Fresh pumpkin leaf is popular vegetable for its rich nutrition. The pleasant taro-like odour is important aroma quality of crops, and mostly contributed by 2-acetyl-1-pyrroline in pumpkin. Element Zn can impact metabolite biosynthesis in plants, including aroma formation. However, Zn-induced biochemical responses, especially 2-acetyl-1-pyrroline formation in pumpkin, haven’t been elucidated.This study integrated metabolome and transcriptome to explore molecular fluctuations in pumpkin leaves at different time intervals after foliar Zn treatment.P5CS and proline biosynthesis, which, therefore, affect 2-acetyl-1-pyrroline production. This research reveals molecular mechanisms of 2-acetyl-1-pyrroline formation in pumpkin, which will provide the molecular basis for desired aroma compound production through metabolite engineering.We first identified more than one thousand metabolites from pumpkin leaves by integrating different mass spectrometry methods according to the form in which a metabolite exists. Comparative metabolomic analysis revealed there were separately 25 out of 50 and 286 out of 963 metabolites that were respectively identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, differentially regulated by Zn treatment. Our findings revealed that 50mg/L of Zn significantly enhanced 2-acetyl-1-pyrroline production by more than 38%, which was contributed by increased biosynthesis of its precursors, including ornithine and proline. The following transcriptome analysis discovered 30,574 genes, including 953 novel genes. Zn treatment induced the differential expression of 41.6% of identified genes which were supposed to regulate the downstream metabolite changes in a time-dependent manner. Pathway analysis indicated that alternations in primary metabolism, including carbon metabolism and biosynthesis of amino acids, were vital to the fluctuated aromatic compound generation. Phytohormones and transcription factors may regulate the expression of gene  Only a 4) treatment (2) application positively impacted the yield, together with the aroma of fragrant rice, especially the accumulation of key aroma contributor 2-AP  were involved in the regulation of 2-AP biosynthesis. Our research elucidated that Zn application could be an effective technique to improve the aromatic profile of the taro-like flavours of pumpkin leaves. The illustrated molecular mechanisms underlying Zn-induced increase of 2-AP will be of great value to the quality breeding of pumpkins associated with aroma formation.22.1Cucurbita moschata D., was selected as the experimental material. This material is the selfed progeny of one important pumpkin germplasm that has the taro-like aroma and is widely used for pumpkin fragrance-associated breeding in China. Seeds were germinated at 30°C in the dark for two days and then cultured in Hoagland’s hydroponic medium which contains all essential elements at proper levels for growth. Treatment was carefully applied to one-month-old pumpkin through evenly spraying ZnCl2  on each leaf surface according to the previous experiments (2 was determined according to previous studies as the specified application significantly improved the 2-AP abundance of pumpkin (not published). After foliar Zn application, fresh pumpkin leaves at top three nodes were separately collected at 24 hrs, 72 hrs, and 120 hrs. Plant samples sprayed with MilliQ water were taken as the controls. Three biological replicates were prepared for each condition, and three plants were collected for each replicate. The three experimental conditions , together with the control (Ctrl), constituted the experimental materials in this study. Collected tissues were snap-frozen in liquid nitrogen tank, followed by storage inside an ultra-low temperature freezer at -80°C for the subsequent experiments.The pumpkin variety NO.44, belonging to the eriments . The tre2.2via the headspace solid-phase microextraction (HS-SPME) combined with GC-MS analytical technology. Samples were freeze-dried in a vacuum and ground into powder. Half gram of lyophilized and powdered leave sample was weighed and carefully placed in a 20 mL vial. 3-nonanone and 2,4,6-trimethylpyridine  were added to the vial as the dual internal standards . The freeze-dried leaves were then grounded . 100 mg of leave power was weighed and used for metabolite extraction. Metabolites were extracted using 70% cold methanol. Samples were vortexed for 30 sec every 30 min, after six times of vortex, the samples were kept in the extraction solution at 4°C for overnight. High speed centrifuge  was followed after the overnight extraction. The supernatant was carefully transferred onto the filter membrane (0.22 μm pore size). The filtered sample was kept in vial before loading to the instrument for mass spectrometer analysis  coupled to an ultra performance liquid chromatography . Metabolites were characterized using the multiple reaction monitoring method, and then searched against the MetWare database. The downstream data analysis was carried out using Analyst 1.6.1 software. Metabolites were considered with changed abundance if the result of statistical analysis was significant .2.4Cucurbita moschata reference genome (http://cucurbitgenomics.org/). Differentially expressed genes were analysed using DESeq2 package. The statistical significance testing approach was applied to identify genes that express differently   was used to measure the RNA concentration. The RNA quality was further confirmed using the Agilent 2100 Bioanalyzer  followed by cDNA library construction. The constructed cDNA library was validated through quality control assays. Library concentration and size distribution were confirmed through Qubit dsDNA high sensitivity assay and Agilent ScreenTape assay, respectively. Kapa BioSystems qPCR method was applied to calculate molarities and normalize libraries prior to pooling and sequencing. The sequencing data was uploaded to the CNGB Sequence Archive (CNSA) of China National Genebank DataBase (CNGBdb) (accession number CNP0003866). Clean reads were aligned to the an 0.05) . The fun2.5-ΔΔCT method  was utilized to detect metabolites in the pumpkin leaves at different time intervals after foliar Zn treatments, with the aim to integrate with the GC-MS generated data and explore the intermediates or precursors associated with the identified 2-AP and its biosynthesis. By contrast, more abundant metabolites (963 metabolites) were identified from pumpkin leave using LC-MS/MS Table S2https://www.metaboanalyst.ca/). Arginine biosynthesis, galactose metabolism, and citrate cycle were the top three significantly enriched pathways , pyrroline-5-carboxylate reductase (P5CR), and delta-1-pyrroline-5-carboxylate synthetase (P5CS), were up-regulated, which results basically explained the increased abundance of 2-AP or related metabolites.As mentioned above, the production of 2-AP involves several pathways, including biosynthesis of amino acids, TCA cycle, biosynthesis of arginine, and biosynthesis of arginine and proline, indicating the complex regulatory networks that involved during the process of 2-AP biosynthesis in the foliar Zn primed pumpkin. Those pathways were integrated and carefully organised to illustrate the process of 2-AP formation, and visualise the dynamic changes of associate intermediate metabolites and genes after different time intervals Figure 43.5via phytohormones homeostasis and their signaling networks. Phytohormone regulation initiates the biosynthesis of associated metabolites, including the volatile metabolites 2-AP and its precursor proline, by modulating genes involved in the corresponding biosynthetic pathway, thereby, affecting crop performance  was up-regulated with more than three-fold changes at the initial stage (24 hrs), while the other three genes  were all down-regulated, representing the incompatible and compatible responses to Zn treatment. The third proposed pathway in SA synthesis is associated with mandelonitrile. Mandelonitrile is another SA precursor and derived from L-phenylalanine through cytochrome P450 (CYP) and mandelonitrile lyase (MDL)  to SA is a presumed enzyme benzoic acid hydroxylase (BA2H) (SAMT genes (CmoCh03G007580 and CmoCh07G004720) decreased in this study at 24 hrs and 72 hrs, which result was consistent with the down-regulated MeSA. Another vital gene related to SA biosynthesis was salicylic acid-binding protein 2 (SABP2)  after Zn application. Hence, it is supposed that the increased conversion from MeSA to SA may mostly contribute to the increased concentration of SA. Another mechanism responsible for the SA accumulation may be the decreased conversion of SA to SA 2-O-beta-D-glucoside and the glucose ester of SA (SGE) by SA-glucosyltransferase (SAGT). SAGT is the key factor that negatively regulates SA levels  at 120 hrs, which may be the stress tolerance mechanisms induced at the later stage.Exogenous applications of stimulates can trigger the activation of downstream pathways formance . As rougse (MDL) . Either e (BA2H) , but wase (BA2H) . The exp (SABP2) . The metA levels . There wNCED, 10 ABA2, and three AAO3 genes were found with differential expression after Zn treatment. The high number of regulated ABA2 implied there may be functional redundancy. Two of three AAO3 were up-regulated at both 24 hrs and 72 hrs, whereas the third AAO3 gene had increased expression only at 120 hrs. Interestingly, the three AAO3 genes were closely distributed on the same chromosome 3  was up-regulated at all time-points when compared to the control condition. Additionally, it was notable that five abscisate beta-glucosyltransferase (AOG) coded genes were regulated by Zn. In the initial of Zn treatment, three of them were regulated, and all with increased expression levels. AOG catalyzes the active ABA forming the inactive ABA conjugate ABA-glucose ester (ABA-GE), which, therefore, controls the ABA concentration and associated hormone signal in plants (ABA is synthesized in plants through the carotenoid pathway in which the conversion of violaxanthin to xanthoxin by 9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting step . Followimosome 3 . Five ann plants . Althoug3.6MYB, EREBP and WRKY were the three biggest TFs groups that were regulated in Zn-treated pumpkin. KEGG enrichment analysis identified significantly enriched pathways . Interestingly, there were separately 30 and 40 regulated TFs classified into plant hormone signal transduction and MAPK signalling pathway, indicating the involvement of these TFs in signalling after Zn induction , and WRKY TFs (21 genes), respectively. The overlapped 16 regulated MYC2 were further analyzed. Different regulations of various MYC2 were identified (MYC2 in pumpkin could be homologs of MYC2 (AT1G32640) in Arabidopsis thaliana which has been proven to involve in the negative regulation of the rate-limiting enzyme P5CS1 in the biosynthesis of proline under biotic stress  elucidated that the gene was located in the nucleus of the cell, which further demonstrated the potential activities of this MYC2 on the nuclear DNA in pumpkin. The homologous sequence alignment was further confirmed using the online tool Clustal Omega  , 204 , and 168 TFs regulated  at 24 hrs, 72 hrs, and 120 hrs, respectively  genes were identified as differentially expressed at different time intervals after the Zn application. Four of them were significantly up-regulated at 72 hrs, which was coincidence with the increased proline at 72 hrs.Illustrating the molecular mechanism of 2-AP production is important in improving the aroma quality of pumpkin leaves. 2-AP biosynthesis involves in important precursors or intermediates, and key enzymes . Prolinet stress . Six supP5CR and P5CS genes had increased expression after the Zn treatment in this study. It is remarkable that ornithine was the alternative source for proline biosynthesis  were mostly up-regulated in pumpkin leaves by Zn, and therefore, positively catelyse the production of associated 2-AP precursors or intermediates. As previously described, 2-AP formation may occur in both leaves and grains of rice . Increased levels of proline, citrulline, N-α-acetyl-L-ornithine, ornithine and arginine were particularly identified at 72 hrs, as mentioned above. Nitrogen-metabolizing pathways in plants generate ornithine, arginine, proline and polyamines through glutamate metabolism. During this process, plants produce intermediates which are vital to plant development and responses to various environmental changes . Underst4.4Plant hormones are important signal molecules in sorts of signaling pathways as these pathways are directly or indirectly correlated in a wide of abiotic and biotic stress responses . The exoP5CS, and increased formation of proline in wheat under drought stress , therefore, regulate the proline and 2-AP synthesis. Additionally, other differentially expressed TFs in Zn treated pumpkin leaves, such as MADS-box TFs, heat shock TFs, ethylene-responsive TFs, and bHLH TFs, are also reported to be involved in the corresponding regulatory mechanisms that integrate phytohormone and redox signaling in the plant response to a number of stresses, therefore, directly or indirectly impact the 2-AP biosynthesis  in Camellia sinensis, while in turn, this component could considerably improve the hyperosmotic stress tolerance through reducing the stomatal conductance and MDA, increasing ABA and proline generation  with decreased expression, which may result in the decreased production of cis-2-hexen-1-ol. The cis-3-hexen-1-ol with green aroma, geraniol with rose-like fragrance, 2-AP with popcorn flavour, together with other regulated aromatic compounds, contributed to the aroma profile in pumpkin leaves after Zn priming and foliar application.Except for increased 2-AP, the cis-3-hexen-1-ol was detected by GC-MS, but significantly down-regulated at all three time internals compared to the control. The cis-3-hexen-1-ol, as the most influential component of green aroma from plants, is a valuable resource for imparting fruit and vegetable aromas. It was reported that the hyperosmotic stress can induce the accumulation of cis-3-hexen-1-ol and up-regulation of most associated genes , were deemed to play the critical role in the elevation of 2-AP production in the metabolite profile. It is supposed that Zn application regulated the primary metabolism, which contributed to the change of aromatic components. Phytohormones may interact with Zn, and act as the signal molecule and regulate the aroma formation relevant biological processes. TFs that control specific pathways appear to be the promising tool for metabolic engineering with the aim to improve the aroma profile of pumpkins.Aroma of the fresh pumpkin leaves can affect the perception of consumers, and therefore the determination of their preference. The interplay of theoretical and practical have summarized that the character improvement of crops can be achieved by artificially changing the environmental conditions surrounding the plants appropriately. It is manifested that foliar application of Zn was efficient in improving the 2-AP formation in pumpkin leaves. We hypothesis that Zn modulates the 2-AP biosynthesis in a complex way at transcriptional level. The up-regulated The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: CNGB Sequence Archive (CNSA) of China National Genebank DataBase, CNP0003866.JxL and XY designed and planned the experiments. JxL performed the GC-MS experiment. YQ, XY, JnL, HG, HW and XZ prepared the samples. LD, XL, GZ and JxL analyzed the data. LD and JxL wrote and reviewed the article. All authors contributed to the article and approved the submitted version.


Glycyrrhiza uralensis in DTF inhibited eotaxin, Th2 cytokine and IgE production in vitro and in vivo.Eosinophilic Esophagitis (EoE) is a chronic condition characterized by eosinophilic inflammation of the esophagus which leads to esophageal dysfunction with common symptoms including vomiting, feeding difficulty, dysphagia, abdominal pain. Current main treatment options of EoE include dietary elimination and swallowed steroids. Diet elimination approach could lead to identifying the trigger food(s), but it often requires repeated upper endoscopy with general anesthesia and potentially could negatively affect nutrition intake and growth of the child and individuals’ quality of life. Although the swallowed steroid treatment of effective, the EoE will universally recur after discontinuation of the treatment. Digestive Tea formula (DTF) has been used by the Traditional Chinese Medicine (TCM) practice to improve GI symptoms in EoE patients, including abdominal pain, GE reflux, and abnormal bowel movement. Previously, a flavonoid small molecule compound 7, 4 dihydroxy flavone (DHF) from This study comprehensively evaluates the potential therapeutic and immunological mechanisms underlying DHF improvement of symptoms related to EoE using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein-protein interaction analyses, in silico molecular docking and dynamic simulation followed by ex-vivo target validation by qRT-PCR using cultured human esophagus biopsy specimen with or without DHF from patients with EoE.Computational analyses defined 29 common targets of DHF on EoE, among which TNF-α, IL-6, IL1β, MAPK1, MAPK3 and AKT1 were most important. Docking analysis and dynamic simulation revealed that DHF directly binds TNF-α with a free binding energy of -7.7 kcal/mol with greater stability and flexibility. Subsequently, in the human esophagus biopsy culture system, significant reduction in levels of TNF-α, IL-6, IL-8 and IL1-β was found in the supernatant of biopsy sample cultured with DHF. Furthermore, the gene expression profile showed significant reduction in levels of TNF-α, IL1-β, IL-6, CCND and MAPK1 in the esophagus biopsy sample cultured with DHF.Taken together, the current study provides us an insight into the molecular mechanisms underlying multi-targeted benefits of DHF in the treatment of EoE and paves the way for facilitating more effective EoE therapies. EoE is a chronic, immune/antigen- mediated allergic disease of the esophagus that is characterized clinically by esophageal dysfunction and histologically by eosinophil-predominant inflammation (> 15 eosinophils/HPF). The common symptoms of esophageal dysfunction include vomiting, feeding difficulty, dysphagia, and abdominal pain. The incidence of EoE is increasing with estimated prevalence in the US ranging from 40-90 cases/100000  and middThe current therapy for EoE includes the use of swallowed steroid and dietary elimination . The dieGlycyrrhiza uralensis is one the most commonly used herbs in traditional Chinese medicine (TCM). We have shown that G. uralensis significantly inhibits TNF-α production - a flavonoid purified from oduction . Likewisoduction  and chemoduction . The regoduction  Recentlyoduction . Based oin silico molecular docking to estimate the potential therapeutic mechanisms underlying the efficacy of DHF in EoE. The entire workflow of the study is shown in ex vivo biological validation allowed us for the first time to identify potential therapeutic targets of DHF for EoE. This study provides guidance for our future in vivo experimental studies and future clinical trials.Given the complexity of pathological mechanisms of EoE and multiple targets of DHF on immunological responses, we utilized computational modeling—target mining, gene ontology enrichment, protein-protein interaction analyses, and Biological targets of DHF were identified from literature reports , 15 and Target enrichment gene ontology, pathway, and protein-protein interaction (PPI) analyses provided a molecular-level mechanistic insight into biological function. GO was introduced by mapping potential targets to the DAVID database , 36. TheWith obtained targets and significant pathways, C-T-P-D biological networks were constructed using Cytoscape (v3.2.1). This network, containing DHF, its related targets for EoE, and significant principal pathways were constructed to comprehensively elucidate the complex relationship among compound, targets, and disease related pathways. This analysis provided general information about pharmacological mechanisms of DHF for the treatment and management of EoE at a molecular level. The properties of C-T-P-D networks were validated by NetworkAnalyzer , a plugiThe binding modes of DHF with critical targets were predicted through molecular docking by AutoDock Vina . ProteinThe molecular dynamic simulations were carried out by Groningen Machine for Chemicals Simulations (GROMACS) with amber99sb-ildn force field and tip3p water model. 50 ns molecular dynamics simulation was performed for protein-ligand complex. The Root Mean Square Deviation (RMSD) analysis, and the toot-mean-square fluctuation (RMSF) were carried out using Xmgrace software.Pediatric subjects (aged 0-18 years old) with potential or known EoE were recruited under an IRB approved protocol at Mount Sinai Medical Center when undergoing an endoscopy as part of their routine clinical care. All subjects were determined to have active EoE based on clinical criteria including symptoms consistent with EoE and biopsies showing >15 eosinophils per high power field. Subjects ‘characteristics are listed in Biopsies were digested with DNases I and Collagenase IV as previously described48 and divided equally into two wells then cultured with or without DHF (0.02mg/ml) in complete RPMI with 10% FBS overnight. Cytokines were measured by ELISA  as per the manufacturer’s instructions.RNA was extracted from precipitation of esophageal biopsy culture using TRIZOL  followed by isopropanol precipitation. RNA was then reverse transcribed to complementary DNA (cDNA) using a PrimeScriptTM RT Reagent Kit . Real-time PCR was performed using SYBRTM Green Master Mix  as previously described . The tarThe Venn diagram shows that 440 target genes for EoE were selected. Likewise, 95 biological targets of DHF were collected from the literature , 15, andFurther, to clarify the potential pharmacological effects of DHF on EoE from the pathway level, the pathway enriched analysis was performed by connecting the predicted targets to DAVID database. The top 15 significant KEGG pathways with False Discovery Rate (FDR) <0.01 and genes associated with each pathway were ranked and picked out by enrichment score –log (FDR) as shown in The GO biological process terms in DAVID database were obtained with the identified targets as an enriched gene-set. The top 15 biological processes GO terms with False Discovery Rate (FDR) <0.01 were ranked by enrichment score (–log FDR) (The C-T-P-D network containing DHF, selected targets, top 15 pathways and EoE was developed to interpret the potential pharmacological mechanisms of DHF for management and treatment of EoE at the molecular level (The PPI network was constructed by mapping potential targets to the String database . The sizWe assumed that DHF regulated the crucial targets by interfering in gene expression or binding with proteins directly. Therefore, a molecular docking was introduced to calculate the binding energy and evaluate binding favorability between DHF, and crucial targets. The binding energy indicated that DHF might be a promising regulator for each of the selected targets with binding affinities between -6.3 to -7.7 Kcal/mol Table S2To provide experimental support and validate the claim that DHF could be an effective alternative for the management of EoE patients, we evaluated the effect of DHF on key therapeutic targets obtained from the computational analysis. We evaluated the levels of different pro-inflammatory cytokines by ELISA and their expression by qRT-PCR in human esophageal biopsy cultures with or without the presence of DHF. In presence of DHF, we found significantly lower levels of pro-inflammatory cytokines- TNF-α, IL-8, IL-6, IL-1B respectively (In this study we, for the first time, used a computational approach that integrated target mining, network technology and pathway analysis to reveal the potential therapeutic pathways and targets to understand pharmacological mechanisms of DHF in treating EoE. The cutting-edge technologies allowed us to uncover key mechanisms involved in the pathogenesis of EoE and further advance our knowledge into new therapeutic applications. It is believed that mechanism of EoE is strictly driven by complex interplay between genetic and early life environmental risk factors , interacAt present, dupilumab is the only the FDA approved treatment for EoE , and higThe current study uses computational approach to identify key targets which are potentially regulated by DHF in EoE. However, the key targets obtained using this approach are not primary drivers of eosinophilic esophagitis but based on the evidence on the literature the role of TNF in EoE has been actively investigated. It has been reported that the inflamed epithelial cells prime esophageal fibroblasts to secrete the profibrogenic cytokines IL-1β and TNF-α, which in turn promote epithelial-to-mesenchymal transition and esophageal fibrosis . AdditioThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by IRB approved protocol at Mount Sinai Medical Center. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Cnidium monnieri, has long been used to treat atopic dermatitis (AD) in China. However, the anti-AD compounds and mechanisms of CF are not fully understood. In this study, we evaluated the active compounds and molecular targets of CF in treating AD.Atopic dermatitis (AD) is a common inflammatory skin disease that compromises the skin's barrier function and capacity to retain moisture. Cnidii Fructus (CF), the dried fruits of The Traditional Chinese Medicine Systems Pharmacology database was used to acquire information regarding the compounds that occur in the herb. Targets of these compounds were predicted using the SwissTargetPrediction website tool. AD-related genes were collected from the GeneCards database. Gene ontology (GO) enrichment analysis and KEGG pathway analysis of proteins that are targeted by active compounds of CF and encoded by AD-related genes were performed using Database for Annotation, Visualization, and Integrated Discovery Bioinformatics Resources. A “compound-target” network was constructed and analyzed using Cytoscape Software. Molecular docking was performed using BIOVIA Discovery Studio Visualizer and AutoDock Vina.We identified 19 active compounds in CF, 532 potential targets for these compounds, and 1540 genes related to AD. Results of GO enrichment indicated that CF affects biological processes and molecular functions, such as inflammatory response and steroid hormone receptor activity, which may be associated with its anti-AD effects. KEGG pathway analyses showed that PI3K-Akt signaling, calcium signaling, Rap1 signaling, and cAMP signaling pathways are the main pathways involved in the anti-AD effects of CF. Molecular docking analyses revealed that the key active compounds in CF, such as (E)-2,3-bis(2-keto-7-methoxy-chromen-8-yl)acrolein, ar-curcumene, and diosmetin, can bind the main therapeutic targets AKT1, SRC, MAPK3, EGFR, CASP3, and PTGS2.Results of the present study establish a foundation for further investigation of the anti-AD compounds and mechanisms of CF and provide a basis for developing modern anti-AD agents based on compounds that occur in CF. Both males and females are afflicted, and the incidence varies according to race and ethnic group. In the United States, for example, the frequency is much greater among black children (19.3%) than among white children (16.1%) [Atopic dermatitis (AD)), also known as atopic eczema, is an inflammatory dry skin disease , 2. The  16.1%) , 6. Arou.1% , 6.  (16.1%) –9. The i (16.1%) , 11.AD is thought to be caused by a disruption of the epidermal barrier that allows allergens to penetrate the epidermis and activate dendritic and innate lymphoid cells, which then attract and activate Th2 cells . These aAD has no definitive cure, and current treatment consists primarily of topical anti-inflammatory agents such as corticosteroids and calcineurin inhibitors. Corticosteroids, powerful anti-inflammatory medicines, have been demonstrated to be beneficial for both acute and chronic AD , 16; howShe Chuang Zi in Chinese; common name, Monnier's snow parsley; henceforth referred to here as CF) is the dried fruit of Cnidium monnieri (L.) Cuss. The use of CF in China can be traced back to Shennong's Classic of Materia Medica (Shennong Bencao Jing) written in the Eastern Han Dynasty (25–220 AD), i.e., close to 2000 years ago. CF is traditionally used for treating female genitals, kidney deficiency, male impotence, and skin-related diseases [Cnidii Fructus  was used to obtain the active phytomolecules of CF [Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  [The potential targets of the active compounds of CF (with a probability score > 0) were predicted using the SwissTargetPrediction website tool  .https://www.genecards.org/, accessed on December 10, 2021) by searching using the keywords “atopic dermatitis”, “atopic dermatitis inhibiting” and “anti-atopic dermatitis” [The AD-related genes were collected from GeneCards®: The Human Gene Database . The common molecules were considered to be potential anti-AD targets of CF.Proteins that are compound targets and are encoded by AD-related genes were identified using the Venny 2.1 online software  [The protein–protein interaction (PPI) between the potential anti-AD targets of CF was then analyzed by using the STRING database  . The con2, 2021) .To analyze the interaction between active compounds and the anti-AD targets of CF, compound-target networks were constructed using Cytoscape Software  .https://david.ncifcrf.gov/, accessed on December 16, 2021) [http://www.bioinformatics.com.cn/), bubble plots of bioprocesses and pathways were drawn. Classical hypergeometric test was used to determine statistical significance. The adjusted p-value < 0.05 was utilized as the significance threshold in our investigation after utilizing the Benjamini–Hochberg method to control the false discovery rate (FDR) for multiple hypothesis testing [Gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out using the DAVID database for annotation, visualization, and integrated discovery  –37. The  testing .https://pubchem.ncbi.nlm.nih.gov/) online database. Their three-dimensional (3D) structures were constructed using BIOVIA Discovery Studio Visualizer 2021 and were saved in PDB format. Protein crystal structures of the potential targets  were obtained in PDB format from the Protein Data Bank (https://www.rcsb.org/) [Two-dimensional (2D) structures of active compounds of CF were acquired in Spatial Data File (SDF) format from the NCBI PubChem (sb.org/) . PDB IDssb.org/) , 40. Thesb.org/) . The docsb.org/) . A bindisb.org/) .A total of 114 compounds of CF were extracted from the TCMSP. Further active compound screening was carried out based on OB ≥ 30% and DL ≥ 0.18; from this, 19 active compounds were obtained Fig. a as listTo obtain direct targets of the 19 active compounds of CF, ligand-based target prediction was performed using the SwissTargetPredictaion website tool. A total of 1387 potential targets (probability score > 0) were obtained. After eliminating redundancy, 532 predicted potential targets were selected for further analysis  file and then imported to Cytoscape software 3.9.0 to obtain the PPI network. The results demonstrated that the network involved 175 nodes and 3748 edges, in which the average shortest path length value between all node pairs in the network was 2.148 with a network radius value of 3. Figure A network displaying the 19 active compounds  was conducted. Results are presented in Table Binding affinity between a compound and a protein is represented by bingding energy score. The lower the energy, the better the ligand-receptor protein binding is . DockingThe present study explored the active compounds and molecular mechanisms of CF in treating AD. AD is the most common inflammatory skin condition, and multiple signaling pathways potentially contribute to the disease's aetiology simultaneously.Out of a total of 114 compounds, 19 active compounds with OB ≥ 30% and DL ≥ 0.18 were selected Table . The tenThe PPI analysis revealed that numerous genes are involved in CF’s effects in treating AD, including AKT1, MAPK3, SRC, EGFR, CASP3, and PTGS2. Reports have demonstrated that inhibiting AKT1 in the skin results in a shift in protease expression, a drop in filaggrin expression, and ultimately, the disintegration of the barrier function of the skin , 43. IL-GO enrichment analysis demonstrated that compounds in CF may affect a variety of biological processes, including inflammatory response, response to lipopolysaccharides, protein phosphorylation, positive regulation of the ERK1 and ERK2 cascade, and negative regulation of the apoptotic process, all of which may be associated with its anti-AD effects Fig. . AlthougThe KEGG pathway analysis results demonstrated that anti-AD key active compounds of CF act on several signaling pathways, including PI3K-Akt signaling pathway, calcium signaling pathway, Rap1 signaling pathway, cAMP signaling pathway, chemokine signaling pathway, TNF signaling pathway, and inflammatory mediator regulation of TRP channels Fig. . PI3K-AkThe present study explored the compounds and molecular mechanisms active in the therapeutic application of CF to treat AD. In this research, 10 key active compounds of CF and 66 anti-AD core targets were identified. Our study revealed that the underlying mechanisms of anti-AD effects of CF are likely to involve inhibition of the inflammatory response, response to lipopolysaccharides, protein phosphorylation, negative regulation of apoptotic processes, and positive regulation of the ERK1 and ERK2 cascades. Further, we found that four key signaling pathways are likely to be involved: PI3K-Akt signaling pathway, calcium signaling pathway, Rap1 signaling pathway, and cAMP signaling pathway. Our results justify the conclusion that the anti-AD effects of CF might be due to the direct or indirect synergistic effects of multi-target and multi-pathway efforts. Molecular docking results showed that key active compounds of CF can potentially bind to AKT1, SRC, MAPK3, EGFR, CASP3, and PTGS2. Results of the present study establish a foundation for further investigation of the anti-AD compounds and mechanisms of CF and provide a basis for developing modern anti-AD agents based on compounds that occur in CF.


Paeonia lactiflora Pall.(PLP) is a traditional Chinese medicine with various pharmacological effects such as anti-tumor, anti-inflammatory, and immune regulation. Previous studies have shown that PLP has potential anti-allergic activity. However, there is still no comprehensive analysis of the targeted effects and exact molecular mechanisms of the anti-allergic components of PLP. This study aimed to reveal the mechanism of PLP. in the treatment of type I allergy by combining network pharmacological methods and experimental verification.The incidence of allergic reaction is increasing year by year, but the specific mechanism is still unclear. First, we used the traditional Chinese medicine systems pharmacology (TCMSP) database and analysis platform to screen the main components and targets of PLP, and then used databases such as GeneCards to retrieve target information related to ‘allergy’. Protein–protein interaction (PPI) analysis obtained the core target genes in the intersection target, and then imported the intersection target into the David database for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis. Furthermore, the therapeutic effect of paeoniflorin, the main component of PLP, on IgE-induced type I allergy was evaluated in vitro.GO analysis obtained the main biological processes, cell components and molecular functions involved in the target genes. KEGG analysis screened out MAPK1, MAPK10, MAPK14 and TNF that have a strong correlation with PLP anti-type I allergy, and showed that PLP may pass through signal pathways such as IgE/FcεR I, PI3K/Akt and MAPK to regulate type I allergy. RT-qPCR and Western Blot results confirmed that paeoniflorin can inhibit the expression of key genes and down-regulate the phosphorylation level of proteins in these signal pathways. It further proved the reliability of the results of network pharmacology research.The results of this study will provide a basis for revealing the multi-dimensional regulatory mechanism of PLP for the treatment of type I allergy and the development of new drugs.The online version contains supplementary material available at 10.1186/s12906-022-03677-z. The incidence and mortality of allergic diseases is increasing, and has become a common disease, which greatly affects people's life and physical health. But so far, people have not revealed its exact pathogenesis, and there is no ideal treatment method. At present, glucocorticoid and antihistamine are commonly used clinically to treat allergy, but the curative effect is short and there are many adverse reactions after long-term use . TherefoTCM has been used for the treatment of allergic diseases with long history. But due to the complex chemical components and pharmacological effects of TCM, its specific effective substance basis and mechanism are still unclear, which brings huge challenges to the study of the mechanism of TCM to treat allergy. For the past few years, with the in-depth research of TCM and the development of related technologies, the use of TCM in treatment of allergic diseases has gained great recognition and breakthroughs. It has been found that many TCMs and their components have therapeutic effects on allergy, such as Polydatin, Glycyrrhizic acid and Quercetin –5. TreasThe medicinal part of PLP is its dried root, and it has many pharmacological effects such as protecting liver, nerve and heart, anti-tumor, anti-inflammatory and immune regulation. The main active ingredient of PLP is Paeoniflorin (Pae) , 7. StudNetwork pharmacology is a research method based on multi-directional pharmacology and systems biology, which can analyze the relationship between drugs and diseases at the overall level. Network pharmacology is based on the drug-target-disease network, so as to systematically explore the specific mechanisms of drug to treat diseases. Its greatest advantage is the integration of holistic, dynamic and analysis, which is consistent with the holistic and dialectical treatment principles of TCM .Consequently, our research was based on the network pharmacology to systematically analyze the active ingredients of PLP, allergy-related targets and their pathways to identify potential drug targets and mechanisms. Type I allergy is the most common type of allergy in clinical practice. We used cell models and in vitro experiments to explore the effects and related mechanisms of Pae, the main active ingredient of PLP, in treating type I allergy. Most reports on the relationship between Pae and allergy only focused on showing the inhibitory effects of this compound and lacked in-depth exploration of the underlying mechanism , 12.TherRBL-2H3 cells were obtained from the ATCC. PrimeScript™ RT reagent Kit, TB Green Kit were purchased from Takara . The finished product of Paeoniflorin  were purchased from Solarbio .https://tcmspw.com/tcmsp.php), and all known chemical components contained in the PLP have been retrieved and screened for potential activities, that is, OB ≥ 30%, DL ≥ 0.18. According to the active ingredients obtained after screening, the TCMSP database is used again to retrieve its target.Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) is a database established based on the framework of TCM system pharmacology, providing 12 important pharmacokinetic properties, such as oral bioavailability (OB) and drug-likeness (DL), which is mainly used to screen and evaluation of pharmaceutical compounds. OB is an important indicator for evaluating whether a drug can be developed, and OB ≥ 30% is considered to have better oral bioavailability. DL can evaluate the possibility of a compound becoming a drug, and DL ≥ 0.18 is considered to have high drug-likeness and may become a new drug . Our methttps://www.genecards.org/) database is not only a database that can provide concise genome, proteome, transcription, inheritance and function of all known and predicted human genes, but also an analytical database that combines retrieval, integration, search and display of the information of the human genome [http://omim.org/) catalogs the genetic components of all known diseases and links them with related genes in the human genome when possible. It provides a reference for further research and genomic analysis tools of cataloging genes [The GeneCards (n genome . The OMIng genes . In theshttp://bioinformatics.psb.ugent.be/webtools/Venn/) to take the intersection of the targets obtained in 2.2.1 and 2.2.2, and imported it into the String database (https://string-db.org/). Then used ‘Multiple proteins’ function to establish the PPI network, selected the species as ‘Homo sapiens’, and clicked ‘SEARCH’ and ‘CONTINUE’ options to get the PPI network.Used the Draw Venn database (https://david.ncifcrf.gov/) can be used for enrichment analysis of a great quantity of sample genes and proteins, also can simultaneously provide systematic and comprehensive biological information. Through the integration and analysis of information, we can intuitively show the pathway enrichment of target genes, which has become one of the indispensable tools of bioinformatics research. Imported the target obtained in 2.2.3 into the David database for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. GO analysis is a description of genes in different dimensions and levels, which includes three aspects: biological process (BP), cell component (CC) and molecular function (MF). KEGG is a database that links gene catalogs obtained from fully sequenced genomes with system functions of higher-level cell, species, and ecosystem. KEGG analysis discovers the pathways of drug targets by enriching target genes, thereby obtaining the mechanism of drug treatment of diseases [The David database  for 24 h, each group was sensitized with 1 mL of DNP-IgE (0.2 μg/mL). After 12 h, drug groups were replaced with 2 mL of the corresponding drug respectively . After 1 h, in addition to the normal group, 400 μL of DNP-BSA (0.4 μg/mL) was added for stimulation. After 30 min, extracted total protein and measured its concentration.Our experimental method was performed with reference to relevant literature , 20, andThe experiment used 8% separating gel, 4% stacking gel, and loaded 30 μg protein sample. After electrophoresis, the cut gel was transferred to the PVDF membrane. The PVDF membrane was blocked with shaking at room temperature for 1 h. After incubation with primary antibodies of Lyn, p-Lyn, Syk, p-Syk and β-actin at 4℃ overnight, the secondary antibodies were incubated at room temperature for 1 h. Visualization was performed by using the ChemiScope Mini 3300 and density analysis was performed with Image J software.The steps were the same as 2.3.1. Then extracted total RNA, removed gDNA from RNA and performed reverse transcription by using PrimeScript™ RT reagent Kit. Used TB Green kit for qPCR reaction. The key genes tested include: Lyn, Syk, Fyn, PLCγ, PI3K, Akt, p38, ERK, JNK, p65 and GAPDH.p < 0.05).Results were expressed as the mean ± SD. ANOVA in SPSS 17.0 software was used to assess significant differences between groups .Imported the two target sets obtained in 2.2.1 and 2.2.2 into the Draw Venn database to obtain the intersection Fig. .Fig. 1In2+ and triglycerides and so on. The process with the number of genes ≥ 10 is binding of Ca2+. The visual processing was showed in Fig. Imported the obtained 50 intersection targets into the David database for GO and KEGG analysis. As shown in Table http://metascape.org/gp/index.html), and obtained the bubble chart of related pathways of PLP anti-allergic effect . The inhibitory effect of 5 μg/mL Pae on ERK, p38 and p65 was stronger than Keto group Fig. .Fig. 10EThe characteristics of multi-component, multi-target and the interaction of each component of TCM make it a complex system, and network pharmacology is a more comprehensive and systematic research technology that aims to reveal the complexity of biological systems, drugs and diseases, which has certain similarities with TCM, and is becoming a hot spot in TCM research . Zhang ZAs one of the TCMs that can be used in dietary supplement, PLP has been found to have anti-inflammatory, anti-tumor and immune regulation effects. So it has been widely used to treat many diseases. PLP is often combined with other TCMs in the treatment of allergy. Shaoyao Gancao Decoction and Xiaoqinglong Decoction are classic prescriptions with anti-allergic effects and have good therapeutic effects, and both contain PLP. Therefore, it is speculated that PLP may have anti-allergic activity, but the mechanism of its treatment of allergy has not been fully understood. However, considering that PLP has the characteristics of multiple components and multiple targets based on the theory of TCM, experimental research alone cannot systematically reveal the biological mechanism of PLP anti-type I allergy, and the holistic characteristics of network pharmacology are suitable for this research. Different from previous studies, this research used network pharmacology to predict the efficacious ingredients and key mechanisms of PLP anti-type I allergy, and then conducted in vitro experiments for verification.2+ and triglycerides and so on, among which the Ca2+ concentration is closely related to the occurrence of type I allergy. KEGG analysis obtained 31 related pathways of PLP anti-allergy, including the FcεR I signal pathway that is closely related to type I allergy, which researchers are familiar with, indicated that PLP has the potential to treat allergy, and also verified the reliability of network pharmacological analysis. The results concurrently showed that PLP may regulate allergy through signal pathways such as MAPK, TNF, PI3K/Akt, apoptosis and Th cell differentiation.The TCMSP database contains 499 TCMs included in the Chinese Pharmacopoeia and their 29,384 components, 3311 targets and 837 related diseases. Each component provides pharmacokinetic data, as well as potential targets and related disease information, so that the relationship network of drug-target-disease can be obtained, which provides a new platform for the in-depth study of the pharmacological mechanism of TCM . In ordeThe obtained network of drug-target-pathway contains 52 nodes and 153 edges, among which MAPK 1, MAPK 10, MAPK 14 and TNF have high topological metrics and may be key targets. Combined with the results of KEGG analysis, it is found that these four important targets are distributed in the FcεR I signal pathway. MAPK 1, MAPK 10, and MAPK 14 belong to the MAPK family and are the integration points of many biochemical signals. They regulate cell proliferation, differentiation, and transcriptional regulation, and are closely related to multiple signal pathways involved in the regulation of allergy. TNF is related to various diseases such as allergy, autoimmune diseases, and tumors. Therefore, it is speculated that PLP may exert its inhibitory effect on allergy mainly through these targets and FcεR I signal pathway, and Pae, as the main component of PLP, may also inhibit the degranulation of mast cells (MC) by acting on these targets and pathways, and then play a therapeutic effect on type I allergy. Furthermore, the research on the chemical components and mechanism of PLP used for immune regulation and anti-inflammation is mainly focused on Pae , 25, so max) of Pae and PM-I were 0.21 and 2.05 mg/L, respectively. In addition, the study also found that Baishao decoction  was administered to rats, and the Cmax of PG was as high as 8 mg/L. The peak time (Tmax) of PM-I and PG were 3.0 h and 10 min, respectively. Pae has strong hydrophilicity, weak lipophilicity, and weak transmembrane absorption ability, but it can quickly reach the brain tissue through the blood–brain barrier. The mean AUC of Pae was 615.7 mg/min·L. Pae is less affected by liver metabolism, but can be degraded by glycosidases and anaerobic bacteria in intestine [In addition to the OB values mentioned above. Studies have reported that the absorption permeability and absorption rate of Pae are approximately the same between various sites in the small intestine. And the absorption mechanism is passive diffusion. After oral administration of Pae, it is mainly absorbed in the form of metabolites of paeonimetabolin-I (PM-I) and paeoniflorgenin (PG). Shaoyao Gancao Decoction  was administered to rats, and the peak plasma concentrations (Cntestine . At pres2+ channels and inhibit IgE synthesis. Thus, it is often used as a positive control drug in anti-allergy experiments.RBL-2H3 cells possess the biological characteristics of MCs. And RBL-2H3 cells are used as the classic model for studying degranulation reaction in vitro. Therefore, after considering various factors, we finally chose RBL-2H3 cells as the cell model. To improve the reliability of the results, we chose Keto as the positive control drug. It has a strong anti-allergic effect, and can inhibit the release of allergic mediators from MCs and stabilize their membranes. Keto can also block CaAccording to different pathogenesis, allergy can be divided into 4 types, among which type I allergy is the most common in life . The patThe classic IgE/FcεR I signal pathway includes Syk, Lyn and Fyn, among which Lyn and Syk as initial signals to participate in the activation of MC, and they have become key therapeutic targets for allergic diseases. Activated Syk can finally activate PLCγ and PI3K, which can cause the degranulation of MC , 29. FynWhen the IgE/FcεR I signal pathway is activated, it will directly or indirectly activate the MAPK and NF-κB signal pathways , 33. MAPIn summary, it was speculated that MAPK 1, MAPK 10, MAPK 14 and TNF may be the key targets of PLP to treat allergy. By interacting with these targets, PLP regulates FcεR I, MAPK, TNF, PI3K/Akt and Th cell differentiation and other signal pathways to participate in the occurrence and development of type I allergy Fig. . MoreoveAdditional file 1: Fig.S1. Original image of the expression of Lyn in RBL-2H3 cells detected by Western Blot.Additional file 2: Fig.S2. Original image of the expression of p-Lyn in RBL-2H3 cells detected by Western Blot.Additional file 3: Fig.S3. Original image of the expression of Syk in RBL-2H3 cells detected by Western Blot.Additional file 4: Fig.S4. Original image of the expression of p-Syk in RBL-2H3 cells detected by Western Blot.Additional file 5: Fig.S5. Original image of the expression of β-actin in RBL-2H3 cells detected by Western Blot.


Based on network pharmacology and molecular docking, this study discusses the potential mechanism of Gancao Xiexin decoction in the treatment of Behçet disease (BD) to provide a more reliable theoretical basis for the treatment of BD.The effective components and action targets of Gancao Xiexin decoction were obtained by searching the traditional Chinese medicine systems pharmacology database and analysis platform database, and the genome annotation database platform (GeneCards) database Search BD related targets in Online Mendelian inheritance in man database, pharmacogenetics and pharmacogenomics knowledge base database, therapeutic target database and drugbank database, Venny analysis tool was used to overlap drug targets and disease targets; The “active drug active ingredient target gene” network was constructed with the help of Cytoscape 3.8.2 software, and the protein–protein interaction (PPI) network was constructed with string database; R language was used for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis; Target prediction based on pubchemp platform.A total of 163 active components were identified, with 730 corresponding targets, including 56 common targets of the active components and BD. GO enrichment analysis yielded 1126 entries for biological processes (BP), 17 entries for cellular components, and 89 entries for molecular functions. The significant items of BP enrichment mainly included reaction to lipopolysaccharide, reaction to bacteria-derived molecules, exogenous apoptosis signal pathways, and biological metabolism processes of reactive oxygen species. KEGG pathway enrichment analysis identified 118 significantly enriched pathways. The molecular docking technology verified that its effective components can effectively bind to the corresponding BD related target proteins.Gancao Xiexin decoction synergistically treats BD through multi-component, multi-target, and multi-channel mechanisms, which provides a basis for further study of the active components and mechanism of Gancao Xiexin decoction. At present, it is considered to be closely related to genetic, infectious, immune, and environmental factors. There is no effective cure for this disease. The traditional use of drugs, including glucocorticoids, immunosuppressants, non-steroidal drugs, and biological agents, is to control existing symptoms and disease activities, prevent and treat damage to important organs, reduce disease recurrence, and delay the development of the disease. However, BD often cycles from onset to remission and recurrence. The long-term use of hormones and immunosuppressants will inevitably have an impact on the body. Therefore, traditional Chinese medicine methods are sought to further understand the characteristics and curative effects of the disease. There is a discussion on “Fox confusion disease” in Treatise on febrile diseases, which holds that BD is similar. When the heat evil is multiplied, the throat swells and aches, and when wet evil is injected, the two Yin will fester. This can also be seen in Article 10 of the synopsis of the golden chamber. Treatment of Yin-Yang toxin disease and pulse syndrome of lily and fox: “Fox confusion is a disease. Gancao Xiexin decoction is the main”. This disease is characterized by the accumulation of dampness and heat. Dampness blocks heat depression and damages blood collaterals. This disease is based on the accumulation of dampness and heat. Dampness blocks heat depression and heat hurts blood collaterals.The “Gancao Xiexin decoction” is based on the theory of the Qi mechanism mediated by Zhongjiao, pay attention to the regulation of the lifting hub of the coke gas machine in the spleen and stomach. In modern clinical application, Gancao Xiexin decoction has significant curative effects on BD, recurrent oral ulcer, ankylosing spondylitis, rheumatoid diseases and other diseases have significant curative effects.Behçet disease (BD) is a chronic systemic vascular inflammatory disease involving the nervous system, digestive tract, lungs, kidneys, epididymis, and other organs, and is characterized by recurrent oral ulcers, vulvar ulcers, ophthalmia, and skin damage.Based on the traditional Chinese medicine systems pharmacology and analysis platform database, this study screened out the main effective components of traditional Chinese medicine in Gancao Xiexin decoction and potential intersection targets with BD and used network pharmacology to explore the mechanism of Banxia Xiexin decoction in the treatment of BD, providing a more reliable theoretical basis for treatment.The main traditional Chinese medicine components of Gancao Xiexin decoction “licorice,” “Scutellaria baicalensis,” “ginseng,” “Coptis chinensis,”“dried ginger,” “jujube” and “Pinellia ternate” were searched through traditional Chinese medicine systems pharmacology and analysis platform database. They were screened according to the criteria of bioavailability (OB) greater than 30% and drug likeness (DL) >0.18. The active components and corresponding targets of seven traditional Chinese medicines were obtained by using the Perl language.The disease target genes related to BD were retrieved from the GeneCards database, Online Mendelian inheritance in man database, pharmacogenetics and pharmacogenomics knowledge base database, therapeutic target database, and DrugBank database with “Behcet disease” as the keyword. The related genes were input as potential targets for analyzing the active components in Gancao Xiexin decoction in the treatment of BD.Using R’s Venndiagram package, the drug disease common target obtained by the above method is drawn into a Venn diagram. The active components in Gancao Xiexin decoction and the action target for the treatment of BD were introduced into Cytoscape 3.8.2 at the same time, and the active component and action target network were constructed.The potential target genes of the Gancao Xiexin decoction for BD were introduced into the string database. The species was defined as “Homo sapiens,” and the target protein interaction relationship was retrieved and saved in TSV format. The TSV file into Cytoscape 3.8.2 software again, and the network analyzer tool for network analysis was used to obtain the degree value. A protein‐protein interaction (PPI) network was drawn using Cytoscape 3.8.2 software.P < .05) were screened, and the top 20 BP and pathways were selected according to the gene proportion (gene ratio), gene enrichment histogram and bubble diagram were drawn, and pathway information map was obtained to find relevant targets of Gancao Xiexin decoction in the treatment of BD.Using the dose, clusterprofiler, ggplot2, and pathview packages of R, Gene Ontology (GO) enrichment analysis and metabolic pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) were carried out for the component target proteins of Gancao Xiexin decoction. The biological processes (BP) and pathways  subtype processes, 1126 biological processes (BP) subtype processes and cellular component (CC) related to the treatment of BD with Gancao Xiexin decoction There are 17 subtype processes. Select the top 20 BP in each subtype according to the degree value, and list the go gene enrichment degree histogram in  in human peripheral blood monocytes-α). The proliferation and gene level of IL-1 are decreased, while the expression of inflammatory factors such as IL-6 and matrix metalloproteinases (MMP3 and MMP9) can inhibit the production of inflammatory mediators and improve inflammatory symptoms. Animal experiments have shown that quercetin can improve pain symptoms by inhibiting the activation of MMP-9 and MMP-2 in microglia in the mouse spinal cord. Levels of interleukin-17 (IL-17) and monocyte chemoattractant protein-1 (MCP-1). Quercetin has antioxidant, free radical scavenging, anti-apoptotic, and immune regulatory functions. Baicalein is an inhibitor, of antibacterial xanthine oxidase, antioxidants, and immune regulation. It can effectively inhibit the itching of free radicals produced during xanthine oxidation. Its hydroxyl structure also has free radical scavenging activity, and through the regulation of the peanut tetraenoic acid metabolic pathway, it plays a powerful antipyretic and analgesic effect by inhibiting the production of cyclooxygenase, dehydrogenase, nuclear factor, and cytokines. Animal experiments have shown that baicalein can inhibit the macrophage inflammatory response induced by lipopolysaccharide and adenosine triphosphate. Ginsenoside Rh2 is a tetracyclic triterpene saponin monomer. In addition to promoting the apoptosis of tumor cells, it also has significant anti-inflammatory effect, which can inhibit the inflammatory response induced by bacterial endotoxin (LPS). Bi reported that rh2-b2 modified with water-soluble groups significantly inhibited the TNF of RAW264.7 macrophages-α, IL-6, IL-1β. In addition, Rh2 can inhibit the production of PGE2, ROS, and MMP-9 in LPS stimulated RAW264.7 macrophages and TNF in keratinocytes-α induced MMP-9 gelatinase activity.Quercetin, wogonin, baicalein, and ginsenoside are the main active components of the Gancao Xiexin decoction. Quercetin is a flavonoid with chemopreventive and therapeutic effects in a variety of diseases, including inhibition of oxidative stress, cytokines and COX2 production. it promotes inflammatory cell drive and plays a key role in the pathogenesis of BD. PTGS2, also known as cyclooxygenase (cox1/2), is a key rate limiting enzyme for the conversion of arachidonic acid to prostaglandins. Meltem believes that the increased activity of (cox1/2) in plasma may be the cause of intravascular inflammation in patients with BD. IL1β is a proinflammatory cytokine, that can continuously promote the release of endogenous arachidonic acid, resulting in the persistent hypersensitivity of mechanical nociceptors, and the increase in inflammatory cytokines is involved in the process of vascular injury caused by BD. MMP-9 plays an important role in immune inflammation, cell migration, proliferation, and apoptosis. It was found that MMP-9 polymorphism is related to BD susceptibility. In the inflammatory state, immune cells adhere to the surface of endothelial cells by secreting inflammatory factors and vascular cell adhesion molecule 1, inducing their expression of MMP-9, and promoting the cross endothelial cell surface of immune inflammatory cells, upregulating of MMP-9 activity. As a chemotactic protein, CCL2 can chemotactic monocytes to specific tissues to eliminate foreign pathogenic microorganisms, produce antibodies and promote the repair of tissue damage; Found its polymorphism in erythrocytes of patients with BD.Through the analysis of drug disease action targets, 56 common targets were obtained from the intersection, including 19 main targets and five key targets, namely CXCL8, PTGS2, IL1β, MMP9, and CCL2, and docking them with the active components of Gancao Xiexin decoction. CXCL8 is an inflammatory chemokine that has several of biological functions. Studies have shown thatFrom the enrichment analysis results, it can be seen that the bioactive components of Gancao Xiexin decoction act on multiple targets to treat BD in a variety of ways, involving a variety of signaling pathways, such as lipid and atherosclerosis, fluid shear stress, atherosclerosis, AGE-RAGE signal pathway, TNF signal pathway, and IL-17 signaling pathway. Studies have shown that the AGE-RAGE pathway can participate in the regression of inflammation, maintenance of intracellular homeostasis, and repair and regeneration after injury and that TNF-α mediates cell necrosis and apoptosis in a variety of ways and plays an important role in the occurrence and development of BD.In summary, this study applied network pharmacology to screen the bioactive components of Gancao Xiexin decoction and its effective action targets in BD, which reflects that the Gancao Xiexin decoction has the characteristics of multi-target and multi-channel treatment, provides ideas for the follow-up study of its action mechanism in the treatment of BD, and provides new scientific connotations to traditional Chinese medicine compounds at a new level. In addition, more clinical trials and animal trials are needed for further verification.Conception and design of study: ZX.Data collection: ZX.Drafting the manuscript: ZX.The author reviewed the results and approved the final version of the manuscript.


Explore the potential molecular mechanisms behind the therapeutic functions of Gualou Niubang decoction (GLNBD) in the treatment of plasma cell mastitis (PCM) by network pharmacology and molecular docking.  GLNBD is a formula of Chinese traditional medicine consisting of 12 herbs. The potential active ingredients of GLNBD and their target genes were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database, and PCM-related target genes were obtained from GeneCards, OMIM, and NCBI databases, using R language to obtain intersection targets; then, the STRING database and Cytoscape software were used to establish protein-protein interaction networks and herb ingredient target networks. DAVID was used to perform GO and KEGG pathway enrichment analyses on the intersection target. PyMoL-2.5.0 and AutoDock Tools-1.5.6 were used to verify the molecular docking.  164 ingredients and 58 intersection targets were obtained in the treatment of PCM by GLNBD. Four key active compounds and four key proteins were identified. Then, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that biological functions of potential target genes were associated with negative regulation of the apoptotic process, response to hypoxia, positive regulation of transcription, and DNA-templated, with related pathways involving the pathway in cancer, phosphatidylinositol 3-kinase (PI3K) Akt signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. Moreover, the binding activities of key target genes and essential active compounds of Chinese herbal medicines in GLNBD were further validated by molecular docking. The results showed that the docking results were stable and had good binding ability.  This study suggested that four potential key active components, including quercetin, luteolin, fisetin, and kaempferol, were identified in GLNBD, which could interact with ALB, EGFR, IL-6, and VEGFA modulating the activation of the pathway in cancer, PI3K-Akt pathway, and AGE-RAGE signaling pathway in diabetic complications. Plasma cell mastitis (PCM), also known as mammary gland duct dilation or occlusive mastitis, is a kind of chronic nonbacterial mammary gland inflammation. The incidence of PCM is mainly in nonpregnant and nonbreastfeeding women and occasionally occurs in males; the incidence rate of breast benign diseases is 4.1%~5.5 . The incGualou Niubang decoction (GLNBD) is derived from “Yizong Jinjian,” which is used to treat breast cellulitis and acute mastitis. The original formula is composed of Gua Louren (Fructus Trichosanthis), Niu Bangzi (Fructus arctii), Tian Huafen (Trichosanthin), Huangqin , Zhizi (Gardenia), Lianqiao (Forsythia suspensa Vahl), Zaoci (Gleditsia sinensis), Jin Yinhua (honeysuckle), Gancao (licorice), Chenpi (dried tangerine peel), Qingpi (Pericarpium Citri Reticulatae Viride), and Chaihu (Radix bupleuri). Modern pharmacological research shows that Niu Bangzi has antibacterial, antiviral, antitumor, and other functions . HuangqiNetwork pharmacology was first proposed by British pharmacologist Andrew T·Hopkins in 2007 and described as a next-generation drug development model in 2008 , which ihttps://old.tcmspe.com/tcmsp.ph) [http://bidd.group/TCMID/) database [https://pubchem.ncbi.nlm.nih.gov/) [All chemical ingredients and their related targets of each herb of GLNBD were collected from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) (cmsp.ph)  and TCM-database . Ingrediih.gov/) .https://www.genecards.org/) [https://www.ncbi.NLM.Nih.gov/) [www.omim.org/) database [“Plasma cell mastitis”, “mastitis obliterans”, and “mammary duct ectasia” were searched as the search terms in GeneCards (ds.org/) , NCBI (hih.gov/) , and OMIdatabase , which wValidated human species-associated target genes were selected in the Uniprot database , and genhttps://string-db.Org/) [The intersection target genes were imported into the STRING database (db.Org/) , and a pdb.Org/) . To map The network of the visualization of GLNBD was generated by importing the two files above into Cytoscape 3.7.2. The top four active ingredients were selected as key ingredients based on the degree. Degree indicated the number of edges between a single node and other nodes in a network.DAVID was used to analyze the GO biological process and KEGG signal pathway enrichment of intersection target proteins and obtain GO and KEGG signal pathways of GLNBD in the treatment of PCM.http://www.pdb.org/) database [The top four key active compound mol2 format files were downloaded from the ZINC database, and small molecules were extracted using Python. After removing the redundant water molecules from the protein crystal structure and adding hydrogen atoms, we batch processed them into pdbqt files with Openbable to complete ligand preparation. Receptor files were prepared by downloading pdb format files of four key targets from the Protein Data Bank (PDB)  determined by topology analysis , Table 5In this study, the main active components of 12 traditional Chinese medicines of GLNBD were obtained according to TCMSP and TCM-ID database, ingredient screening, and target prediction; 164 ingredients of GLNBD were collected, with 222 drug corresponding treatment targets and 997 PCM disease-related targets. After intersection, 58 target genes were obtained, and then, GO and KEGG pathways were further enriched and analyzed.κB signaling pathway, thus playing a role in controlling bovine mastitis [κB and MAPK signaling pathways mediated by TLR4-MyD88 [κBα and NF-κB P65, regulate the expression of MMP2 and MMP9, and prevent mastitis [α by enhancing the expression of arginase (ARG-1) IL-10. The expression of IL-6 iNOS can also change the polarization of macrophages, thus playing an anti-inflammatory role [κB inflammatory pathway, thereby reducing the expression level of TNF-α and IL-6 and controlling the occurrence of inflammation [α and ANGPTL2 in cells, prevent the occurrence of mastitis in mice, inhibit the phosphorylation of NF-κB P65 subunit and the degradation of IκBα, and play a therapeutic role in mastitis [The active components of quercetin, luteolin, fisetin, and kaempferol were predicted by network visualization. Quercetin is a dietary flavonoid with anti-inflammatory, antioxidant, and antifungal properties and has certain effects on inflammation, angiogenesis, and vascular inflammation. Experiments have confirmed that quercetin can enhance the transcriptional activity of NRF2-ARE by inhibiting the NF-mastitis . QuercetR4-MyD88 . The antR4-MyD88  Luteolinmastitis . Luteoliory role . Luteoliory role , 33. Expory role . Fisetinory role . Fisetinammation . In vivoammation –40. Kaemmastitis .PPI network analysis showed that the key genes were ALB, IL-6, EGFR, and VEGFA. ALB has been mentioned in many inflammatory studies, and the expression of the ALB gene can also be detected in peripheral blood monocytes. For example, ALB is lower than normal in patients with lymph node tuberculosis, and neutrophil ratio and red blood cell distribution width are closely related to ALB in patients with systemic lupus erythematosus (SLE); the expression of the ALB gene can reflect the function of differentiated liver cells in patients with hepatitis , 43. In According to GO function enrichment analysis, BP is negative regulation of apoptotic process, response to hypoxia, positive regulation of transcription, DNA-templated, extracellular matrix disassembly, positive regulation of gene expression, and wound healing. MF is protein binding, identical protein binding, protein heterodimerization activity, enzyme binding, sequence-specific DNA binding, and BH3 domain binding. It can be seen that the treatment of PCM with GLNBD is related to the binding of protein; the inflammatory process is related to a variety of binding proteins, such as the binding protein is related to the airway inflammatory response of children's asthma . Bindingα, and causes the destruction of tissue cells [κB nuclear translocation, while inflammatory factors such as TNF-α and IL-6 were also inhibited [The enrichment analysis of the KEGG pathway showed that the possible signal pathways for the treatment of PCM by GLNBD were the AGE-RAGE signaling pathway in diabetic complications and PI3K/Akt signal pathway. Studies have confirmed that after activation of the AGE-RAGE signaling pathway, a large amount of VEGF will be secreted and released to promote the generation of new blood vessels and contribute to the repair of PCM tissue. It also causes the expression and release of a large number of proinflammatory cytokines, such as IL-6 and TNF-ue cells , 54. AGEue cells . Akt actnhibited ; therefonhibited . PCM tisnhibited . The actnhibited . RNA-SEQnhibited . OverallIn this study, the interactions of 164 bioactive compounds and 58 potential targets for GLNBD treatment of PCM were identified through network pharmacological analysis. These targets are closely related to biological processes such as negative regulation of apoptosis, hypoxia response, and wound repair. The AGE-RAGE signaling pathway and PI3K/Akt signal pathway in diabetic complications are involved in these biological processes. The binding ability of quercetin, luteolin, fisetin, and kastricol to ALB, IL-6, EGFR, and VEGFA was verified by molecular docking, while kaempferol showed a strong affinity for targets. This study provides important clues for further exploring the mechanism of GLNBD in the treatment of PCM. However, in vivo or in vitro experiments are needed to verify the mechanism of GLNBD in treating PCM by regulating the abovementioned core targets and pathways.


Diabetes mellitus (DM) is a major risk factor for the development of heart failure (HF). Sodium-glucose co-transporter 2 (SGLT2) inhibitors have demonstrated consistent benefits in the reduction of hospitalization for HF in patients with DM. However, the pharmacological mechanism is not clear. To investigate the mechanisms of SGLT2 inhibitors in DM with HF, we performed target prediction and network analysis by a network pharmacology method.We selected targets of SGLT2 inhibitors and DM status with HF from databases and studies. The “Drug-Target” and “Drug-Target-Disease” networks were constructed using Cytoscape. Then the protein–protein interaction (PPI) was analyzed using the STRING database. Gene Ontology (GO) biological functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed to investigate using the Bioconductor tool for analysis.There were 125 effective targets between SGLT2 inhibitors and DM status with HF. Through further screening, 33 core targets were obtained, including SRC, MAPK1, NARS, MAPK3 and EGFR. It was predicted that the Rap1 signaling pathway, MAPK signaling pathway, EGFR tyrosine kinase inhibitor resistance, AGE-RAGE signaling pathway in diabetic complications and other signaling pathways were involved in the treatment of DM with HF by SGLT2 inhibitors.Our study elucidated the possible mechanisms of SGLT2 inhibitors from a systemic and holistic perspective based on pharmacological networks. The key targets and pathways will provide new insights for further research on the pharmacological mechanism of SGLT2 inhibitors in the treatment of DM with HF.The online version contains supplementary material available at 10.1007/s10557-021-07186-y. Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia due to partial or complete insulin deficiency and/or insulin resistance, which affects 8.5–9.3% of the population worldwide, rising to an expected 10.2% 578 million) by 2030 and 10.9% (700 million) by 2045 8 million. TherefoSodium-glucose co-transporter 2 (SGLT2) inhibitors, a class of glucose-lowering therapies, including dapagliflozin, canagliflozin, empagliflozin and ertugliflozin, have been approved by the US Food and Drug Administration for the treatment of type 2 diabetes mellitus . SGLT2 iWith the development of high-throughput sequencing and computer technology, huge new bioinformatics networks have emerged. Network pharmacology, one such bioinformatics network, aims to construct a multilevel network through various database searches, high-throughput omics data analysis and computer simulations to analyze the relationship of medicines, diseases and targets . Comparehttps://pubchem.ncbi.nlm.nih.gov/), which is an open chemistry database with 96,502,248 compositions, of which 3,151,393 have been tested. Then SwissTargetPrediction (http://www.swisstargetprediction.ch/), a tool for target prediction according to two-dimensional and three-dimensional similarity measures with known ligands, was selected to predict potential targets for four SGLT2 inhibitors by putting their chemical structures into this platform [https://www.drugbank.ca/), which is a unique bioinformatics and chemical informatics database, containing 11,628 drugs and related chemical information, drug targets, protein data, and so on [http://www.uniprot.org/), all the SGLT2 inhibitor-related genes were normalized into consistent symbols for subsequent analysis.The chemical structures of four SGLT2 inhibitors, namely canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, were obtained using Pubchem (nd so on . With fuhttps://www.genecards.org) and OMIM (https://www.omim.org) databases. The GeneCards database includes more than 7000 human genes, and each gene has an approved gene symbol. The OMIM database is a knowledge base of human genes and hereditary diseases. The two methods are a good reference for the collection of disease targets. Meanwhile, we collected disease targets from the studies reporting existing network analysis on actual patients with DM status with HF [https://bioinfogp.cnb.csic.es/tools/venny/).With the keywords of “heart failure”, “diabetes”, and “diabetes mellitus”, target genes related to HF and DM were found in the GeneCards  network between four SGLT2 inhibitors  and their corresponding targets; (2) network between four SGLT2 inhibitors, intersection targets, and diseases (DM status with HF). Cytoscape is a software program that can efficiently express the interaction between protein and protein, protein and DNA or gene, in order to visualize network relationships.https://string-db.org) dataset is now one of the largest PPI datasets, including co-expression data, biomedical literature data, high-throughput data and genomic background data. In the platform, “Multiple proteins” was selected and the organism was selected as “Homo sapiens.” The intersection targets of SGLT2 inhibitors and DM status with HF were then imported to construct the protein–protein interaction network. In order to ensure the high confidence of information, the scoring condition was set to > 0.90, and the isolated proteins in the figure were hidden. The file was exported in “TSV” format, and then Cytoscape v3.6.1 was used to analyze the topological properties of the PPI network. To detect the core targets in the common targets between drugs and diseases, the degrees of targets were calculated using the Cytohubba plugin based on Cytoscape.The STRING  and GO (Gene Ontology) were analyzed for pathway functional enrichment using the clusterProfiler software package on the R platform. The interaction network was constructed using the Top-Go package of the R platform.The PubChem platform was used to acquire the molecular structure of dapagliflozin empagliflozin, canagliflozin and ertugliflozin. The details of their structure are shown in Table After repetition was removed, a total of 13,523 targets related to HF and 15,291 targets corresponding to DM were collected from the GeneCards and the OMIM databases. In addition, 283 targets in the context of existing network analysis on actual patients with DM with HF were also obtained. Among these, 125 common targets were shared among potential targets of SGLT2 inhibitors, known HF-related targets and DM-related targets by an online Venn diagram-drawing platform, seven of which came from actual patients with DM with HF in the studies Fig. . A totalA total of 125 common targets were imported into the STRING online platform, and a file in “TSV” format was exported. It was then imported into Cytoscape v3.6.1, and the function "Cytohubba" was used to calculate the degree of targets to find the core targets. Thirty-three core targets of SGLT2 inhibitors in the treatment of DM with HF were screened  selected for analysis in Fig. In addition, we performed KEGG analysis on the targets of the four SGLT2 inhibitors to find their pathways separately. We intersected the pathways of the four drugs in a Venn diagram to explore the same and different signaling pathways among them Fig. . We idenHeart failure (HF) is a major complication of diabetes mellitus (DM). Therefore, the prevention of HF in patients with DM is critical. In clinical studies, SGLT2 inhibitors were found to be effective in treating HF among patients with DM, which aroused great interest and attention. However, the mechanism is not clear. Based on network pharmacological analysis, this study explored the mechanism of SGLT2 inhibitors in the treatment of DM with HF. In the present study, four SGLT2 inhibitors  were obtained to analyze the effects on DM with HF based on the network pharmacology analysis. According to the drug-disease target network and PPI network, 33 core genes of the SGLT2 inhibitors acting on DM with HF were obtained from 125 common genes. Among the 33 core genes, the top five hub targets SRC, MAPK1, NRAS, MAPK3 and EGFR were screened according to the degree. The module analysis confirmed that SGLT2 inhibitors have the potential to influence a variety of biological pathways that play an important role in the pathogenesis of DM with HF. The key pathways were screened by KEGG analysis, mainly involving the Rap1 signaling pathway, MAPK signaling pathway, EGFR tyrosine kinase inhibitor resistance and AGE-RAGE signaling pathway in diabetic complications.Among the top five core genes, the first, SRC, is a non-receptor tyrosine kinase that plays a role in numerous biological processes including cell adhesion, cell cycle and cell migration. It was demonstrated that SRC kinases could be activated by Ang II, which plays an important role in Ang II-mediated processes , includiThe next two hub genes, MAPK1 (mitogen-activated protein kinase 1) and MAPK3 (mitogen-activated protein kinase 3), also known as ERK1 and ERK2, respectively, are members of the MAP kinase (MAPK) family. MAPK acts as an integration point for multiple biochemical signals and is involved in a wide variety of cellular processes including proliferation, differentiation, transcription regulation and development. As one of the MAPK family members, the activation of ERK1/2 has also been known to be involved in cardiac hypertrophy and dysfunction . In addiThe next gene is NRAS, an N-ras oncogene encoding a membrane protein that shuttles between the Golgi apparatus and the plasma membrane, and works in various differentiation processes and signal transduction involving the regulation of cell survival and growth, T cell activation and apoptosis. Katoh et al. reported that NRAS was one of the representative targets on cardio-miR-214 that were upregulated in human heart failure, showing that NRAS might be associated with the progression of heart failure . It was 1 receptors was considered to be one of the mechanisms leading to this result [The last of the top five hub genes is EGFR, epidermal growth factor receptor, a cell surface protein binding to epidermal growth factor that regulates cell growth, proliferation and survival, and is involved in blood pressure regulation, neointimal hyperplasia, atherogenesis and reactive oxygen. Xu et al. revealed that the enhanced myogenic constriction of the mesenteric artery in heart failure might be related to the loss of plasmalemmal caveolae in mesenteric vascular smooth muscle cells, and the increased activity of the EGFR and ATs result . Belmadas result . Zhang es result . These fMoreover, the targets of our findings have some overlap with those in existing network analysis on actual patients with DM with HF, that is ALB, FDFT1, SLC22A8, ABCG2, MME, LGALS3 and FUCA1, which act as biomarkers in HF patients with DM , 27. TheGO functional enrichment and KEGG pathway analysis have illustrated the role of the SGLT2 inhibitors in the gene function and signaling pathway. According to the biological processes that mainly reflect the GO functional enrichment, these core targets are focused on kinase activity, primarily in positive regulation of MAP kinase activity, positive regulation of protein serine/threonine kinase activity and so on. Among them, positive regulation of protein serine/threonine kinase activity is a core biological process that may affect the function of cGMP in the regulation of the physiological response of the natriuretic peptide (NP) system. This is because cGMP-dependent protein kinases (cGK) are exactly serine/threonine kinases widely distributed in eukaryotes . The phyIn the enrichment of the KEGG pathway, the Rap1 signaling pathway, MAPK signaling pathway, EGFR tyrosine kinase inhibitor resistance and AGE-RAGE signaling pathway in diabetic complications were found to be the significant pathways.The Rap1 signaling pathway is implicated in a wide range of biological processes from cell proliferation and differentiation to cell adhesion. Rap1 has been proven to play a part in the regulation of integrin affinity, adhesion, and migration in postnatal neovascularization, mainly mediating the angiogenesis pathway and serving in an important role in cardiac hypertrophy. Furthermore, Rap1B can prevent excessive vascular leakage in early diabetes mellitus by inhibiting VEGF signal transduction. By controlling telomere length, Rap1 can decrease the occurrence and development of diabetes-related cardiovascular disease . DownregConsistent with the core target results above, the MAPK pathway is also predicted to play a role in DM with HF. It was demonstrated that the MAPK signaling pathway cascade is initiated in cardiomyocytes through activation of G protein-coupled receptors, receptor tyrosine kinases and stress stimulation. Zhang et al. reported that the MAPK signaling pathway was found to regulate cardiomyocyte apoptosis in mice with heart failure after MI, indicating that this pathway has a potential role in heart failure disease . During EGFR tyrosine kinase inhibitor resistance acts on EGFR tyrosine kinase, also identical to the core genes predicted based on the PPI. Previous studies had indicated that inhibition of EGFR activity protected against progressive DN in T1 DM and T2 DM . Zeng etThe AGE-RAGE signaling pathway is a well-studied cascade in DM. It is found that the AGE-RAGE signaling pathway can directly mediate vascular calcification in diabetes. Additionally, the pathway can also impact diabetic complications, as it leads to oxidative stress, increased inflammation, and enhanced extracellular matrix accumulation resulting in diastolic and systolic dysfunction . Fukami Meanwhile, other KEGG analysis results that explore the same and different signaling pathways between four SGLT2 inhibitors showed that the four key pathways mentioned above were contained in 125 pathways overlapping in two or more SGLT2 inhibitors, which also indicated that the four pathways might play a key part in the treatment of DM with HF by SGLT2 inhibitors. In addition, according to the same and different mechanistic pathways of four SGLT2 inhibitors, we suggest that even though the four SGLT2 inhibitors have a few different pathways, a large part of their pathways of action are similar. The four SGLT2 inhibitors have a common parent nucleus, although their side-chain substituents are somewhat different, leading to small differences in the efficacy of drugs . ConsideNetwork pharmacology is indeed a new method for studying the relationship between drugs and diseases. In our study, network pharmacology revealed the potential targets of SGLT2 inhibitors, as well as targets of DM with HF, and bioinformatics was used to study the main enrichment pathways. Based on the network pharmacology, our study predicted the potential therapeutic targets of SGLT2 inhibitors in DM with HF and revealed their action on the main pathways through core genes, which explained the mechanisms of SGLT2 inhibitors in DM with HF and provided scientific evidence for SGLT2 inhibitors to treat DM with HF. However, the main limitation of this study is the lack of experimental verification. Consequently, pharmacological studies will be critical to further elucidate the relationship of SGLT2 inhibitors and DM with HF. In addition, validation of the molecular levels of our findings is necessary for the future.Taken together, our study systematically predicted, screened and analyzed the targets and pathways that might play a vital role in the biological process, which elaborated the possible mechanisms of SGLT2 inhibitors in DM status with HF. Most importantly, these results provide evidence and new insights for further research on the pharmacological mechanism of SGLT2 inhibitors.Supplementary Table S1The predicted targets of four SGLT2 inhibitors from SwissTargetPrediction. (CSV 35 kb)Supplementary Table S2The predicted targets of four SGLT2 inhibitors from DrugBank. (CSV 2 kb)Supplementary Table S3The related targets of heart failure from GeneCards. (CSV 1692 kb)Supplementary Table S4.The related targets of heart failure from OMIM. (CSV 49 kb)Supplementary Table S5.The related targets of diabetes mellitus from GeneCards. (CSV 1880 kb)Supplementary Table S6The related targets of diabetes mellitus from OMIM. (CSV 69 kb)Supplementary Table S7The detailed results of GO biological process analysis. (CSV 317 kb)Supplementary Table S8The detailed results of KEGG analysis. (CSV 26 kb)Supplementary Table S9The related targets of HF-DM from the network analysis studies conducted on patients with DM status with HF. (XLSX 13 kb)Supplementary Table S10The detailed results of KEGG analysis on canagliflozin. (XLSX 21 kb)Supplementary Table S11The detailed results of KEGG analysis on dapagliflozin. (XLSX 23 kb)Supplementary Table S12The detailed results of KEGG analysis on empagliflozin. (XLSX 11 kb)Supplementary Table S13The detailed results of KEGG analysis on ertugliflozin. (XLSX 23 kb)