected, and according to the p-value, Q-value of each item, and the number of genes enriched on it, the ggplot2 package of the R language was used to draw the bubble map of GO analysis. The abscissa represents the number of targets, the left represents BP, CC, and MF, and the color represents the p-value. The smaller the p-value, the more red the color is; the larger the p-value, the more blue the target is, as depicted in In order to further explore which pathways are mainly affected by the shared targets of diseases and TCM, we conducted GO and KEGG enrichment analysis on the shared targets. A total of 1,634 GO entries were enriched. There were 217 cell components (CCs), which mainly located membrane rafts, membrane microdomains, neuronal cell bodies, synaptic membranes, postsynaptic membranes, and components of presynaptic membranes. There were 3,477 biological processes (BP), which mainly regulated and participated in the processes of positive regulation of kinase activity, positive regulation of MAPK cascade, calcium homeostasis, peptidyl-serine modification, peptidyl-serine phosphorylation, peptidyl-tyrosine phosphorylation, peptidyl-tyrosine modification, and protein self-phosphorylation. There were 426 molecular functions (MFs), mainly manifested in protein serine/threonine/tyrosine kinase activity, transmembrane receptor protein tyrosine kinase activity, nuclear receptor active ligand-activated transcription factor activity, and non-transmembrane protein tyrosine kinase activity. The top eight enrichment items with p-value were selected, and according to the p-value, Q-value of each item, and the number of genes enriched on it, the ggplot2 package of the R language was used for visual analysis of the results. The abscissa represents the number of genes enriched, the left represents the pathway name, and the color represents the p-value. The smaller the p-value, the more red the color, and the larger the p-value, the more blue the color. As shown in A total of 191 KEGG pathways were enriched. The top 20 enrichment items with http://www.rcsb.org/). AutoDock software was used to prepare the ligands and proteins required for molecular docking, remove water molecules, hydrogenate, modify amino acids, optimize energy, adjust force field parameters for the crystal structure of the target protein, and then meet the low-energy conformation of the ligand structure. Finally, the five key target structures and five key active components were used for molecular docking, and the affinity  value represents the binding ability of the two. Finally, Discovery Studio and PyMol software were used to analyze and observe the docking results. The key active components stigmasterol, kaempferol, quercetin, beta-sitosterol, and corymbosin were used to perform molecular docking verification with the key targets SRC, TNF, CASP3, GAPDH, and ERBB2. The results showed that all 25 groups had good binding activity. The top five results were selected and analyzed. In order to clarify the interaction between active components and targets and to predict the binding mode and affinity of complexes, AutoDock software was used to dock the aforementioned five key components and five targets. The compound names, molecular weights, and 3D structures of the active ingredients were determined from the PubChem database, and then, the corresponding 3D structures of the active ingredients were downloaded from the RCSB PDB database  was used to measure the stability of the system, and the RMSD fluctuation range was small in the three groups of dynamics simulations, which could quickly stabilize to 0.2 nm after the beginning of the simulation . RMSF cap > 0.05). As shown in p > 0.05).As shown in p < 0.01). In comparison with the CLP group, p-ERBB2 was sharply reduced after RDN intervention (p < 0.01).To confirm the cell type to which Reduning applied, IF was used to investigate the expression and localization of p-ERBB2 in each group. As shown in 7A, p-ERBB2 was co-located with F4/80. As shown in p < 0.001). There is no significant difference between the Reduning group and the sham group (p > 0.05).To further detect the underling mechanism of RDN, AMs were isolated from the lung tissue. We detected the ERBB2-p38 MAPK signaling pathway-related proteins in each group. As illustrated in p < 0.001).To clarify the impact of RDN on the polarization of AMs, flow cytometry was used to measure the expression of CD206. As shown in ARDS is a common clinically acute critical illness with an average mortality of 30–40% . Sepsis Reduning has pharmacological effects such as clearing heat, dispersing wind, and being antiviral and antibacterial, and has been mainly used to treat infectious emergencies in clinical practice. In recent years, Reduning and its active components have been widely studied in the prevention and treatment of pulmonary fibrosis. For example, it was identified that beta-sitosterol could significantly suppress epithelial–mesenchymal transition (EMT) by the governing TGF-β1/Snail pathway, thus downregulating the TGF-β1-induced fibrotic proteins in human alveolar epithelial cells . In addiIn this experiment, we explored the possible targets of Reduning in the prevention of sepsis-induced pulmonary fibrosis using network pharmacological methods. We identified key active components, stigmasterol, kaempferol, quercetin, beta-sitosterol, and corymbosin, and key targets, SRC, TNF, CASP3, GAPDH, and ERBB2, by PPI. We performed molecular docking between key targets and key active components and selected the top five according to the absolute value of binding energy for molecular docking display. As can be seen in the table, the binding energies of stigmasterol, quercetin, and beta-sitosterol complexes with ERBB2 were in the top five, indicating that many active components of Reduning had good binding activities with ERBB2. Molecular docking and MDs can explain the mechanism of interactions between molecules in depth, and provide an important tool for predicting the binding types and interaction modes of biomacromolecule complexes. Currently, they have become an important research method for explaining biological mechanisms . MDs werGO and KEGG enrichment analysis showed that the shared targets of diseases and drugs were mostly enriched in the MAPK cascade signaling pathway, protein phosphorylation, and kinase activation pathway. The ERBB2–MAPK pathway has been extensively studied in cancer. P38γ MAPK is downstream of ERBB2 and plays an important role in alcohol-enhanced aggressiveness of breast cancer . Prolactin vivo and in vitro experiments were performed to verify the assumption. CLP operation was applied to mimic a sepsis-induced pulmonary fibrosis mice model. Masson staining and IHC showed that Reduning could attenuate CLP-induced fiber deposition in vivo. Next, we explored the cell types in which RDN improved the progression of pulmonary fibrosis by downregulating the activation of ERBB2. As illustrated in the IF results, p-ERBB2 is co-localized with AMs. M1 macrophages are pro-inflammatory cells that have bactericidal and tumor-killing activities, while M2 macrophages are involved in tissue remodeling and promoting fibrosis and mainly secrete anti-inflammatory factors such as TGF-β1 and IL-10. Studies have shown that pulmonary fibrosis is directly related to the abnormal polarization of AMs (Then, n of AMs . In co-cn of AMs . For exan of AMs . Abnorman of AMs . Therefoin vivo and in vitro experiments were used to verify the results of network pharmacology, this study still fell short of unfolding the underlying mechanism between AMs and fibroblasts. We could further explore it in the future.This study innovatively proposed that Reduning could prevent sepsis-induced pulmonary fibrosis through ERBB2 as a target. Although in vivo and in vitro experiments, and the kinetic characteristics of the complexes were explained. This study provides a new clue to the molecular mechanism of Reduning in the prevention of sepsis-induced pulmonary fibrosis and also provides a basis for its clinical application.In conclusion, downregulating the ERBB2-p38 MAPK signaling pathway in AMs may prevent sepsis-induced pulmonary fibrosis, pointing to a potential therapeutic target. In this study, molecular docking and molecular dynamics simulation were used for the first time to explore the possibility of Reduning in the prevention of sepsis-induced pulmonary fibrosis. Network pharmacology and molecular docking results showed Reduning may regulate ERBB2 phosphorylation and the MAPK signaling pathway through stigmasterol, quercetin, and beta-sitosterol, thereby preventing sepsis-induced pulmonary fibrosis. The reliability of the aforementioned results was verified by


Alpinia officinarum Hance (A. officinarum) in improving type 2 diabetes mellitus (T2DM).  We used network pharmacology, molecular docking, and cellular analysis to explore the pharmacodynamic components and action mechanism of A. officinarum toward improving T2DM. The first 9 core targets and potential active compounds were docked using Discovery Studio 2019. Finally, IR-HepG2 cells and qPCR were applied to determine the mRNA expression of the top 6 core targets of the PPI network.  The protein-protein interaction (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to predict the potential targets and mechanism of A. officinarum were obtained. T2DM-related targets overlapped with 176 targets. The core targets of the PPI network were identified as AKT serine/threonine kinase 1 (AKT1), an activator of transcription 3 (STAT3), tumor necrosis factor (TNF), tumor protein p53 (TP53), SRC proto-oncogene, nonreceptor tyrosine kinase (SRC), epidermal growth factor receptor (EGFR), albumin (ALB), mitogen-activated protein kinase 1 (MAPK1), and peroxisome proliferator-activated receptor gamma (PPARG). A. officinarum performs an antidiabetic role via the AGE-RAGE signaling pathway, the HIF-1 signaling pathway, the PI3K-AKT signaling pathway, and others, according to GO and KEGG enrichment analyses. Molecular docking revealed that the binding ability of diarylheptanoid active components in A. officinarum to core target protein was higher than that of flavonoids. The cell experiments confirmed that the A. officinarum extracts improved the glucose uptake of IR-HepG2 cells and AKT expression while inhibiting the STAT3, TNF, TP53, SRC, and EGFR mRNA expression.  A total of 29 active ingredients and 607 targets of A. officinarum Hance improves T2DM by acting on numerous components, multiple targets, and several pathways. Our results lay the groundwork for the subsequent research and broaden the clinical application of A. officinarum Hance. Type 2 diabetes mellitus (T2DM) has gradually become a global epidemic disease, influencing global public health, and its incidence is rising. According to recent figures issued by the International Diabetes Federation, 783 million adults are estimated to develop diabetes by 2045 . NeverthAlpinia officinarum Hance (A. officinarum), also known as smaller galangal, is a dry rhizome of A. officinarum, a member of the Ginger Family Alpinia from Guangdong, Hainan, Guangxi, and Taiwan. The main functions are to warm the stomach and stop vomiting, disperse cold, and relieve pain [A. officinarum is commonly used in China and Europe to improve blood sugar levels, stomach pain, swelling, and cold [A. officinarum and its extract can alleviate T2DM by promoting glucose metabolism and lowering the blood sugar levels [A. officinarum against diabetes is unknown.eve pain , 7. A. oand cold . It has and cold , 10. Modr levels –13. HoweOwing to its characteristics of integrity and systematization, as well as based on the principle of traditional Chinese medicine (TCM) holistic view and syndrome differentiation, network pharmacology has gradually become one of the important means of predicting the mechanism of TCM treatment for various diseases , 15. TheA. officinarum were identified using a method based on network pharmacology by integrating molecular docking in this study. IR-HepG2 cells and the qPCR method were employed to validate the proposed molecular mechanism. These findings provide some predicted and inferred evidence for elucidating A. officinarum's material basis and the action mechanism in improving T2DM.The primary active components, prospective targets, and pharmacological mechanisms of A. officinarum were screened through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (https://old.tcmsp-e.com/tcmsp.php) [https://bionet.ncpsb.org/batman-tcm/) [https://www.tcmip.cn/ETCM/index.php/Home/Index/index.html) [https://www.swissadme.ch/) database, which could predict the components based on their chemical structures. In vivo pharmacokinetics-related information includes gastrointestinal (GI) absorption and Lipinski's 5 rules as the most important indicators [A. officinarum were screened preliminarily.The active compounds of msp.php) , the Bioan-tcm/) , and theex.html) . Furtherex.html) –23. Aftedicators . Based ohttps://www.swisstargetprediction.ch/) [A. officinarum. Subsequently, the target protein was transformed into the corresponding target gene through the UniProt database (https://www.uniprot.org/) [A. officinarum compounds and their targets was constructed. Finally, the resultant components and the targets were employed to construct a component-target (C-T) network diagram of A. officinarum by using the Cytoscape 3.8.2 software (https://apps.cytoscape.org) [TCMSP, BATMAN-TCM, ETCM, and the SwissTargetPrediction database (ion.ch/)  were refot.org/) . The datape.org) .https://www.genecards.org) [https://www.pharmgkb.org/) [https://db.idrblab.net/ttd/) [https://www.drugbank.ca/) [https://www.omim.org/) [A. officinarum to improve T2DM. Finally, the results were visualized using the EVenn network tool (https://www.ehbio.com/test/venn/#/) [Using “Type 2 diabetes mellitus” as the keyword, T2DM-related genes were screened from the GeneCards database (rds.org) , PharmaGkb.org/) , Therapeet/ttd/) , DrugBanank.ca/) , and theim.org/) . Then, tvenn/#/) .A. officinarum common target in the amelioration of T2DM, the EVenn network tool was applied to construct the Venn diagram of the A. officinarum target genes and the T2DM-target genes, and the overlaps were considered as the potential targets of A. officinarum toward improving T2DM.In order to acquire the A. officinarum and T2DM were obtained from the STRING database (https://string-db.org) [https://apps.cytoscape.org) to create the PPI network diagram. Then, the core targets of the network were screened by applying the degree (D), betweenness centrality (BC), and closeness centrality (CC) in the CytoNCA analysis [PPI data for the intersection targets of -db.org) . The proanalysis , 36.https://metascape.org/gp/index.html#/) [P < 0.05 was used as the screening condition, and the top 20 signaling pathways and the top 10 GO terms were shortlisted. Moreover, the GO terms were classified into 3 aspects, including cellular component (CC), molecular function (MF), and biological process (BP). Finally, the bioinformatics software platform (https://www.bioinformatics.com.cn/login/) was applied to visualize the ultimate outcomes.GO functional analysis and the KEGG pathway enrichment analysis were performed on the potential targets by using the Metascape database (.html#/) . P < 0.0A. officinarum to improve T2DM was constructed by the Cytoscape 3.8.2 software. The nodes of the network were mainly composed of A. officinarum, active components, potential targets, the top 20 signaling pathways, and their targets screened by the KEGG pathway enrichment analysis. Moreover, the core active component was further determined based on the degree value of the node.The “component-target-pathway” (C-T-P) network of https://www.RCSB.org). The proteins were added to the Discovery Studio software to remove water and ligands from the environment to define their binding sites. After preparing the receptors and ligands, the (LibDock) docking mode was selected to perform semiflexible and fast molecular docking, with the conformation set to “BEST” and the remaining parameters set to default. After the docking was complete, the successfully docked compounds were sorted in descending order by score. The CDOCKER mode was selected to better understand the ligand-binding mechanism, and the protein receptor was docked with the ligands and positive drugs with the highest LibDock scores for flexible molecular docking. The “Pose Cluster Radius” was set to 0.5, and the other parameters were left alone. After the docking was complete, the binding force of the successfully docked compounds was expressed as “CDOCKER ENERGY.”The structures of the 29 components were painted in the Discovery Studio 2019 . The cryA. officinarum rhizomes were procured in Haikou, China, in July 2013. Professor Niankai Zeng performed the authentication, and a sample (No. 20130716) was deposited in the Medicinal Chemistry Laboratory of Hainan Medical University. The dried and crushed A. officinarum (1.5 kg) was heated and refluxed with 80% ethanol thrice for 1 h, each time. The solution was filtered and concentrated at room temperature (200 g) to obtain the A. officinarum extract (AOE). The components of the AOE were identified using high-performance liquid chromatography (HPLC), and the representative chromatogram of phytochemical compounds in Alpinia officinarum extract is shown in 2 cell incubator. The cells in the log phase were used for subsequent experiments. To investigate the effect of AOE on IR, the cells from the past methods were grown in 50 mM glucose for 48 h to establish an IR-HepG2 cell model [HepG2 cells were procured from the Zhong Qiao Xin Zhou Biotechnology  and cultured in high-glucose Dulbecco's modified essential medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  under 37°C in a 5% COll model . In addi4 cells/well in a 96-well plate and cultured in the DMEM medium supplemented with 10% CS. After 24 h of cell adhesion, the cells were treated with different concentrations of AOE (1–200 µg/mL) for 48 h. Then, 10 μL of the CCK8 solution was added to each well and the plate was incubated at 37°C for 1 h. The absorbance was measured at 450 nm by using the Spectra Max plus Automatic Plate Reader .Cell viability was measured by using the Cell Counting Kit-8 assay . Then, the HepG2 cells were seeded at the concentration of 1 × 10The glucose uptake assay was performed using the fluorescence probe , and the HepG2 cells were cultured in a 6-well plate until the cells reached >70% confluence. After IR induction and AOE treatment, the cells were stimulated in a low-glucose DMEM containing 100 nmol/L insulin for 30 min, followed by treatment with 25 mM of 2-NBDG/well for 30 min. Next, the cells were washed with cold PBS until they became colorless. The fluorescence intensity was then read with the Gemini XPS fluorescence microplate (Molecular Devices) reader with the excitation and emission wavelengths of 485 and 535 nm, respectively.AKT, STAT3, TNF, TP53, SRC, and EGFR was determined by qPCR. HepG2 cells were seeded in a 6-well plate until the cells reached >70% confluence. After IR induction and AOE treatment, respectively, total RNA was extracted from HepG2 cells by using the Eastep® Super Total RNA Extraction kit . The cDNA template was then reverse transcribed using the HiScript® II Q RT Super Mix for qPCR Kit under the following PCR amplification reaction conditions: initial activation of thermostart DNA polymerase for 5 min at 95°C, the second step of 40 cycles . Then, the melting curve phase was followed, and finally, the amplification primers were obtained. All primer sequences were synthesized by Sangon Biotech : AKT ,STAT3 ,TNF ,TP53 , SRC ,EGFR , and GAPDH . The relative expression of each gene was analyzed by the 2−ΔΔCt method, and all genes were normalized to their GAPDH levels.The mRNA expression of n ≥ 3). Differences between the samples were analyzed by one-way ANOVA with GraphPad Prism 8.0.1. P < 0.05 was considered to indicate statistical significance.The data were expressed as the mean ± SD  and 1,168 edges , STAT3 , TNF (tumor necrosis factor), TP53 (tumor protein P53), SRC , EGFR , ALB , MAPK1 (mitogen-activated protein kinase 1), and PPARG (peroxisome proliferator-activated receptor gamma). Furthermore, the magnitude of the degree value indicates the importance of the node, and A PPI network was built and evaluated using the Cytoscape 3.8.2 software after predicting reliable interaction information for 176 potential targets from the STRING database. After hiding the unconnected nodes, a PPI network consisting of 170 nodes and 2,814 edges was obtained . The CytA. officinarum and T2DM, yielding 2,631 GO items, including 2,324 BPs, 118 CCs, and 189 MFs terms. Considering P < 0.05 as the screening criterion, the top 10 BPs indicated that the common genes of A. officinarum and T2DM were mainly involved in hormone response, peptide response, organic nitrogen compound response, and circulatory system process  and 29 active compounds. A. officinarum active components exhibited a high biological affinity for potential target molecules and the potential to exert a high pharmacodynamic activity. The strongest binding force to the 9 core target proteins is 5 diarylheptanoids, including hexahydrocurcumin (COM-21), octahydrocurcumin (COM-27), 1,7-diphenyl-5-hydroxy-3-heptanone (COM-3), 5-hydroxy-7-(4″-hydroxy-3″-methoxy-phenyl)-1-phenyl-3-heptanone (COM-5), and 5R-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl)-3-heptanone (COM-8). Furthermore, 9 diarylheptanoids  exhibited the highest binding scores to core targets, all >100, out of the 29 active components. Second, the binding scores of 7 flavonoids  and the core targets were all >80. As a result, this indicated that the principal medicinal components of A. officinarum are diarylheptanoids and flavonoids, with diarylheptanoids being more active.To better explain the binding activity between the active compounds of A. officinarum active compounds, we selected the compounds with the highest ligand-binding scores from each core protein and docked them again in the CDOCKER mode. A. officinarum binds to the core protein primarily carbon-hydrogen bond, conventional hydrogen bond, van der Waals, and others. To compare the molecular docking results, we selected resveratrol, napabucasin, thalidomide, 5-fluorouracil, bosutinib, celecoxib, ulixertinib, warfarin, and rosiglitazone as positive drugs with their corresponding proteins [A. officinarum has a beneficial effect in the treatment of IR.To better understand the ligand-binding mechanism of proteins –48. The A. officinarum exerted an antidiabetic effect by using numerous components, multiple targets, and several pathways.Overall, the molecular docking results were consistent with the network pharmacology results, indicating that μg/mL had no obvious toxic effect on the HepG2 cells. Therefore, a dose of 50 μg/mL of AOE was selected for subsequent analyses.We analyzed the viability of HepG2 cells at different AOE concentrations to determine the AOE concentrations that did not alter cell viability. As shown in P < 0.001) (μM AOE might reverse the high glucose-induced decline caused by glucose uptake (P < 0.05).To explore the effect of AOE on IR-HepG2 cells, 50 mM glucose was used to induce IR, and intracellular glucose uptake was assessed by using the 2-NBDG method. The glucose uptake level of 3T3-L1 adipocytes was significantly decreased after 48 h of 50 mM glucose induction (< 0.001) , indicatThe first six-core targets  were determined by qPCR to validate the core targets predicted by network pharmacology. As shown in Figures A. officinarum were obtained, including quercetin, kaempferol, galangin, medicarpin, 5-hydroxy-1,7-diphenyl-3-heptanone, octahydrocurcumin, 5-hydroxy-7-(4″-hydroxy-3″-methoxy-phenyl)-1-phenyl-3-heptanone, 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-hept-4-en-3-one, among others -1-phenyl-3-heptanone, 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-hept-4-en-3-one, and 1,7-diphenyl-5-hydroxy-3-heptanone are important active ingredients.We employed the molecular docking method to verify the binding fraction of each active component to the core target protein in order to further verify the relationship between the main active components of A. officinarum were identified to improve T2DM in this investigation. According to GO enrichment analysis, A. officinarum mainly exerts its anti-T2DM effect via hormone response, peptide response, organic nitrogen compound response, and others. The KEGG enrichment analysis identified the AGE-RAGE signaling pathway, HIF-1 signaling pathway, PI3K/AKT signaling pathway, and TNF signaling pathway as important signaling pathways in the network. Past studies have demonstrated that the AGEs-RAGE signaling pathway is closely related to DM and its complications. If IR occurs, excessive glucose levels in the blood can cause nonenzymatic glycosylation of proteins, lipids, and DNA in the body, resulting in the formation of excessively advanced glycosylation end-products (AGEs), which are widely distributed throughout the body [α, which is one of the major proinflammatory mediators and induces low-leveltissue-specific inflammation by activating IKKβ and promoting the development of insulin resistance and the onset of T2DM [A. officinarum ameliorates T2DM by promoting insulin signaling, improving inflammatory responses, and attenuating diabetic markers.A total of 167 potential targets of the body . HIF-1, the body . The PI3the body . The TNF of T2DM . These rA. officinarum exhibited good binding ability to the core target protein. More importantly, in vitro experiments revealed that AOE may significantly increase the AKT mRNA expression while inhibiting the STAT3, TNF, TP53, SRC, and EGFR mRNA expression. Phosphorylation of AKT1 by phosphoinositide 3-kinase (PI3K) promotes the translocation of GLUT4 to the cell surface, which regulates glucose uptake, and GSK3B regulates gluconeogenesis [β-cells metabolic-secretory coupling [in vitro experiments indicated that AOE improves insulin resistance in IR-hepG2 cells by regulating the abovementioned core targets, further demonstrating that A. officinarum exerts anti-T2DM effects through multiple target multipath ways.AKT1, STAT3, TNF, TP53, SRC, EGFR, PPARG, ALB, and MAPK1 were identified as the main targets after a topological analysis of the PPI network. The molecular docking results showed that the active components of ogenesis . Phosphoogenesis . TNF indogenesis . In STZ-ogenesis . Excessicoupling . In typecoupling . Therefoin vitro experimental validation were performed. Therefore, subsequent validation should be combined with animal models. Second, we only verified the first 6 core targets by qPCR. Therefore, other targets (such as PPARG and ALB) or specific pathways  can be selected for further verification. We plan to pursue these gaps as a focus of our future work so as to provide more reliable data supporting the elucidation of the treatment of T2DM with A. officinarum.This study has some limitations. First, only molecular docking and A. officinarum Hance were used for network pharmacological analysis and cell experimentation in this study, and the main active components, action targets, and main signaling pathways of A. officinarum Hance in the improvement of type 2 DM were predicted. The main targets were AKT1, STAT3, TNF, TP53, SRC, EGFR, ALB, MAPK1, and PPARG, and the core pathways were the AGE-RAGE signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, and MAPK signaling pathway. Furthermore, cell experiments revealed that AOE may increase glucose uptake and AKT mRNA expression, while inhibiting the STAT3, TNF, TP53, SRC, and EGFR mRNA expression. These results suggest that A. officinarum Hance plays a role in the amelioration of T2DM through a variety of components, multiple targets, and several pathways, providing sufficient reference and guidance for future experimental research and clinical applications of A. officinarum Hance.The active ingredients of


This study aimed to investigate the molecular regulatory mechanisms underpinning Duchenne muscular dystrophy (DMD). 2 fold change| > 1 and false discovery rate (FDR) < 0.05. Then, protein-protein interaction (PPI), coexpression network of lncRNA-mRNA, and DMD-related lncRNA-mRNA pathway networks were constructed, and functional analyses of the genes in the network were performed. Finally, the proportions of immune cells infiltrating the muscle tissues in DMD were analyzed, and the correlation between the immune cells and expression of the DELs/DEMs was studied.  Using microarray data, differentially expressed long noncoding RNAs (DELs) and DMD-related differentially expressed mRNAs (DEMs) were screened based on the comparative toxicogenomics database, using a cutoff of |logSTAT1, VEGFA, and CCL2 to be the top three hub genes. The DMD-related lncRNA-mRNA pathway network comprising two pathways, nine DELs, and nine DMD-related DEMs showed that PYCARD, RIPK2, and CASP1 were significantly enriched in the NOD-like receptor signaling pathway, whereas MAP2K2, LUM, RPS6, PDCD4, TWIST1, and HIF1A were significantly enriched with proteoglycans in cancers. The nine DELs in this network were DBET, MBNL1-AS1, MIR29B2CHG, CCDC18-AS1, FAM111A-DT, GAS5, LINC01290, ATP2B1-AS1, and PSMB8-AS1.  A total of 46 DELs and 313 DMD-related DEMs were identified. The PPI network revealed  The nine DMD-related DEMs and DELs identified in this study may play important roles in the occurrence and progression of DMD through the two pathways of the NOD-like receptor signaling pathway and proteoglycans in cancers. Duchenne muscular dystrophy (DMD), a severe and progressive X-linked disease that is characterized by muscular dystrophy, is caused by mutations in the dystrophin gene and has a global prevalence of approximately 0.005% of male births . DeficieLong noncoding RNAs (lncRNAs) are noncoding transcripts longer than 200 nucleotides that are not translated into proteins . LncRNAsIn the present study, gene expression profiles of DMD samples were analyzed using a series of bioinformatics tools to identify differentially expressed mRNAs (DEMs) and DELs in DMD. Next, protein-protein interaction (PPI) networks of the DMD-related DEMs and DMD-related lncRNA-mRNA pathway were constructed. Additionally, alterations in different types of immune cells and the correlation between immune cells and crucial factors in DMD were also analyzed. Finally, the expression of the important factors (lncRNAs and mRNAs) identified in the study was verified in the training and validation sets. Thus, the study identified several potential biomarkers and therapeutic targets for DMD and has significant implications for the development of novel treatment strategies based on them.https://www.ncbi.nlm.nih.gov/geo/). The GSE38417 dataset, which was used as a training set, included muscle tissue samples from 16 patients with DMD and six normal control individuals (Affymetrix Human Genome U133 Plus 2.0 Array platform). However, the GSE6011 dataset, which served as a validation set, contained 36 muscle tissue samples  and was analyzed using the GPL96 detection platform. Then, the mRNA and lncRNA expression was reannotated according to the annotation files from the Ensembl genome browser database (https://asia.ensembl.org/index.html).The gene expression profiles from the GSE38417 and GSE6011 datasets were downloaded from the NCBI Gene Expression Omnibus , the raw expression datasets were subjected to log2 transformation and then normalized using the “between array normalization” function. Subsequently, DEMs and DELs were screened with a strict cutoff of false discovery rate (FDR) < 0.05 and |log2 fold change (FC)| > 1. Then, bidirectional hierarchical clustering analysis based on the centered Pearson correlation was performed on the identified DEMs and DELs using the pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html) in R 3.6.1.Using the limma package of the https://ctdbase.org/), with the inference score threshold set at >3. Following comparison with the identified DEMs, the overlapping DMD-related DEMs were selected for gene ontology (GO) terms [https://david.ncifcrf.gov/). An FDR of <0.05 was set as the threshold for a significant difference.Using the keyword “Duchenne muscular dystrophy,” DMD-related genes were searched in the comparative toxicogenomics database (CTD)  (https:/O) terms  and KyotO) terms  enrichmehttps://string-db.org/), and the PPI pairs with a combined score >0.6 were retained to construct a PPI network [https://apps.cytoscape.org/apps/centiscape). Degree refers to the number of interactions (edges) at a node (protein), and node genes with the highest degrees were defined as hub genes.The overlapping DMD-related DEMs identified using the screening were subjected to searches for interactions of the proteins they encoded using the search tool for the retrieval of interacting genes   plugin in the Cytoscape software was used to mine the functionally related and highly interconnected modules using the following parameters: degree cutoff = 2, node score cutoff = 0.2, and K-core = 2. Then, the functional analysis of the genes in these modules was performed using the BINGO plugin  in the Cytoscape software with FDR <0.05.The molecular complex detection (MCODE) version 1.4.2; https://stat.ethz.ch/R-manual/R-devel/library/stats/html/cor.test.html) using the R software. Then, the lncRNA-mRNA pairs with P < 0.05 and PCC > 0.9 were chosen to construct a lncRNA-mRNA coexpression network, and the network was visualized using Cytoscape software. Next, the genes in the coexpression network were subjected to GO term and KEGG pathway enrichment analyses using the DAVID online tool (version 6.8) with FDR < 0.05.Correlations between the DELs and overlapping DMD-related DEMs were analyzed by calculating Pearson's correlation coefficient (PCC) using the cor.test , and the differences between the two sets of samples were compared using t-tests. Finally, the correlation between the significantly different immune cell types and the expression of the factors (mRNAs and lncRNAs) in the DMD-related lncRNA mRNA pathway network was analyzed by calculating the PCC.The infiltration of immune cells in muscle tissues is an important feature of DMD . The pro2 FC| > 1, 46 DELs and 2125 DEMs were screened between DMD and normal control samples , including “positive regulation of ERK1 and ERK2,” “inflammatory response,” “response to hypoxia,” “TNF signaling pathway,” “NOD-like receptor signaling pathway,” “PI3K-Akt signaling pathway,” “p53 signaling pathway,” “FoxO signaling pathway,” “Ras signaling pathway,” and “MAPK signaling pathway” Figures .STAT1 (degree = 41), VEGFA (degree = 32), and CCL2 (degree = 28).The 313 DMD-related DEMs were subjected to a search for interactions between the proteins encoded by these DMD-related DEMs using the STRING database, and 695 interaction pairs with a combined score >0.6 were identified and used to build a PPI network, which contained 216 nodes and 695 edges . After cFurthermore, we extracted four network clusters using MCODE , includiP < 0.05 and PPC > 0.9, 308 lncRNA-mRNA pairs were obtained, and a coexpression network of lncRNA-mRNA was constructed , and nine DELs   . From thP=0.0139), activated natural killer (NK) cells (P=0.0461), and neutrophils (P=0.0251) were significantly decreased, whereas the proportions of M2 macrophages (P=0.02) and resting myeloid dendritic cells (P=0.0007) were significantly increased  and one DMD-related DEM (MAP2K2), whereas the cells were negatively correlated with the expression of the other five DELs and eight DMD-related DEMs. However, resting myeloid dendritic cells and M2 macrophages showed an opposite trend , whereas that of CCDC18-AS1, DBET, MBNL1-AS1, and MIR29B2CHG was significantly decreased , whereas mRNA MAP2K2 was markedly downregulated in DMD samples  in GSE6011 was consistent with that in GSE38417  and validation (GSE6011) sets. In GSE38417, the expression of lncRNA ATP2B1-AS1, FAM111A-DT, GAS5, LINC01290, and PSMB8-AS1 significantly increased  assembles a protein complex called the NLR family pyrin domain-containing 3 (NLRP3) inflammasome in response to certain infectious and sterile stimuli [PYCARD encodes the inflammasome adaptor apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), a component of the NLRP3 inflammasomes, which is involved in downstream signaling of the inflammasome pathway [RIPK2) regulates NLRP3 and inflammation [CASP1, encoding caspase 1, is a key component of NLRP3 inflammasomes [PYCARD, RIPK2, and CASP1 may be involved in regulating DMD-related inflammation via the NLR signaling pathway.Lack of dystrophin results in chronic inflammation, which is closely associated with muscle degeneration in the pathogenesis of DMD , 20. In  stimuli . NLPR3 i stimuli . The NLR stimuli . PYCARD  pathway . Receptoammation , and theammation . CASP1, mmasomes  and is pmmasomes . Our resMAP2K2 encodes mitogen-activated protein kinase 2, an important component of the MAPK pathway. The MAPK pathway is an important regulator of myofiber death [RPS6) controls mRNA translation, and phospho-RPS6 has been identified as a surrogate marker of the activated PI3K/AKT/mTORC1 pathway found in many cancer types [PDCD4) mediates inflammation and regulates the MAPK pathway [LUM), a member of the small leucine-rich proteoglycan family, is present in muscle tissues and plays dual roles as an oncogene and tumor suppressor [TWIST1 has been reported to regulate inflammatory genes in skeletal muscle [α (encoded by HIF1A) is an important cause of vascular dysfunction and impaired angiogenesis in DMD [HIF1A and TWIST was observed in the DMD-related lncRNA-mRNA pathway network. HIF1α silencing reportedly leads to downregulation of TWIST1 [MAP2K2, LUM, RPS6, PDCD4, TWIST1, and HIF1A may play important roles in DMD pathophysiology by regulating the MAPK pathway and proteoglycans in cancer.The dystrophin protein is a component of the dystrophin-associated glycoprotein complex that connects actin filaments, intermediate filaments, and microtubules to transmembrane protein complexes to stabilize muscle cells . Proteoger death . MAPK/ERer death . Ribosomer types . Program pathway . Wang et pathway  demonstrppressor . TWIST1 l muscle  and medil muscle . The acts in DMD . Additiof TWIST1 . Taken tκB signaling is activated during DMD pathogenesis and leads to inflammation and muscle degeneration [κB signaling pathway [κB signaling. However, the role of FAM111A-DT has not been identified. Therefore, these nine DELs may play important roles in the occurrence and development of DMD through the NLR signaling pathway and proteoglycans in cancers. However, the specific mechanisms of these nine lncRNAs in DMD pathogenesis need to be investigated further.In addition, nine DELs, including DBET, MBNL1-AS1, MIR29B2CHG, CCDC18-AS1, FAM111A-DT, GAS5, LINC01290, ATP2B1-AS1, and PSMB8-AS1, were identified in the DMD-related lncRNA-mRNA pathway network. The lncRNA DBET is associated with the epigenetic etiology of facioscapulohumeral muscular dystrophy . Li et aneration . A subst pathway , 51, whiCCDC18-AS1 is positively correlated with activated myeloid dendritic cells and neutrophils [CCDC18-AS1 was positively correlated with the proportion of activated NK cells, neutrophils, and Tregs but negatively correlated with resting myeloid dendritic cells and M2 macrophages. Our study also showed that the proportions of Tregs, activated NK cells, neutrophils, M2 macrophages, and resting myeloid dendritic cells were significantly different between patients with DMD and normal control individuals.In DMD, the immune system is activated and several types of immune cells, such as CD4+ and CD8+ T cells, Tregs, and NK cells, invade the skeletal muscles . A recentrophils , in concHowever, this study has some limitations. First, the number of samples in the GSE38417 dataset was small, and additional validation cohorts need to be analyzed in future studies. Second, as this study involved secondary analysis of microarray data, further experimental studies are needed to verify the results of our study. Additionally, the specific mechanisms of the identified DMD-related DEMs and DELs need to be further explored in vitro and in vivo.PYCARD, RIPK2, CASP1, MAP2K2, LUM, RPS6, PDCD4, TWIST1, and HIF1A), and two KEGG pathways  were identified to play a crucial role in the pathogenesis of DMD. Our findings provide novel insights into the regulatory relationships between lncRNAs, mRNAs, and pathways in the pathogenesis of DMD. The DMD-related DEMs and DELs identified in this study need to be further investigated for their clinical use as potential biomarkers and therapeutic targets for DMD.By performing a series of bioinformatics analyses on microarray data, a DMD-related lncRNA-mRNA pathway network was constructed. Nine DELs , nine DMD-related DEMs (


Radix Salviae and Panax notoginseng, is a patented traditional Chinese medicine that is widely used in multiple countries for relieving coronary heart disease (CHD), but its pharmacological mechanism has not been fully elucidated. In this study, we screened the key pharmacological pathways and targets of CDDP that act on CHD using a network pharmacology-based strategy, and the angiogenic activity of CDDP was directly visually investigated in zebrafish embryos in vivo.The compound Danshen Dripping Pill (CDDP), which is a mixture of extracts from The potential therapeutic targets and pathways were predicted through a bioinformatics analysis. The proangiogenic effects of CDDP were examined using vascular sprouting assays on subintestinal vessels (SIVs) and optic arteries (OAs) as well as injury assays on intersegmental vessels (ISVs). Pharmacological experiments were applied to confirm the pathway involved.PI3K/AKT and VEGF/VEGFR pathways. An in vivo study revealed that CDDP promoted angiogenesis in SIVs and OAs in a dose-dependent manner and relieved the impairments in ISVs induced by lenvatinib, a VEGF receptor kinase inhibitor (VRI). In addition, Vegfaa and Kdrl expression were significantly upregulated after CDDP treatment. Furthermore, the proangiogenic effect of CDDP could be abolished by PI3K/AKT pathway inhibitors.Sixty-five potential therapeutic targets of CDDP on CHD were identified and enriched in the VEGF/VEGFR and PI3K/AKT signaling pathways. These results suggest a new insight into the cardiovascular protective effect of CDDP.CDDP has a proangiogenic effect, the mechanism of which involves the The online version contains supplementary material available at 10.1186/s12906-022-03589-y. Current data from the World Health Organization show that cardiovascular diseases (CVDs) are still the leading cause of death among noncommunicable diseases and account for approximately one-third of all deaths worldwide . Among CVEGF/VEGFR signaling [Angiogenesis is a process by which new blood vessels sprout from pre-existing blood vessels and is affected by ignaling . Endotheignaling . Drugs tignaling . In receignaling , and proignaling . EnhanciCompound Danshen Dripping Pill (CDDP), a patented traditional Chinese medicine (TCM), is widely used for relieving AP, ischemic heart diseases and coronary arteriosclerosis in China. It was first launched in 1995 by the China Food and Drug Administration (CFDA) and was approved by the US Food and Drug Administration (FDA) in 2016 to enter phase III NCT01659580) clinical trials . These a0 clinicaVEGF/VEGFR and PI3K/AKT signaling for ischemic CVDs.The zebrafish has become an ideal animal model to study angiogenesis, in part because its responses to many pro- or antiangiogenic drugs are comparable to those observed in mammalian systems –12. Remahttps://old.tcmsp-e.com/tcmsp.php) with the filters OB  ≥ 30% and DL (drug-likeness) ≥ 0.18. Target prediction of the active compounds was then performed using TCMSP and confirmed through the DrugBank database (https://go.drugbank.com/). For CHD-related target screening, we searched the keyword ‘coronary heart disease’ in the GeneCards database (https://www.genecards.org/). The target genes obtained were then pooled using R software, and the genes in common were regarded as hub genes to be further screened. The protein–protein interactions of these hub genes were constructed using the STRING database (http://string-db.org/) with a minimum required interaction score of 0.950. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) [The main active ingredients of CDDP, Radix Salviae and Panax notoginseng, were recognized based on traditional medicine guidelines and the Chinese Clinical Trial Registry up to April 30, 2020 . The constituents were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (s (KEGG)  pathway CDDP extracts were purchased from Tasly Pharmaceutical Group Co. Ltd.  and dissolved using dimethyl sulfoxide (DMSO)  to make different concentrations. In previous research, the chemical profile of CDDP had been fully investigated using HPLC . The finTg(fli1: EGFP), were obtained from the Laboratory of the Department of Cardiology, Shanghai Changhai Hospital, maintained at 28 °C on a 14/10-h light/dark cycle and staged as previously described [Transgenic zebrafish embryos, escribed . To prevescribed . The embTwenty-four hpf WT zebrafish were used to perform toxicity screening of CDDP. After being dechorionated with 0.5 mg/mL Pronase E , 24 hpf Tg(fli1:EGFP) embryos were incubated with CDDP at safe concentrations  for 48 h. DMSO (≤ 0.5%) served as the vehicle control and was equivalent to no treatment. For each concentration, three replicates of fifteen embryos were set up and exposed to 1 mL of test medium. The entire screening system was performed in a 24-well tissue culture plate. At 72 hpf, the embryos were examined using a fluorescence microscope  and each group tested was scored for mortality and morphological defects. The numbers of sprouting vessels in SIVs and abnormally developed vessels in OAs were photographed and counted for quantitative analysis. For the ISV injury and restoration assay, twenty-four hpf zebrafish embryos were treated with VRI (100 nM) for 24 h and then incubated with or without various concentrations of CDDP for another 24 h. LY and AKTi were added to the system 30 min before CDDP when used. The intact and deficient vessels of intersegmental vessels (ISVs) were calculated for each embryo. The ISV index was calculated as: (the number of intact vessels × 1) + (the number of defective vessels × 0.5).Total RNA was isolated from CDDP-treated embryos using TRIzol reagent  and reverse transcribed using a FastQuant RT Kit . Quantitative RT–PCR assays were performed using SYBR Green Real-time PCR Master Mix . The primers used for mRNA detection are listed in Supplementary Table P < 0.05 was considered statistically significant.All experiments were conducted more than three times. The data are presented as the mean ± SEM and were analyzed using GraphPad Prism 7.0 software. Normality was evaluated using the Shapiro–Wilk test. The Mann–Whitney test or Kruskal–Wallis test followed by Dunn's multiple comparisons test was then performed for the data that did not fit a normal distribution. Whereas for the data that followed a normal distribution, Levene's test of homogeneity of variance was further performed. When the data exhibited homogeneity of variance, a one-way ANOVA was applied. A Radix Salviae and Panax notoginseng, were screened using the TCMSP database. A total of 65 compounds were identified in Radix Salviae , Kaposi sarcoma-associated herpesvirus infection related (16 proteins), and Hepatitis B related signaling pathways (15 proteins), , which specifically expresses enhanced green fluorescence protein (EGFP) in vascular endothelial cells, to evaluate the proangiogenic effect of CDDP. The toxicity of CDDP in zebrafish embryos was evaluated as follows. Twenty-four hours post-fertilization (24 hpf), embryos were treated with various concentrations of CDDP for 48 h because angiogenic vessel development begins at approximately 20 hpf and is completed at 72 hpf [We employed the transgenic zebrafish line t 72 hpf . Figure Twenty-four hpf embryos were treated with CDDP as illustrated in Fig. Vegfaa (an isoform of VEGFA in zebrafish) and its receptors, Flt1, Kdrl and Flt4, in the whole tissue homogenate of CDDP-treated embryos. Figure Vegfaa and Kdrl mRNA were upregulated in the 1.2 mg/mL CDDP treatment group.We also observed thickening of the walls of OAs Fig. A-E and nFGF signaling pathway in regulating angiogenesis in zebrafish [FGF pathway  and can be used to establish a defective angiogenesis model in zebrafish . In the PI3K/AKT signaling pathway and promotes angiogenesis [VEGF/PI3K/AKT signaling pathway probably affected angiogenesis, so we speculated that CDDP also promotes the angiogenesis process by activating the VEGF/PI3K/AKT signaling pathway. Based on this inference, because CDDP reversed the vascular development disorder caused by VRI, when the activation of the PI3K/AKT pathway was inhibited, the protective effect of CDDP on vascular development might also be blocked. Hence, the PI3K inhibitor LY (0.3–3 µM) and AKT inhibitor AKTi (0.03–0.3 µM) were employed to evaluate this prediction in vivo , which expresses enhanced fluorescent protein in vascular endothelial cells, to test whether CDDP actually has proangiogenic activity in vivo and in the involved pathways. This animal model, with the advantages of high genetic similarity to humans and low cost, was convenient for vascular system observation under a fluorescence microscope. We found that CDDP did not affect the survival rate or early global development below a concentration of 1.2 mg/mL . Interestingly, when treated with LY or AKTi alone, ISV growth was slightly enhanced (Fig. PI3K/AKT alone was inhibited, some other pathways may be regulated in vivo to some extent, maintaining a physiological balance of angiogenesis. Direct treatment with CDDP also promoted ISV development, which could be diminished by LY or AKTi cotreatment (Fig. VEGF/VEGFR and PI3K/AKT pathway-mediated angiogenesis.Several reported key genes and pathways affecting angiogenesis such as the ted Fig. A-B. Howeted Fig. C. This ited Fig. . We havetivation . Becauseted Fig. B, which ced Fig. C. This ment Fig. D. These However, our study still has many limitations. For example, fold changes of the genes and pathways mentioned in this manuscript was not further detected on the protein level due to the lack of specific antibodies against zebrafish. Hence, our results could provide a reference for TCM researchers and clinicians but still need further verificationVEGF/VEGFR and PI3K/AKT  signaling pathways. Our results provide new evidence for the application of CDDP in the treatment of CHD.In conclusion, our study demonstrates that CDDP, a patented TCM formula, promotes angiogenic effects in zebrafish embryos through the activation of the Additional file 1. Fig. S1. CDDP promotes sprouting angiogenesis. Schematic diagram of CDDP promoting angiogenesis by activating the VEGF/VEGFR and PI3K/AKT signaling pathways.Additional file 2. Table 1. Composition of CDDP. The main 2 components ofCDDP are listed in the table. Additional file 3. Table 2. Primers usedfor RT–qPCR in this study. The primers used in this study are listed in thetable.Additional file 4. Table 3. Pharmacokinetic parameters of theingredients reported in Radix Salviae. The 65 ingredients and their pharmacokinetic parameters in RadixSalviae were obtained from the online database TCMSP.Additional file 5. Table 4. Pharmacokinetic parameters of theingredients reported for Panax notoginseng. The 8 ingredients and their pharmacokinetic parameters in Panaxnotoginseng were obtained from the online database TCMSP.Additional file 6. Table 5. Genes related to active ingredients of Radix Salviae. The screening of ingredient-related genes of Radix Salviae wasperformed using TCMSP and confirmed through the DrugBank database Additional file 7. Table 6. Genes related to the active ingredients of Panax notoginseng. The screening of ingredient-related genes of Panax notoginsengwas performed using TCMSP and confirmed through the DrugBank database.Additional file 8. Table 7. Key genes related to coronary heart disease.The 7,100 coronary heart disease-related genes reported were screened outthrough the GeneCards databaseAdditional file 9. Table 8. Screenedtarget genes of CDDP for CHD treatment. In total, 65 potential target genes of CDDP forCHD treatment were identified.


To identify the active chemical in Wenshen Huatan Quyu Decotion (WHQD) and to explore its possible network interactions with the polycystic ovary syndrome (PCOS).  The Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP) and the Bioinformatics Analysis Tool for Molecular Mechanisms in Chinese Medicine (BATMAN-TCM) were used to decompose compound formulations, detect active chemicals and their corresponding target genes, and then convert them into UniProt gene symbols. Meanwhile, PCOS-related target genes were collected from GeneCards to construct a protein-protein interaction (PPI) network, which was further analyzed by STRING online database. Gene Ontology (GO) functional analysis was also performed afterwards to construct the component-target gene-disease network to visualize the correlation between WHQD and PCOS. We then performed an in silico molecular docking study to validate the predicted relationships.  WHQD consists of 14 single drugs containing a total of 67 chemical components. 216 genes were predicted as possible targets. 123 of the 216 target genes overlapped with PCOS. GO annotation analysis revealed that 1968 genes were associated with biological processes, 145 with molecular functions, and 71 with cellular components. KEGG analysis revealed 146 pathways involved PPI, and chemical-target gene-disease networks suggest that PGR, AR, ADRB2, IL-6, MAPK1/8, ESR1/2, CHRM3, RXRA, PPARG, BCL2/BAX, GABRA1, and NR3C2 may be key genes for the pharmacological effects of WHQD on PCOS. Molecular docking analysis confirmed that hydrogen bonding was the main interaction between WHQD and its targets.  WHQD exerts its pharmacological effects by improving insulin sensitivity, subfertility, and hormonal imbalance, increasing ovulation rates, which in turn may increase pregnancy rates in patients with significant efficacy. Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive women, with a worldwide prevalence of 6-8% . It is aAlthough there is no medical term equivalent to polycystic ovary syndrome in traditional Chinese medicine, there are formulations used to improve symptoms similar to those of polycystic ovary syndrome, such as oligomenorrhea and subfertility , 7. WHQDIn this study, we introduced a network pharmacology approach to establish a multilevel study to determine the possible relationship between WHQD and PCOS. Network pharmacology is a new strategy for studying the effects and interactions between drugs and diseases. It was originally proposed by Hopkins in 2007 . This aphttps://www.tcmspw.com/tcmsp.php) [http://bionet.ncpsb.org/batman-tcm/) [We identified all fourteen herbs of the formula from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) (msp.php) . Each sian-tcm/) .http://www.UniProt.org) under the species of “Homo sapiens.”All target genes were then converted into gene symbols after searching in UniProt Knowledgebase (https://www.genecards.org/) and got the potential disease-related genes after eliminating candidates whose scores are lower than the median level.We used “Polycystic Ovary Syndrome” as the keyword to explore the disease-related genes at GeneCards  to show common target genes.All predicted target genes of Wenshen Huatan Quyu Decotion were collected together with the projected target genes of PCOS. They were then imported to the Venn diagram (https://string-db.org/). The organism is limited to “Homo sapiens.” The software gives scores to represent the confidence of the interaction between the proteins. We selected high confidence data > 0.7 to ensure the reliability of the analysis. The network was then exported to Cytoscape (version 3.8.0), an open-source free software to facilitate further exploration of the multirelationship among target genes.Protein-protein interaction (PPI) diagram was drawn after shared target genes were uploaded to STRING database (https://www.kegg.jp/) were applied to the STRING online database (https://string-db.org/) to annotate and classify common targets [P value cutoff of 0.05, we collected and analyzed the data by RStudio 3.6.3 .The results of pathway enrichment analysis from Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes . In the figure (see http://www.rcsb.org). The docking simulation was conducted via AutoDock Vina 1.5.6 with the selected key proteins, e.g., adrenoceptor beta 2 (ADRB2), gamma-aminobutyric acid receptor (GABRA1), nuclear receptor subfamily 3 group C member 2 (NR3C2 or MR), and progesterone receptor . The binding affinities of molecules to proteins were predicted based on the docking score. Lower score indicates higher affinity. The results were saved in pdbqt file. All modelling and screening were analyzed and demonstrated via Ligplot.We rank the compounds according to their degree in the network and pick up some important molecules: quercetin , kaempferol , beta-sitosterol , stigmasterol , and isorhamnetin . The structures of the molecules were downloaded from TCMSP, while the structure of the receptors were downloaded from the website of RCSB Protein Data Bank  common target genes were extracted out of a total of 2905 genes. A Venn diagram was drawn accordingly see .The shared target genes were uploaded to STRING online database to form the protein-protein interaction network. 122 nodes (genes) and 1944 edges (interactions) were identified, representing the main genes targeted by the active constitute of WHQD formula . ImportaGO enrichment analysis was performed subsequently. There are 1968 enrichment results related to biological process (BP), 145 related to molecular function (MF), and 71 related to cell component (CC). The top 10 results of the 3 respective sections are shown in The related pathway of WHQD was obtained through KEGG enrichment analysis. 146 signaling pathways were discovered, and the top 20 were shown in Visualization of the complex interactions among WHQD, corresponding target genes, and PCOS was made available via Cytoscape as shown in The validation study of molecular docking was conducted via AutoDock Vina. The results revealed that docking scores of quercetin , isorhamnetin , kaempferol , beta-sitosterol , and stigmasterol  with key targets were listed in Polycystic ovary syndrome is one of the most common disorders in women during the reproductive years . It can Previous studies have found that some TCM medicines and formulations are effective in the treatment of polycystic ovary syndrome . The claβ-sitosterol , β-carotene , formononetin , and isorhamnetin  may be the WHQD treatment for key and active components of PCOS, as they function as antioxidants and may alleviate the symptoms of PCOS [In the present study, we explored the possible interactions between WHQD and PCOS in the network using newly developed bioinformatics technologies . We foun of PCOS . Common  of PCOS , 29.Pathway analysis shows that the AGE-RAGE pathway is significantly active, which may promote inflammation, apoptosis, and vascular dysfunction , 31. In To our knowledge, this is the first time to reveal the active ingredients of Wenshen Huatan Quyu Decotion (WHQD) and its pharmacological effects on PCOS. This helps researchers and pharmacologists to understand the mechanism of WHQD. However, further in vitro experiments should be conducted to verify the predicted course.Wenshen Huatan Quyu Decotion (WHQD) is a TCM formula that is effective in ameliorating the symptoms of PCOS. However, further experiments are awaited to verify the causal relationship between WHQD and PCOS.


Primary membranous nephropathy (MN) is an important cause of nephrotic syndrome and chronic kidney disease (CKD) in the adult population. Although the discovery of different autoantibodies against glomerular/podocytic antigens have highlighted the role of B cells in the pathogenesis of MN, suboptimal response or even resistance to B cell-directed therapies occurs, suggesting that other pathophysiological mechanisms are involved in mediating podocyte injury. The complement system plays an important role in the innate immune response to infection, and dysregulation of the complement system has been observed in various kidney diseases. There is compelling evidence of complement cascade activation in primary MN, with the mannose-binding lectin (MBL) and alternative pathways particularly implicated. With appropriate validation, assays of complements and associated activation products could hold promise as adjunctive tools for non-invasive disease monitoring and prognostication. While there is growing interest to target the complement system in MN, there is concern regarding the risk of infection due to encapsulated organisms and high treatment costs, highlighting the need for clinical trials to identify patients most likely to benefit from complement-directed therapies. Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in the adult population worldwide. Up to 12 per million adults are affected each year, although there is significant regional variation , a chronic infection (such as hepatitis B or C), or drugs . Accordingly, the term “primary MN” was used to describe cases that were thought to be idiopathic . Recent The identification of target glomerular antigens in primary MN has revolutionized disease diagnosis, classification, prognostication and treatment. MN is now conceptualized as a predominantly B cell disorder with disease-causing antibodies. Some of these antibodies, such as anti-PLA2R antibodies, are directly correlated with the clinical disease course. Anti-PLA2R antibodies have been used as a “liquid biopsy” for patients presenting with proteinuria, obviating the need for kidney biopsy in select patients; anti-PLA2R antibodies may also be used to track the clinical course and detect relapses at an earlier time –13. AccoDisease-specific antibodies lead to the clinical manifestations of primary MN through a variety of downstream pathways, of which the complement system has been the best characterized. A growing body of research is re-evaluating the role of complement pathways in primary MN associated with particular glomerular antigens, in light of the plethora of new antigens being discovered that challenge the previous monolithic understanding of idiopathic primary MN. This narrative review will review the evidence regarding the role that complement pathway activation plays in the pathogenesis of primary MN, and highlight its potential role in disease diagnosis, prognostication, monitoring and treatment.The complement system is a tightly regulated component of the innate immune system intended to enhance the abilities of antibodies and phagocytic cells to attack cell membranes of pathogens, clear microbial organisms and damaged cells, and promote inflammation. Three pathways, namely the classical pathway, alternative pathway and mannose-binding lectin (MBL) pathway, can activate the complement cascade. All three pathways converge on a common terminal pathway to result in the synthesis of a membrane attack complex (MAC), which can cause pores in pathogen cell membranes resulting in cell lysis , 15. TheThe classical pathway of complement is triggered by the interaction between C1q, which is part of a C1qrs complex with 2 C1r molecules and 2 C1s molecules, and the Fc receptors of immunoglobulin molecules such as IgM or IgG. A conformational change of C1s allows for the cleavage of C4 into C4a and C4b, and C2 into C2a and C2b. The larger fragments of these 2 key cleavages combine to generate the C3 convertase C4b2b, which further cleaves C3. C3b generated from cleavage of C3 then binds to C3 convertase to form the C5 convertase (C4b2b3b) that cleaves C5. Among these cleaved complement activation products, C3a and C5a are anaphylatoxins that attract phagocytic cells to sites of inflammation, while C3b may also function as an opsonin. C5b interacts with other terminal pathway proteins, including C6, C7, C8 and C9 to form the C5b-9 complex, better known as the MAC , 15.The MBL pathway of complement is analogous to the classical pathway, and is triggered by the interaction between MBL and carbohydrate motifs on bacteria. Like C1r and C1s, mannose-binding lectin serine proteases (MASPs) can cleave C4 and C2 to generate the C3 convertase C4b2b –16.The alternative pathway of complement, in comparison, is constitutively active at a low level in normal humans, and can amplify rapidly in response to pathogens. The alternative pathway is triggered when C3b binds to a water molecule. Factor B and D are recruited, and factor D enzymatically cleaves factor B to form Bb, an active serine esterase that can cleave C3 into C3a and C3b. C3b binds with Bb to form C3bBb, the alternative pathway C3 convertase which can break down C3 further. This creates an amplification loop whereby more C3 products are formed that can further propagate the pathway. C3bBb forms a multimeric complex with C3b to form the C5 convertase C3bBbC3b, thereby cleaving C5 to generate the terminal complement pathway , 15.To prevent the alternative pathway from spiraling out of control, several regulatory proteins and pathways are normally present to keep a check on alternative pathway activity. These include both regulatory proteins in the blood and those that are membrane-bound. Decay-accelerating factor (DAF), a membrane-bound regulator catalyzes the degradation of both classical pathway and alternative pathway-associated C3 convertases (C4b2b and C3bBb). Membrane cofactor protein (MCP) is a surface-expressed protein that serves as a cofactor for factor I, which irreversibly cleaves C3b to iC3b thus preventing formation of the alternative complement C3 convertase. It has an equivalent murine homologue in the form of Crry , 15. FacThe classical and MBL pathways are also subjected to regulation, although this is less important than alternative pathway regulation since these pathways require an antigenic trigger. The classical pathway C3 convertase C4b2b undergoes decay mediated by the membrane-bound DAF and CR1, and by C4 binding protein (C4BP) in the fluid phase . C1 inhiThe archetypal example of a kidney disease caused by complement dysregulation is C3 glomerulopathy. C3 glomerulopathy is a rare kidney disease characterized by dysregulated activation of the alternative pathway. This could be due to development of a C3 nephritic factor, an autoantibody that binds to the alternative pathway C3 convertase C3bBb to stabilize it against factor H-mediated decay acceleration. Mutations in the factor H gene or other components of the alternative pathway of complement could also result in complement pathway dysregulation . HoweverThere is unequivocal evidence from animal and human cell line models, as well as patients with primary MN, implicating complement pathway activation in the pathogenesis of proteinuria and kidney injury. Bioinformatics analysis in MN has shown that differentially expressed genes in MN were preponderantly associated with complement cascade-related immune responses . Regardlin vitro cytotoxicity of anti-PLA2R antibodies derived from donor sera on human kidney cells was greatly enhanced by the addition of rabbit complement, highlighting the pathogenic role of complement pathways  initiative . The choIn this regard, the largest trial of the C5 inhibitor eculizumab in MN did not show clinically meaningful improvement with two different dose regimens, although side effects were minimal. This trial has been criticized for using doses far smaller than those used for other complement-mediated diseases, including C3 glomerulopathy or atypical hemolytic-uremic syndrome, and adequate blockade of the complement pathway was demonstrated only in a small fraction of patients . Given tUse of the small-molecule factor B inhibitor LNP023, also known as iptacopan, in a murine model of MN led to improved proteinuria and halted further disease progression. After the mice were sacrificed, histological analysis showed improvement in glomerulopathy and tubular injury, and absence of C3 staining, confirming successful blockade of the alternative pathway of complement. Importantly, these effects on proteinuria and histology were demonstrated regardless of whether complement blockade was delivered prophylactically prior to induction of MN in mice, or therapeutically after proteinuria had already occurred . A recenThere have been trials of other complement-based therapies in MN . In thisActivation of the complement system is a key contributor to the development of MN. There is a growing understanding of the interplay between disease-causing antibodies, their target antigens, and the complement pathways, in the pathogenesis and clinico-pathological correlations of MN. While there is promising preliminary experience with the use of complements and their activation products in disease monitoring and prognostication, these approaches will have to be validated in well-defined patient cohorts. Therapeutic modulation of the complement system has emerged as a promising treatment strategy in various kidney diseases, and the results in MN are eagerly awaited.BS conceptualized the work and drafted the original manuscript. GC, DY, and TC reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.DY received research donations from the Wai Im Charitable Foundation and the Chan Sui Kau Family Benefits and Charitable Foundation. DY and TC received research funding support from the Mr and Mrs Tam Wing Fun Edmund Renal Research Fund. TC received research funding support from the Wai Hung Charitable Foundation and Mr S Ho. 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.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Heart failure (HF), the final stage of cardiovascular diseases, is a clinical syndrome of cardiac structural or functional abnormalities. QiShenYiQi Dripping Pills, short for QSYQ, showed effectiveness and safety in the treatment of HF according to modern pharmacological research and clinical studies, but the mechanism remains unclear. This study aims to clarify the mechanism of QSYQ in treating heart failure through the analysis to critical biomarkers, targets and pathways.Materials and Methods: In this study, the efficacies of QSYQ in non-human primates and rodents were evaluated, and the mechanism was demonstrated by integrating network pharmacology and metabolomics analysis. Furthermore, the targets from network pharmacology and the metabolites from targeted metabolomics were jointly analyzed to screen the critical pathways.Results: In rhesus monkeys with spontaneous chronic heart failure, nasogastric administration of QSYQ for 12 weeks caused profound improvement of systolic and diastolic function as evidenced by echocardiography detection. Consistently, QSYQ administration especially with higher dose lowered the blood pressure and improved the ventricular remodeling, collagen deposition and fibrosis markedly in Spontaneous Hypertension Rats (SHR) model. Computational prediction showed that QSYQ exhibited anti-HF effects possibly through HIF-1 signaling pathway, FoxO signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway and other enriched paths. Metabolomics analysis obtained 23 significantly altered metabolites, revealing that QSYQ significantly regulated the abnormal levels of fatty acids, carnitines, organic acids pyridines, nucleosides, which were mostly involved in myocardial energy metabolism related pathways.Conclusion: Based on serum and myocardium metabolomics and network pharmacology, the present study revealed that the actions of QSYQ in treating HF depend on multi-components, multi-targets and multi-pathways. Cardiac dysfunction is associated with a complex spectrum of pathophysiological changes, including metabolic disorders, inflammation, cellular senescence, cell death, and fibrosis . AccordiAstragalus membranaceus (AM), Salvia miltiorrhiza (SM), Panax notoginseng (PN), and Dalbergia odorifera (DO), is approved by the State Food and Drug Administration of China in 2003 for treatment of coronary heart disease.Traditional Chinese Medicine (TCM) compound is formed under the compatibility principles of Chinese herbal medicinal formulae and customarily composed of multiple medicinal herbs with different pharmacological properties . QiShenYModern pharmacological research and clinical studies have demonstrated its effectiveness in the treatment of HF. Recent studies verified that QSYQ enhanced the degree of myocardial fibrosis by inhibiting the TGF-β1/Smads pathway and exerted an obvious myocardial protective effect on CHF rats . Chang eIn this study, we reported the protective effect of QSYQ on rodent and non-human primate HF models. By integrating network pharmacology and metabolomics data acquired from rodents’ samples as well as reported results from publications, the molecular mechanism by which QSYQ exerts its pharmacodynamics effects was elucidated. The research flowchart is shown in QSYQ were provided from Tasly group, Tianjin with the batch No. For rhesus Monkeys and rats were 180615 and 190301, respectively and referred to commercial standards and quality control standards. Captopril (batch No. 1901001) as a control in this study was purchased from Tianjin Feiying Yuchuan Pharmacy Co., Ltd.https://old.tcmsp-e.com/tcmsp.php) could provide quantitative and systems information about TCM ingredients, ADME-related properties, targets and diseases. When retrieving the compound information of QSYQ, the screening criteria were set as the oral bioavailability (OB) greater than 30% and the drug-likeness (DL) greater than 0.18. It is worth mentioning that OB values were predicted by constructing a novel chemometric method, a SAR-based in silico models (https://www.uniprot.org/) was adopted to adjust the names of genes to official symbols while DAVID database . All experimental protocols had been reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of Sichuan Primed Shines Biotech Co., Ltd., with approval number AW 1813.Male rhesus monkeys with spontaneous chronic heart failure (CHF)  were proAnimals were divided into QSYQ 50 mg/kg, QSYQ 100 mg/kg and vehicle group according to LVEF, Ea and E/Ea parameters detected in the adaptation period, each consisting of four monkeys. QSYQ and vehicle were orally administrated once a day (QD) for 12 weeks, followed by an additional 4-week washout period, cardiac function was evaluated every 4 weeks during this time.n = 60, 14-week-old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China. They were kept under a light/dark cycle of 12 h at controlled temperature and humidity. The rats were fed normally for 10 weeks and then randomly divided into five groups, each consisting of 13 rats. They were normal group (WKY), model group (SHR), captopril group , QSYQ low-dose group  and QSYQ high-dose group . As indicated, the rats were administrated daily by gavage with vehicle, captopril or QSYQ for 12 weeks. They were weighed once a week and the dosage for the next week was calculated accordingly.Male SHR and WKY (®Vevo®3100) before administration, on the eighth week and after administration.The blood pressure of each rat was measured by KENT noninvasive sphygmomanometer while the cardiac function was evaluated by ultra-high resolution animal ultrasound imaging platform  calculation, partial was adopted for histopathological analysis and partial for metabonomic analysis. All experimental protocols were approved by the local ethics committee of Tasly with approval number TSL-IACUC-2019-07. The relevant experiments were conducted in accordance with the Guidelines for the Animal Care and Use in Tasly.For rats, hearts were fixed with 4% neutral paraformaldehyde solution for 48 h and embedded in paraffin wax. The heart sections (3–4 μm) were cut and mounted on glass slides for hematoxylin and eosin (H&E) staining and modified Masson staining. The histomorphology changes were observed under optical microscope.Targeted metabolomics profiling of serum and heart samples of rats were performed by Metabo-Profile . Six rats were taken out and the left ventricle of myocardial tissue was quickly removed, separated and stored at −80°C. Based on ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), the functional small molecule metabolites were quantitatively determined. Sample preparation and instrument parameter setting can be referred to published studies .p < 0.05 and |log2FC|>0 (Fold Change). Moreover, metabolic pathway analysis was performed by MetaboAnalyst 4.0  to reveal disturbed metabolism. Pathways with the values of p < 0.05 were screened out as the candidate target pathways.For statistical analysis, both principal component analysis (PCA) and orthogonal projection to latent structures square-discriminate analysis (OPLS-DA) were performed. The V-plot of the OPLS-DA model was used to visualize metabolites, while the candidate biomarkers were screened through multivariate and univariate statistical analysis by Variable importance in projection (VIP) > 1, p < 0.05 was considered to be statistically significant.The measurement data for monkeys and rats were expressed as mean ± standard deviation (SD), unless otherwise stated. One-way analysis of variance (ANOVA) was used to analyze significant differences. A value of p < 0.05), which supported its critical role in the mechanism related to cardiovascular disease . The LVEF improvement demonstrated time-dependent and dose-dependent during the administration period.To investigate the role of QSYQ on the cardiac function, a study in rhesus monkeys with spontaneous chronic heart failure was conducted . By Doppp < 0.05)  and Ea (E/Ea) can be adopted to evaluate the diastolic function if under normal state of systolic function . Similar < 0.05) . Left ve < 0.05) . ConsistHypertension is the leading risk factor for numerous cardiovascular diseases, and chronic pressure overload leads to the development of left ventricular hypertrophy (LVH). Progressive hypertrophy and fibrotic changes in the heart lead to progressive diastolic dysfunction ultimately leading to diastolic heart failure . To furtIt has been verified that QSYQ could ameliorated the cardiac function in rhesus monkeys. To further prove the effects in rats, echocardiographic measurements were conducted correspondingly. Compared to WKY group , Table 1Consistent with the above results, compared to SHR, diffused collagen deposition was improved after 12 weeks of QSYQ treatment by H&E staining , althougWhen the heart is damaged, the myocardial structure changes as well as the metabolism of glucose, fatty acid, lactic acid and other substances in myocardial cells are disturbed, which will lead to the abnormality of cardiac function. Herein, based on the results of pharmacodynamics evaluation above, metabolomics was conducted to explore the discrimination of metabolite profiles among six samples from the WKY control, the SHR model and QSYQ high dose treatment groups, respectively.In the PCA score plot , 9, the p < 0.05 and |log2FC|>0), totally 23 metabolites in serum samples and myocardial tissue samples could be significantly altered in SHR group compared with WKY group, while QSYQ dramatically call-backed the expression of these common metabolites to normal levels to different extents  to further explore the possible mechanisms through which QSYQ regulates the cardiac structure and function. By setting the screening threshold as raw p < 0.05, six key metabolic pathways include alanine, aspartate and glutamate metabolism, aminoacyl-tRNA biosynthesis, phenylalanine metabolism, butanoate metabolism, citrate cycle (TCA cycle), biosynthesis of unsaturated fatty acids were screened out as potential target paths among given groups  as well as functional changes. Sustained pressure setting when left uncontrolled will promote the conversion from compensatory to maladaptive phase, named as pathological hypertrophy. Under the condition, cardiomyocyte growth beyond the capacity of the capillaries to fully supply nutrients and oxygen, leading to cardiac hypoxia, cardiac remodeling and fibrosis, generally predisposing to the development of heart failure . As cardThe current study reported that QSYQ treatment once a day for 12 weeks could ameliorate the cardiac systolic and diastolic function in rhesus monkeys with spontaneous chronic heart failure, which was more obvious in the dose of 100 mg/kg. Moreover, administration of QSYQ at higher dose of 465 mg/kg could significantly lower the blood pressure and improve the ventricular remodeling, collagen deposition and fibrosis in SHR-induced myocardial damaged model, namely pre-HF model. Above-mentioned results were in accordance with previous reports in animal models and clinical trials . MoreoveTo further elucidate the mechanism of QSYQ on aforementioned effects, we used the strategy of network pharmacology to predict the potential targets and pathways as well as metabolomics to explore biomarkers and pathways. Through network pharmacological analysis, chemical constituents, target proteins and pathway networks of QSYQ were obtained, which also reflect the coordinated regulation characteristics of multi-target and multi-pathway of TCM. The possible pathways contained commonly accepted HIF-1 signaling pathway, FoxO signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, which were in accordance with previous papers . Furthervia the carnitine palmitoyltransferase (CPT-1 and CPT-2) to undergo β-oxidation. The subsequently produced acetyl-coA enters TCA cycle to produce ATP  β-oxidation and glucose oxidation is an important determinant of cardiac efficiency and function. Under normal conditions, FAs and carbohydrate are the main substrates used by mitochondria to meet the dynamic demands for myocardial energy . In the duce ATP . In uncoduce ATP . It is wvia the pyruvate dehydrogenase (PDH) to continue energy transference. Instead, it is metabolized in the cytosol, primarily reduced to lactate, as well as to other metabolites such as involving in propanoate metabolism  promotes cardiac glycolysis without activation of glucose oxidation . At thistabolism . In prevtabolism , decreastabolism . In our Additionally, as to other types of metabolites such as pyridines, nucleosides play a major role in regulating the cardiovascular function. N-methylnicotinamide, a primary metabolite of nicotinamide, exerts antithrombotic and anti-inflammatory effects through mediating the endothelium function by asymmetric dimethylarginine (ADMA) -dimethylarginine dimethylaminohydrolase 2 (DDAH2) axis and contribute to the vasoprotective properties in mice model . In seveTherefore, according to the metabolomics analysis, the differentiated metabolites involved in aforementioned metabolic pathways can be mapped to the results predicted by network pharmacology, including HIF-1 signaling pathway, AMPK signaling pathway as well as PI3K-AKT signaling pathway and FoxO signaling pathway. These paths verified by reported studies from experimental and clinical studies tended to be the core metabolic pathway for QSYQ in HF.. In this study, QSYQ reinforced Qi efficacy by the regulation of myocardial energy metabolism related pathways, and improved the blood stasis via atherosclerosis intervention and promoted blood circulation by nicotinate and nicotinamide metabolism or SAH regulation.According to the theory of traditional Chinese medicine, QSYQ is used as a compound preparation against disease related to “Qi deficiency and blood stasis”. Qi deficiency mainly reflects in the lack of energy to support a variety of biological functions, while blood stasis is caused by disturbance of blood circulation . In thisIn summary, as evidenced by pharmacology experiments in non-human primates and rodents, our study showed beneficial effects of QSYQ on parameters related to HF. A systematic exploration of mechanism based on network pharmacology and metabolomics integration analysis received HIF-1 signaling pathway, AMPK signaling pathway, PI3K-AKT signaling pathway, FoxO signaling pathway to potentially regulate state of Qi deficiency and blood stasis in patients. Our study and data support the theory of the underlying mechanism of QSYQ in preventing the development of heart failure, hence lays a foundation on clinical application.