Background: Under Chinese medicine theory guidance, Fuzheng Yangxin Recipe (FZYX) is clinically effective for the treatment of heart failure (HF) caused by ischemic heart disease (IHD). This study aimed to investigate the mechanism of the myocardial protective effects of FZYX on HF.Materials and methods: The Gene expression omnibus database was used to identify differential genes of the IHD subtype. Through network pharmacological methods, the targets of the active components of FZYX were obtained. We also constructed IHD-induced HF model rats by ligating the left anterior descending coronary artery. Echocardiography, pathological section staining, enzyme-linked immunosorbent assay, western blotting, immunohistochemistry, and quantitative real-time PCR analyses were performed to verify the protective effects of FZYX on the myocardium.Results: We identified 53 active components and 37 potential targets of FZYX associated with the IHD subtype. Signal transducer and activator of transcription 3 (STAT3) is a key protein in the protein-protein interaction (PPI) network. A total of 146 biological processes, 10 cellular components and 40 molecular function subcategories were identified by Gene Ontology (GO) enrichment analysis, and 18 signalling pathways, including apoptosis, were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In vivo experiments showed that FZYX significantly inhibited cardiomyocyte apoptosis, promoted the expression and phosphorylation of STAT3, and improved cardiac function.Conclusion: FZXY improves cardiac function and protects cardiomyocytes from injury via multi-component, multi-target and multi-pathway action, especially its possible role in regulating STAT3 expression and anti-apoptotic effect. Heart failure (HF) is one of the main public health problems worldwide. In the 2017 Global Burden of Disease Study, the number of HF patients was 64.34 million . Data frYangqi” in the heart, and herbal medicine is widely used in the treatment of patients with HF in China (Panax notoginseng (Burkill) F. H. Chen, 20 g of Panax ginseng C. A. Meyer, 30 g of Rhodiola crenulata (Hook.f.et Thoms.) H. Ohba, 10 g of Aconitum carmichaeli Debx, 20 g of Ophiopogon japonicus (Linn. f.) Ker-Gawl, 30 g of Astragalus membranaceus (Fisch.) Bunge, 15 g of Angelica sinensis (Oliv.) Diels and 15 g of Rehmannia glutinosa Libosch. In clinical practice, FZYX has a beneficial effect on patients with HF caused by IHD , but its clinical symptoms were described more than 2000 years ago. Chinese medicine proposes that the onset of HF is related to impairment of the “in China . Fuzhengd by IHD , but thein vivo experiments. The findings provide a foundation for the treatment of HF by FZYX.Network pharmacology, based on systems biology and multi-directional pharmacology, allows the selection of specific nodes using biomolecular network analysis methods to carry out molecular design and target analysis of drugs . This me2FC (fold change) | > 0.5 and adjPvalue < 0.05 as the cut-off criteria for differentially expressed genes (DEGs). Finally, we selected the top 20 genes with the highest up- and downregulation to draw a heatmap. Genes unaffected and differential genes were used to draw a volcano map.Firstly, we retrieved HF gene expression profiles and selected “Homo sapiens” entries from the gene expression omnibus (GEO) database. After reading the abstract, we selected entries according to the following criteria: 1) The number of samples was >40 ; 2) Healthy people were used as the control group; 3) Clinical samples were myocardial tissue. Secondly, Matrix and Platforms were downloaded after targets were selected, and the R packages BiocManager, Limma and Pheatmap were used to perform a secondary analysis of the chip data, with |logp-value = 0.05 and score cutoff = 20 when using the BATMAN-TCM database, and Druglikeness Grading = ‘Moderate’ or ‘Good’ when using the ETCM database. The obtained components were used by the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) to predict targets, which were organised into a table according to herb-ingredient code-ingredient name-target.The BATMAN-TCM database  and the DEGs in Researchers used “BisoGenet” in Cytoscape and data from six major human PPI databases  to perform PPI network analysis of overlapping genes . The Cytp-value cut-off <0.05, q-value cut-off <0.05), a bubble diagram was generated, KEGG pathways were imported into Cytoscape, and a pathway-gene network was drawn based on degree values.Gene Ontology (GO) provides functional annotation for biological process (BP), cellular component (CC) and molecular function (MF) categories. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to analyse the signalling pathways in which the differential genes are involved. The R packages org.Hs.eg.db, DOSE, clusterProfiler, enrichplot, colorspace, stringi and ggplot2 can be used for visualisation of GO and KEGG results. According to the value and significance of enrichment (https://www.rcsb.org/) (The intersection of key nodes in the PPI network and high relevance genes in pathway-gene network were selected as candidate genes for molecular docking. The receptors 3D format of the core targets were downloaded from the Protein Data Bank (sb.org/) , and opesb.org/) . Autodocsb.org/) .Panax notoginseng (Burkill) F. H. Chen (60100000994), Panax ginseng C. A. Meyer (60100000951), Rhodiola crenulata (Hook.f.et Thoms.) H. Ohba (SA200226005), Aconitum carmichaeli Debx (SA200325013), Ophiopogon japonicus (Linn. f.) Ker-Gawl (60100000925), Astragalus membranaceus (Fisch.) Bunge (60100000921), Angelica sinensis (Oliv.) Diels (SA200530014) and Rehmannia glutinosa Libosch (60100000919) were purchased from China Resources Sanjiu . In addition, angiotensin receptor neprilysin inhibitors (ARNI) were purchased from Novartis  and employed as positive control drugs.This experiment included 40 specific pathogen free male Sprague Dawley rats weighing 200 ± 20 g purchased from Charles River . Animals were raised in the experimental animal room of Xiyuan Hospital. Standard synthetic feed was provided with free access to drinking pure water, the temperature was (24 ± 2°C), the humidity was 45%–50%, and a 12 h/12 h light/dark photoperiod was employed. Animal experiments were reviewed and approved by the ethics committee of Xiyuan Hospital. Rats were randomly divided into four groups; a sham operation group, a model group, an FZYX group (4.2 g/kg) and an ARNI (68 mg/kg) group. Animal drug dosage was based on the equivalent conversion between body surface area and weight of animals and humans , as wellMI-induced HF model rats were established by ligating the left anterior descending coronary artery. Briefly, rates were intubated following being anesthetised with 1% sodium pentobarbital  using an ALC-V8S-P small animal ventilator  at a rate of 70 breaths per min, ribs were separated to expose the heart, and a 4/0 medical silk thread was employed to ligate the artery 2–3 mm below the left atrial appendage. If the electrocardiogram indicated ST-segment elevation and the colour of the ligation area was immediately pale, ligation was considered successful . In the After rats were fed drugs for 42 days, uninformed researchers anesthetized them with 1% sodium pentobarbital (50 mg/kg), and two-dimensional M-mode and B-mode echocardiography were performed using a Vevo2100 instrument  to assess left ventricular function. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular internal dimension end-diastolic (LVIDd) and left ventricular internal dimension end-systolic (LVIDs) were automatically calculated by the echocardiographic system.Heart tissue of rats fed drugs for 42 days was collected and fixed in 4% paraformaldehyde overnight. Myocardial tissue was embedded and fixed in paraffin to prepare 4 μm sections. Different concentrations of ethanol were used for dewaxing, and samples were stained using a modified Masson’s Trichrome Stain Kit  and a hematoxylin-eosin (HE) Staining Kit . Finally, myocardial tissue morphology and fibrosis degree were observed under a 400× microscope. Collagen volume fraction (CVF) was analyzed by Image-Pro Plus 6.0 . CVF = area of collagen/total area ×100%.g for 10 min at 4°C. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the level of N-terminal pro-B-type natriuretic peptide  according to the manufacturer’s instructions.Blood was collected from the abdominal aorta, incubated at room temperature for 1 h, then centrifuged at 1000 The infarction border area of the left ventricular myocardium was rapidly frozen in liquid nitrogen. The RIPA buffer  was added, the tissue was homogenised on ice, then centrifuged at a low temperature to extract total protein. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit . Equal amounts of proteins (50 μg) were separated based on molecular weight by 8%–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with quick block buffer for western blot  at room temperature and incubated overnight at 4°C in the presence of primary antibody . The memTissue sections were dewaxed and rehydrated, then subjected to antigen repair using the citric acid solution (pH 6.0). Hydrogen peroxide (3%) was used to block sections for 10 min, and 5% goat serum  was added and incubated for 1 h. Tissue sections were incubated with primary antibody  overnight at 4°C, followed by secondary antibody . Finally, immunohistochemical reactions were analysed using a DAB Kit  and nuclei were stained with HE . Images were observed under a microscope at 400× magnification. The integrated option density (IOD)/area was analysed using Image-Pro Plus 6.0.−ΔΔCT method . The PrimeScript RT reagent Kit  was used for reverse transcription of RNA samples into cDNA. Primer sequences are listed . Each 20T method .p-value <0.05 was considered statistically significant.Data were statistically analysed by SPSS Statistics software . All data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) with a completely random design was used for comparison between multiple groups, and the least significant difference (LSD) test was used for pairwise comparison between groups. When applying one-way ANOVA was not appropriate, the Kruskal-Wallis rank sum test was used. A The GSE57338 chip and platform files from the GEO database were downloaded to obtain sample data from 136 non-failing (NF), 95 IHD with HF and 82 idiopathic dilated cardiomyopathy (DCM) with HF. To gain a better understanding of the different HF subtypes, we performed differential expression analysis on all types of HF patients based on the data. The patients’ characteristics (age and sex) were shown in Panax notoginseng (Burkill) F. H. Chen, 43 in Panax ginseng C. A. Meyer, 4 in Rhodiola crenulata (Hook.f.et Thoms.) H. Ohba, 8 in Aconitum carmichaeli Debx, 27 in Ophiopogon japonicus (Linn. f.) Ker-Gawl, 2 in Astragalus membranaceus (Fisch.) Bunge, 47 in Angelica sinensis (Oliv.) Diels and 4 in Rehmannia glutinosa Libosch. After deduplication, 405 components were identified in FZYX. In addition, targets were identified by database screening, including 369 ingredients in Panax notoginseng (Burkill) F. H. Chen, 423 in Panax ginseng C. A. Meyer, 124 in Rhodiola crenulata  Ker-Gawl, 180 in Astragalus membranaceus (Fisch.) Bunge, 591 in Angelica sinensis (Oliv.) Diels and 77 in Rehmannia glutinosa Libosch. After deduplication, 923 targets in FZYX were identified  146 BP subcategories including vascular-associated smooth muscle contraction, extracellular matrix disassembly, positive regulation of cytosolic calcium ion concentration, regulation of lipase activity, regulation of protein serine/threonine kinase activity, regulation of inflammatory response, and collagen catabolic process; 2) 10 CC subcategories including nuclear membrane, external side of plasma membrane and nuclear envelope; 3) 40 MF subcategories including protease binding, endopeptidase activity, phosphoric diester hydrolase activity, exopeptidase activity and phospholipase activity. The top 20 BP and MF subcategories and the top 10 CC subcategories are shown in To further understand the mechanism of FZYX, GO and KEGG were performed. GO functional analysis was performed on 37 key intersection genes with KEGG pathway analysis showed that 18 signal pathways were enriched . Based oMolecular docking was performed with the main compounds of FZYX and the intersection targets, namely STAT3. The lower the affinity value in the docking findings, the more stable the contact between the targets and the active component was. Through molecular docking, it was found that STAT3 had good binding activities with the active components of FZYX . These cAt the end of the experiment cycle, two animals in the model group, one in the FZYX group and none in the remaining groups died. Echocardiographic results showed that LVEF and LVFS were decreased and LVIDs, LVIDd, LVESV and LVEDV were increased in the HF model group compared with the sham operation group, suggesting that cardiac structure and function were severely impaired after acute MI . LVEF anTo verify the underlying mechanism of the network pharmacological findings, expression of apoptosis-related genes at mRNA and protein levels in cardiomyocytes was measured by Quantitative real-time PCR (qRT-PCR) and western blotting, respectively . The resThe mRNA expression levels of STAT3 were measured by qRT-PCR, and the abundance of phosphorylated STAT3 and total STAT3 proteins was measured by western blotting according to the results of network pharmacology . CompareBased on pattern identification and disease, TCM uses various herb combinations according to conditions to achieve the goal of holistic treatment. Therefore, TCM prescriptions often contain numerous herbs, resulting in complex chemical composition and numerous targets. Hence, modern research faces great challenges in deciphering their mechanisms and modes of action. Network pharmacology retrieves known chemical components of herbs in prescriptions based on a variety of databases and analysis software. From the data, targets of the chemical components, sites of action, molecules, and pathways can be obtained, allowing comprehensive analysis of the mechanisms of the compounds, and providing a reference for further clinical research on a specific mechanism .. This suggests a strong relationship between inflammation and IHD, although further validation is needed considering that VSIG4 has been poorly studied in HF. Extracellular matrix changes play an important role in the development of DCM. Different from fibrosis triggered by ischemic conditions, DCM manifests as diffuse fibrosis[21]. Secreted modular calcium-binding protein 2(SMOC2), as a matrix protein, is a non-structural component of the extracellular matrix[22]. Whether SMOC2 affects extracellular mechanism components in DCM and plays a key role also needs to be further verifiedThe present study was divided into two parts: network pharmacology and experimental validation. We first used GSE57338 to identify DEGs in HF. GSE57338 includes three groups: NF, IHD and DCM. V-set and immunoglobulin domain containing 4(VSIG4) is specifically expressed in tissue-resident macrophages and can mediate various cellular events. Inhibition of the expression of VSIG4 can significantly increase the secretion of cytokines in macrophages and aggravate the inflammatory responsein vitro and in vivo experiments. Kaempferol, a natural flavonoid compound, possesses anti-inflammatory and antioxidant activities (Rhodiola crenulata (Hook.f.et Thoms.) H. Ohba, and has good biological activity for the treatment of cardiovascular and metabolic diseases (FZXY has a good clinical effect on IHD-induced HF, but its specific active components are still unclear. We used the database to collect and screen the active components of FZYX, and finally obtained 143 compounds. Some of these ingredients exert their cardioprotective effects through known mechanisms determined by tivities . On the tivities ; on the tivities  and modutivities . Salidrodiseases . Salidrodiseases . In addidiseases .Ferulic diseases . Clinicadiseases . In rat diseases .Butylidediseases , which cdiseases . Quercetdiseases .Quercetidiseases . In animdiseases . Quercetdiseases  and inhidiseases . Ligustidiseases .After obtaining the active ingredients of FZYX, we used SwissTargetPrediction to predict the target of the drug. The results were intersected with the DEGs of IHD-induced HF, and 37 intersection genes were finally obtained. PPI and functional enrichment analyses were performed on these 37 genes to discover the underlying mechanisms. Based on the results of PPI and enrichment analysis, STAT3 and apoptosis were selected as the key roles of FZYX prescription for subsequent studies. The results of molecular docking also showed that the main components of FZYX and STAT3 had good binding activity. STAT3 can affect cell-to-cell communication, signal transduction and gene transcription. A large number of previous studies have confirmed the role of STAT3 in tumours . In receApoptosis is the process of programmed cell death, which is primarily mediated by the death receptor pathway and the mitochondrial apoptosis pathway . ApoptosTo test whether FZYX exerts cardiac protective effects through apoptosis and STAT3, we performed animal experiments. The results of echocardiography and NT-proBNP showed that FZYX could improve cardiac function in the HF model. The qRT-PCR and western blotting showed that FZYX could increase the expression of BCL-2 and decrease the expression of BAX, and Caspase-3. The results showed that FZYX had anti-apoptosis ability, which was consistent with the results of enrichment analysis. We also tested the effect of FZYX on STAT3 expression. The qRT-PCR showed that FZYX could upregulate STAT3 mRNA expression levels, and western blotting showed that the pSTAT3:STAT3 ratio was increased, indicating that the anti-apoptotic mechanism of FZYX might be related to STAT3.In summary, this study systematically investigated the mechanism of FZYX in the treatment of HF through a combination of network pharmacology and experimental validation. Network pharmacology revealed that FZYX contains various active components that exert cardioprotective effects through multiple targets and signalling pathways. STAT3 may be the core factor of FZYX action, and the anti-apoptotic activity may be one of the most important effects of FZYX in protecting cardiac function. In addition, we verified the effect of FZYX on STAT3 expression and its anti-apoptotic effects in a rat model of ischemic HF.In this study, we only focused on the apoptotic pathway and STAT3, and did not fully utilize the results derived from network pharmacology. Second, this study is only a preliminary exploratory study, and the inhibitor group is not designed. In our subsequent study, a more reasonable design will be carried out to increase the reliability of the conclusion.


In early chronicity, we observed enrichment of several pathways. Dysregulation of Ephrin/Eph signaling leads to growth cone collapse and impairs neuronal regeneration. Adra2b and Adra2a overexpression in early and full chronicity, respectively, decreased cAMP production and impaired re-epithelialization and granulation tissue formation. Several pathways involving a Smooth-muscle-actin (Acta1) were also enriched with Acta1 overexpression contributing to impaired angiogenesis. During full chronicity, the ‘JAK-STAT’ pathway was suppressed undermining host defenses against infection. Wnt signaling was also suppressed, impairing re-epithelialization and granulation tissue formation. Biomarkers of cancer such as overexpression of SDC1 and constitutive activation of ErbB2/HER2 were also identified. In conclusion, we show that during progression to full chronicity, numerous signaling pathways are dysregulated, including some related to carcinogenesis, suggesting that chronic wounds behave much like cancer. Experimental verification in vivo could identify candidates for treatment of chronic wounds.Previously we have shown that several oxidative stress-driven pathways in cutaneous chronic wounds are dysregulated in the first 48 h post-wounding. Here, we performed an RNASeq analysis of tissues collected up to day 20 after wounding, when we have determined full chronicity is established. Weighted Gene Correlation Network Analysis was performed in R segregating the genes into 14 modules. Genes in the modules significantly correlated ( Thene 2022) . Althoug2O2, is significantly elevated, and consequently, H2O2 is also elevated shortly after injury. Moreover, we also found that Catalase is decreased whereas Gpx is not increased, indicating that OS is high [Wound-healing consists of different phases, starting with homeostasis after the initial injury followed by an inflammatory phase, a proliferation phase, and then the final phase, remodeling. Chronic wounds have increased oxidative stress (OS) from the early stages after injury, due in part to insufficient antioxidant molecules to remove the oxidative radicals. In our previous studies describing the development of the model we determined that superoxide dismutase  an enzyme that produces H is high . In addi is high ,7. In ad is high ,9,10.During the inflammatory phase, chronic wounds show increased numbers of all three types of leukocytes, neutrophils, macrophages, and lymphocytes found in the wound bed simultaneously. Normally, neutrophils come to the wound site a few hours after wounding, then macrophages replace them, and finally, toward the remodeling phase, lymphocytes invade the wound tissue . The fac2O2 to produce various types of hypohalous acids [Neutrophils secrete myeloperoxidase that in turn use Hus acids ,13. Therus acids . Other iIn normal wound-healing, the proliferative phase lasts from a few days to a few weeks. This phase is marked by the proliferation and migration of keratinocytes at the wound edge, resulting in wound closure ,16. FibrLittle is known about the signaling pathways and gene expression patterns that are involved in the progression of wounds to chronicity. In this study we examine the significantly correlated genes and the significantly enriched pathways that lead to chronic wound development and progression using Weighted Gene Correlation Network Analysis (WGCNA). WGCNA divides all the identified genes given as input, into various modules according to correlation based on the expression of the genes. Modules are chosen based on the concept of scale-free topology, which means that the characteristics of the network are independent of the size of the network. The size of the network is measured in the number of input genes also called nodes. However, irrespective of the number of nodes the basic structure of the network remains the same. Networks that follow scale-free topology but are randomized in nature for all other properties are best suited for biological systems because we can see the network as a probability ,19. An iIn visualizing biological data as a network, each gene or protein serves as a node and the data correlating two nodes, for example, co-expression, evidence of interaction, serves as the edge that connects the node and defines the connectivity of the node. The more connected a node is to other participating nodes in the network, the bigger the importance of that node which then becomes a HUB gene. In order to make sure that the connectivity and thereby the importance of the node is relevant to the comparison being addressed in the experiment, selecting an appropriate power for network construction is necessary. To obtain a network with scale-free topology having a co-efficient of correlation greater than 0.8, a power less than 30 is appropriate for signed networks. In our analysis of gene expression data discussed in this paper a signed network is used so that both positive and negative correlation of genes to experimental conditions, can be evaluated ,21.In this study, we found that distinct signature pathways are activated in chronic wounds which create a microenvironment leading to non-healing. We discuss these pathways in terms of early chronicity during the development of chronicity, which ranges from Day 1 to Day 10 post-wounding in our mouse model of chronic wounds and full chronicity at Day 20 post-wounding. Our mouse model develops chronic wounds because of high oxidative stress present in the wound tissue due to reactive oxygen and nitrogen species that are not scavenged due to the decrease of antioxidant molecules and enzymes. Our previously published studies also show that treatment of the wounds with externally applied antioxidants such as NAC and α-tocopherol reverse chronicity. Therefore, the relation between development of chronic wounds and oxidative stress is well established. Here we describe the changes in gene expression resulting from prolonged oxidative stress in chronic wounds. We discuss the significantly identified pathways for early and full chronicity highlighting the genes that act as master regulators for the dysregulation of the pathway. Our rationale for doing this study was to identify the transcriptomic variations that establish chronicity in wounds and find key genes that are dysregulated in chronic wounds to select targets for effective treatment of chronic wounds.All experiments were completed in accordance and compliance with federal regulations and the University of California policy and procedures approved by the UCR IACUC  ethical code 20200024 .2.1 Sample Collection: The db/db−/− mouse model has a knockout mutation in the leptin receptor of the C57BLKS/J background that makes them diabetic and obese. The db/db−/− mice were only used for wounding once they were 5 months old and weigh ≥ 50 g. The mice needed to be this old to be fully diabetic and obese and needed weight > 50 g in order to withstand the burden of wound chronicity. For the creation of chronic wounds, 7 mm diameter full-thickness cutaneous wounds were made, and a one-time treatment with inhibitors of catalase and glutathione peroxidase (GPx) was performed [−/− mice, hair grows back very slowly after shaving; hence the tegaderm remained in place [2. Samples were stored at −80 °C for subsequent RNA extraction. Two biological replicates each were collected for chronic and non-chronic wounds at days 1, 5, and 10 post-wounding, two replicates each of db/db−/− skin and chronic wounds, 20 days post-wounding, were also collected.erformed ,23. Briein place . Control2.2 mRNA Extraction and RNASeq analysis: mRNA was extracted with and purified using the RNeasy Plus Mini Kit, Cat no. 74134. The mRNA was sequenced with Illumina Hi-Seq  with single-end 50 base at UC Riverside Institute for Integrative Genome Biology (IIGB). After the mRNA was sequenced, the FastQC reports were checked for quality. All samples recorded reads with Phred score > 30, which confirmed the quality of reads at 99.9% accuracy, before analysis. Programs available at command line were used to analyze the reads. The fastq files were then aligned to a recent mouse genome assembly  with hisat2 (2.2.1). Kallisto [Kallisto  was used2.3 Weighted Gene Correlation Network Analysis (WGCNA): WGCNA is a comprehensive method of identifying correlation networks. The RNASeq analysis identified, 21,919 genes that showed expression across all samples. The TPM normalized expression dataset was not filtered by differential expression. Using the package WGCNA in R and analyzing scale-free topology with a soft threshold value of 14, an adjacency matrix was calculated using the Pearson correlation method. The adjacency matrix was then used to identify modules according to the correlated co-expression of the genes. The constituent genes of each module were assigned a ‘module membership’ correlation value that gauged if the gene truly belonged to the module . These modules were then correlated to the experimental conditions (dichotomous matrix of whether the wound tissue was collected from chronic or non-chronic wound) via the module eigengenes. Module eigengenes represent the gene expression profile of each module. The module eigengenes that correlated to each timepoint in chronic and non-chronic wounds were then checked for significance . Gene trait significance (Pearson’s correlation method) was calculated for the genes in the significant modules and only genes that showed both significant module membership (p < 0.05) and correlation to traits (p < 0.05) were used for further analysis.2.4 Pathway analysis for significant modules identified by WGCNA: For the discussion of the broader transcriptomic landscape at each timepoint, all the genes from the significantly correlated module were used as input for the R package pathfindR (version 1.6.2) to identify significantly enriched pathways [Mus musculus from STRING was used to identify enriched signaling pathways and to visualize network patterns and interactions between genes. The pathfindR package identified significantly enriched pathways which were precise and mapped out the progression of wound through the timepoints. Only pathways with FDR < 0.05 were reported in the Results section.pathways . The proFor this study, RNASeq was performed during early chronicity (first 10 days post-wounding) and at full chronicity (20 days post-wounding) to provide an overview of the progression of the wound to chronicity, so that we can compare progression to healing in acute wounds to failure to healing in chronic wounds. We discuss our analysis of the RNASeq data using the Weighted Gene Correlation Network Analysis (WGCNA) A. Duringp = 0.006), the turquoise  modules (p < 0.05).When the RNAseq data was analyzed by WGCNA, we found that three modules were significantly correlated to early chronicity: the cyan . The ADRA2B receptor mediates catecholamine-induced inhibition of adenylate cyclase which is the enzyme responsible for producing cAMP, an important second messenger for G-protein coupled receptors. The ADRA2B-mediated inhibition of adenylate cyclase causes decreased production of cAMP which decreases signal transduction by G-protein coupled receptors present in keratinocytes inhibiting re-epithelialization -mediated signaling pathways ,29. FollIn our data Ephrins A1, A3, A4, A5, and receptors EphA1, A2, A4, A7 are overexpressed during very early chronicity . In rodeDrosophila, Robo–Slit interactions lead to growth cone repulsion [Drosophila ROBO2 has two sequences in the cytoplasmic domain in common with mammals, CC0 and CC1. During embryonic development in Drosophila, low levels of Robo2 results in too many axons to cross the midline, whereas overexpression of Robo2 repels axons from crossing the midline. In addition, at higher levels Robo2 also repulses axons away from Slit, where Slit is secreted by the midline and acts as a repellent. In chronic wounds, Robo1 (2.52 log fold), Robo2 (4.35 log fold) and Slit 1 (1.94 log fold) are overexpressed, thereby indicating an abundance of repulsive cues to the growth cone progression leading to decrease of neuronal activity in chronic wounds. The Eph/Ephrin pathways and the Robo/Slit pathways converge during early neurogenesis [Another repulsive cue for growth cones is the overexpression of Robo2 in chronic wounds. ROBO 1–4 are single pass transmembrane receptors and interact with Slits which are present in the extracellular matrix. In epulsion . Drosophogenesis .Proper regulation of the Ephrin/Eph signaling pathways is also required for re-epithelialization and blood vessel morphogenesis, in addition to axon guidance ,34,35. UIt has also been shown that EphrinB-mediated signaling is essential for release of cell-to-cell adhesion junctions. In normal healing this allows for cell migration and thereby facilitates re-epithelialization . HoweverThe overexpression of the receptors for EphrinB in early chronicity also impairs vascularization. Specifically, Ephrin B2 is required for angiogenesis. The process of angiogenesis is driven by hypoxia. However, in diabetic patients, hypoxia signaling is impaired and the activation of the hypoxic pathway via Hif1α is impaired. Under normal conditions when hypoxia is activated by Hif1α, there is also an increase in expression of Ephrin B2 in endothelial cells . In chro2 fold overexpressed, respectively  molecules, including laminin, a component of the basement membrane that underlies the epithelium. Functional integrin receptors are composed of two subunits: 1 alpha and 1 beta. The laminins  and integrins  are overexpressed in chronic wounds . Of part2 fold upregulated in our chronic wounds. In various types of cancer, Sdc1 deregulation has been linked to poor prognosis. For example, in malignant melanoma, treatment with an antibody–drug conjugate that targets Sdc1, showed anti-tumor effects by inhibiting vascular maturation [‘Proteoglycans in cancer’: This pathway is associated with ECM molecules called proteoglycans. Proteoglycans function as supportive matrix molecules that direct spatial, temporal, and contextual information to cells. Syndecans (SDCs), are a type of proteoglycan that facilitate interactions of cells with growth factors such as FGF, VEGF and TGFβ that are involved in acute wound healing, particularly in vascularization. SDC1 also plays an important role in keratinocyte migration by interacting with integrins. During human keratinocyte migration, cells of the basal layer deposit an oriented matrix of laminin 332. Also, α6β4 and α3β1 integrins, expressed in the basal surface of keratinocytes, bind to laminin 332  and mediate their anchorage to the basal lamina. Keratinocyte migration requires phosphorylation of the β4 cytoplasmic domain by ErbB2/HER2 and engaging of the β4 and the SDC1 cytoplasmic domains . In HaCaturation ,46. ThesA.3. The significantly enriched pathways identified in the brown module are: ‘Cardiac muscle contraction’, ‘Adrenergic signaling in cardiomyocytes’, ‘Hypertrophic cardiomyopathy’, ‘Dilated cardiomyopathy’ and ‘Arrhythmogenic right ventricular cardiomyopathy’  and red  modules correlated significantly with full chronicity  cells, which are crucial for host defense response against invading microbes ,55,56. I‘Neuroactive ligand receptor interaction’: this pathway contains genes such as Adra2a, which is upregulated. As mentioned above the Adra2 gene mediates catecholamine-induced inhibition of adenylate cyclase which reduces cAMP production and adversely affects keratinocyte migration . We foun−/− knockout mice, which have impaired healing, high levels of platelets were observed in the wound. Such accumulation of platelets contributes to blood vessel occlusion and enhances platelet adhesion which contributes to impaired healing by depriving the wound of oxygen and nutrients [‘Wnt signaling pathway: Genes involved in this pathway are Wnt2b, Wnt5a, and Notum. In chronic wounds, Wnt2b is upregulated 1.95-fold, Wnt5a 2.99 fold and Notum 3.29 fold. APC and β-catenin expression levels are similar in chronic and non-chronic wounds. Wnt2b upregulation in K562 immortalized lymphoblast cell lines causes differentiation to megakaryocytes. Megakaryocytes give rise to platelets . In TNFSutrients   in keratinocytes, a process that is known to occur during cancer development . Fgf2 inPrevious research from our laboratory used a systems biology approach to analyze Nanostring data using WGCNA to identify signaling pathways during the first 48 hrs post-wounding in chronic wounds. Our current paper focusses on the progression of diabetic wounds towards full chronicity. All pathways are in many ways the result of the increased oxidative stress caused by ROS in the first 24 h after wounding. Much like with the RNAseq approach presented here, our previous studies predicted an upregulation of catecholamine production in chronic wounds from very early on in response to injury. Catecholamines  adversely affect keratinocyte migration during re-epithelialization ,72. OverIt has been shown that, application of DBcAMP  to wounds, results in significantly higher re-epithelialization and granulation tissue formation when compared to saline-treated control wounds . TherefoProteoglycans are components of the extracellular matrix. Sdcs (heparan sulfate proteoglycans), are a type of proteoglycans that facilitate interactions of cells with growth factors such as FGF, VEGF, and TGFβ, which bind to the heparan sulfate chain on the proteoglycans. Sdc1 is expressed in keratinocytes, fibroblasts, and epithelial cells and it has been known to play a role in wound-healing by activating integrins which in turn facilitate cell migration and ECM assembly . Sdc1 isFocal adhesion proteins such as integrins and laminins are important for keratinocyte migration ,78,79. IOne of the most important functions of α6β4 integrin is to bind Lam b3, which is the β-subunit of Laminin 5. Our data shows overexpression of α6β4 and Lam b3 during early chronicity. The integrin α6β4 has been identified as one of the central regulators of tumor angiogenesis. This integrin is only expressed in mature blood vessels and not developing blood vessels. Deletion of the 1355 threonine on the substrate domain of β4 significantly inhibited angiogenesis in 80% of tested tumor xenograft models, further suggesting a crucial role for α6β4 in tumor angiogenesis . Therefo2 fold overexpressed in chronic wounds, which indicates that at the stage when fibroblast mediated repair should be occurring, their proliferation is hampered by Acta1 overexpression.During early chronicity, fibroblast-mediated tissue repair takes place. It has been shown that Acta 1 (the gene for αSMA) overexpression leads to poor proliferation, thereby impairing wound-healing . Our datIn normal wound-healing, the JAK–STAT pathway activates the STAT family of transcription factors, which in turn impact the transcription of genes that regulate recruitment, survival, and activation of immune cells . Our datIn this study, we found the dysregulation of key wound-healing signaling pathways that can potentially lead to progression of a wound to chronicity . One of Another major pathway that is dysfunctional in chronic wounds during early chronicity is the “axon guidance”. The Ephrins/Eph-induced signaling is associated with growth cone collapse that impairs motor nerve regeneration which in turn adversely affects sensory nerve regeneration. Because several Ephrins/Eph are overexpressed growth cone regeneration does not occur in chronic wounds. The Ephrin/Eph pathways are also important for re-epithelialization as well as granulation tissue formation and angiogenesis.Significant modules at full chronicity showed the suppression of JAK–STAT pathway by Socs1 and IL23a, which explains lower immunity and defense against bacterial pathogens colonizing the wound, thus aiding biofilm formation. In addition, genes overexpressed in the ‘Wnt signaling pathway are associated with maintaining a pro-inflammatory state leading to skin disorders, such as psoriasis, as well as affecting the production of hair follicle stem cells that are critical for re-epithelialization.6β4 is overexpressed, blood vessels develop, which is characteristic of tumors. Indeed, similarities in gene expression between tumors and chronic wounds was first introduced by Harold Dvorak in a seminal paper [6β4 can be potentially targeted to treat chronic wounds with drugs that are already on the market.Moreover, during early chronicity, the signaling pathways involving proteoglycans are also dysfunctional. Sdc1 in chronic wounds autoactivates ErbB2, which is a characteristic of melanoma, suggesting that as wounds become chronic, they show many characteristics of cancer. In addition, when the integrin αal paper . TherefoOur findings identify signaling pathways and potential targets that can be manipulated to coax a chronic wound to heal and potentially help in the development of new treatments for chronic wounds.


In the macula-off group, postoperative VD in both whole SCP and whole DCP was lower compared with controls (p < 0.001). In the macula-on group, there was an inverse correlation between axial length increase and SCP VD change . These findings demonstrated microvascular alterations after SB for RRD. However, VD impairment seems to be mitigated after surgery. A greater increase in postoperative axial length was associated with a poorer VD outcome.This retrospective study aimed to investigate macular microvascular alterations after successful scleral buckling (SB) for rhegmatogenous retinal detachment (RRD). Nineteen eyes with macula-on RRD and 18 eyes with macula-off RRD were included. In all cases, an encircling band was placed. Optical coherence tomography angiography (OCTA) was performed at baseline and postoperatively. Changes in the foveal avascular zone (FAZ) area and vessel density (VD) of the superficial capillary plexus (SCP) and deep capillary plexus (DCP) were the primary outcomes. Correlations between OCTA findings and clinical variables were considered secondary outcomes. In both the macula-on and macula-off groups, the FAZ area was comparable with controls. In the macula-on group, VD in the whole SCP was lower compared with controls at both baseline ( Scleral buckling (SB) is a valuable treatment option for rhegmatogenous retinal detachment (RRD), being preferred over vitrectomy in cases of phakic eyes with uncomplicated or medium complexity detachments ,2. Most Optical coherence tomography angiography (OCTA) is a novel, noninvasive imaging method that provides qualitative and quantitative information on macular microcirculation . OCTA haThe purpose of this study was to evaluate macular vessel density (VD) on OCTA following scleral buckling for RRD and to identify which clinical variables might have an influence on macular microvascular status. Additionally, the relationships between microvascular parameters and functional and anatomical outcomes were explored.This retrospective study was conducted at the Eye Clinic of the University of Catania, Italy. The study protocol was in agreement with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board (number 69/2021/PO). Informed consent was obtained from all subjects. Medical charts of all consecutive patients who underwent scleral buckling surgery for primary RRD between September 2017 and December 2020 were reviewed.For inclusion, the following criteria had to be satisfied: (a) scleral buckling procedure as primary surgery for RRD in a phakic eye with healthy fellow eye; (b) follow-up of 6 months or longer; (c) anatomically reattached retina following single surgery with no evidence of intraoperative and postoperative complications; (d) spectral domain-OCT (sd-OCT) and OCTA imaging at baseline and throughout the follow-up, namely at 1 month (±7 days), 3-month (±14 days) and 6-month (±14 days) post-operatively; (e) absence of abnormalities on sd-OCT imaging, such as intraretinal cysts, epiretinal membrane, intraretinal or subretinal spaces, external limiting membrane disruption, ellipsoid zone disruption, and retinal pigment epithelium alterations. Subjects with a history of ophthalmological diseases that could affect visual acuity and vascular density, such as trauma, amblyopia, glaucoma, macular degeneration, macular hole, retinopathy of any type, uveitis and pathological myopia  were excluded. Patients with a history of any prior intraocular surgery, except uncomplicated cataracts, in either eye were also excluded. These exclusion criteria were applied to the fellow eye as well and patients with an anisometropia >2.0 Diopters were excluded since the fellow eye was used as the control eye.®, Mira Inc., Uxbridge, MA, USA) was placed under each rectus muscle. Segmental buckles were inserted underneath the silicone band to ensure retinal tear closure. Evacuative puncture and trans-scleral cryopexy were performed in all cases.All SB surgeries were performed by the same vitreoretinal surgeon under retrobulbar anesthesia. Following a 360° conjunctival peritomy and rectus muscle isolation, all retinal tears were localized. A 2.4 mm encircling silicone band  measurement, slit-lamp examination, dilated fundus examination, and Goldmann tonometry. The BCVA was evaluated using Early Treatment Diabetic Retinopathy charts and converted into the logarithm of the minimum angle of resolution units (logMAR).Axial length was measured preoperatively and 6-month post-operatively by using an IOL Master device  in eyes with macula-on RRD, while an immersion ultrasound biometry  was performed in eyes with macula-off RRD.Spectral domain OCT and OCTA were performed using the XR Avanti AngioVue System . A 6 × 6 mm high-definition (400 × 400) Angio scan pattern, centered on the foveola, was carried out in both eyes at baseline and 1, 3 and 6 months postoperatively. This imaging is based on the split-spectrum amplitude decorrelation angiography (SSADA) method. AngioAnalytic software  automatically calculated the VD in the superficial capillary plexus (SCP) and deep capillary plexus (DCP) of the scanned area, providing these data for the whole image as well as for the foveal, parafoveal (3 mm) and perifoveal (6 mm) ETDRS sub-fields. VD refers to the proportion of the vessel area in the region of interest. The foveal avascular zone (FAZ) area was automatically analyzed. Central macular thickness (CMT) was recorded using the same OCT system. The inbuilt three-dimensional (3D) projection artifact removal (PAR) algorithm improved the depth resolution of vascular layers and distinguished vascular plexus-specific features. The inbuilt software automatically evaluated scan quality and signal strength index (SSI). Two investigators (M.F. and A.L.) independently reviewed OCT and OCTA scans of eligible patients: patients with a scan quality <6 and/or an SSI < 60 and/or residual motion artifacts were excluded. In cases of disagreement in the imaging review process, a third investigator (A.R.) was consulted. Scan segmentation was also checked, and when observed, errors were corrected using the inbuilt editing and propagation tools.For each included patient, demographic characteristics and clinical and OCTA data were collected. The included patients were divided into two subgroups according to the preoperative status of the macula: the macula-on group and the macula-off group. The primary outcome was to assess changes in OCTA VD and FAZ area following SB surgery in the two groups. In the macula-on group, the change between preoperative and postoperative data of the RRD eye was analyzed, while in the macula-off group, given the lack of reliable baseline scans in the RRD eye, only the postoperative change from month 1 to month 6 was analyzed. In both groups, these data were also compared with baseline data of the fellow eye, which was used as a control. BCVA, CMT and axial length changes were considered secondary outcomes in both groups. In the macula-on group, possible correlations between OCTA parameters (FAZ and VD) and changes in BCVA and axial length were also investigated as secondary outcomes.t-test was used to compare continuous variables between the RRD eyes and the control eyes. In the macula-on group, correlations were studied by applying the Pearson regression test. A p value < 0.05 was considered significant. SPSS Statistics software version 21  was used.In the two groups, the values of each parameter detected at different time points were compared by ANOVA; if significant, multiple comparisons were made using Tukey’s HSD test. A A total of 37 eyes were included, of which 19 were in the macula-on group and 18 in the macula-off group. Baseline demographics and ocular characteristics are shown in p < 0.001). The mean baseline FAZ area was 0.227 ± 0.02 mm2 and remained unchanged following SB surgery. The mean baseline CMT was 244 ± 13 microns, with no significant change following SB surgery. Both the mean FAZ area and CMT in the RRD eyes were comparable with controls. Baseline VD in the whole SCP was lower in RRD eyes compared with controls (p < 0.001). A significant increase of VD in the whole SCP was demonstrated in RRD eyes following SB surgery ; however, 6-month VD in the whole SCP was lower compared with controls (p = 0.03). Similarly, SCP VD increased during follow-up in the parafoveal and perifoveal subfields , while it remained unchanged in the foveal subfield (p = 0.07). VD in the foveal SCP was comparable between RRD eyes and controls at both baseline (p = 0.83) and 6 months postoperatively (p = 0.74). In parafoveal and perifoveal subfileds of the SCP, VD was lower in RRD eyes compared with controls at both baseline  and 6 months postoperatively . VD in the DCP did not show any significant change following SB surgery and was comparable with the controls. The clinical and OCTA parameters of the macula-on RRD group are summarized in p = 0.03), perifoveal SCP  and parafoveal SCP . In all cases, an increase in axial length was associated with poorer VD outcomes . The mean axial length increased from 24.9 ± 0.5 mm to 26.2 ± 0.7 mm at 6 months (p = 0.001). Baseline OCTA parameters were not available. Following SB surgery, from month 1 to month 6, no significant change in either the mean CMT or mean FAZ area was found. Both 6-month CMT and FAZ area were comparable with controls .The clinical and OCTA parameters of the macula-off RRD group are summarized in p < 0.001). At 6 months, a lower-than-control VD was found in the parafoveal SCP and perifoveal SCP as well .Following SB surgery, from month 1 to month 6, no significant change in the VD of the SCP was shown. At 6 months, the mean VD of the whole SCP was lower compared with controls (p < 0.001). Such a significant increase was found in the parafoveal DCP as well (p < 0.001). No VD change was seen in the other subfields of the DCP. At 6 months, mean VD in DCP was lower compared with controls . During the follow-up, from month 1 to month 6, VD significantly increased in the whole DCP (This study sought to investigate macular microvascular changes on OCTA imaging after SB for RRD, showing a postoperative reduction in the VD of the SCP in both macula-on and macula-off RRDs. The postoperative VD of DCP seems to be impaired in eyes affected by macula-off RRD, while it seems to be unchanged in macula-on RRDs.Microvascular alterations in retinal and choroidal circulation occurring in RRD eyes have long been studied ,9,18. BeThe introduction of OCTA imaging represents a significant breakthrough in the assessment of retinal microcirculation. Several studies have explored macular microvascular alterations in OCTA imaging following RRD repair surgery ,16,17,23According to the majority of studies on vitrectomy cases, the VD of macular capillary plexi, both deep and superficial, is reduced after RRD surgery ,16,17,23Our findings showed that VD featured a similar increasing trend over a 6-month follow-up after SB surgery for RRD. In eyes with macula-on RRD, baseline VD was reduced in the SCP compared with controls; following the SB procedure, SCP VD increased over a 6-month period. In eyes with macula-off RRD, baseline OCTA was not available; in this group, VD in the DCP significantly improved from month 1 to month 6 postoperatively.Barca et al. hypothesized that the impairment of VD of the SCP in eyes with macula-on RRD might be related to the fact that the SCP is the first vascular layer affected in the case of a rapid increase of vascular resistance secondary to a recent retinal detachment . SCP is We found a reduction in VD in both superficial and deep capillary plexi in the macula-off group at 6 months after SB surgery. DCP has been shown to be more vulnerable to hypoxic injury . VasculaSeveral limitations characterize the present study. First, the retrospective design might have introduced some bias. However, strict eligibility criteria should have reduced this risk. Second, the sample size was relatively small. A larger population would have allowed additional analyses and further investigation of which clinical variables might have an influence on VD. Nonetheless, we included a total of 37 eyes and all surgeries were performed by the same vitreoretinal consultant. With regard to OCTA imaging, our software did not provide two different values of FAZ area for the deep and superficial capillary plexi, but only one value, including both layers. Furthermore, no correlation analysis was conducted in the macula-off group. The reason for this is that in this group, no baseline OCTA images were available because of the macula-off status. A correlation analysis between VD change and other variables would have been of poor reliability, given the absence of baseline OCTA data.Our analyses showed that VD on OCTA was reduced following SB surgery for RRD. These microvascular alterations, to some extent, are similar to those reported after vitrectomy and could be a consequence of the disease rather than the surgical procedure. Axial length increase seems to be associated with worse VD outcome. Whether this is secondary to the presence of a greater amount of subretinal fluid at baseline or to mechanical damage caused by the encircling band needs to be further investigated. Larger studies are warranted to better understand which mechanisms lead to such microvascular impairment and which clinical variables might have an influence on it.


Artemisia frigida willd, in nonalcoholic fatty liver disease (NAFLD).  Herbal medicine discovery is a complex and time-consuming process, while pharmacophore modeling and molecular docking methods enable simple and economic studies. The pharmacophore model provides an abstract description of essential intermolecular interactions between chemical structures, and the molecular docking technology can identify novel compounds of therapeutic interests and predict the ligand-target interaction at the molecular level. This study was based on the two methods to elucidate the mechanism of dehydrovomifoliol, an active ingredient extracted from  Bioinformatics analysis was performed to screen target genes of dehydrovomifoliol in NAFLD treatment, which were thus intersected with NAFLD-related differentially expressed genes (DEGs) and NAFLD-related genes. Venn diagram was used to identify candidate DEGs. A pharmacophore model was then generated, and molecular docking was performed. A protein-protein interaction (PPI) network was constructed to identify core genes, which were evaluated using GO and the KEGG enrichment analyses.  Seven target genes of dehydrovomifoliol in NAFLD treatment were screened out, namely E2F1, MERTK, SOX17, MMP9, SULT2A1, VEGFA, and BLVRA. The pharmacophore model and molecular docking of candidate DEGs and dehydrovomifoliol were successfully constructed. E2F1 was identified as a core gene of dehydrovomifoliol in NAFLD treatment. Further enrichment analysis indicated the regulatory role of E2F1 in fat metabolism was associated with the regulation of the AKT/mTOR signaling pathway.  Overall, this study illustrates the anti-NAFLD mechanism of dehydrovomifoliol, which could be a useful compound for developing novel drugs in the treatment of NAFLD. NAFLD dar level . It is il target . Previoul target  and key l target  in NAFLDArtemisia frigida willd, a traditional Chinese medicine, and can be used as an early lead compound for developing novel drugs for the prevention of NAFLD due to its effects on hepatic lipogenesis and fatty acid oxidation [via activation of the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway [Dehydrovomifoliol is an active ingredient extracted from xidation . Multiplxidation . Howeverxidation . Of inte pathway . In addi pathway . TherefoThe 3D chemical structure map of dehydrovomifoliol was downloaded from the PubChem database and saved in “SDF” format. The SDF file of dehydrovomifoliol was uploaded to the PharmMapper server, after which dehydrovomifoliol was simulated for molecular-target protein docking using the inverse docking tool PharmMapper server, with “Human Protein Targets Only” set as the target range and the remaining parameters were kept at their default values.NAFLD-related gene expression datasets GSE73500 and GSE89632 were downloaded from the GEO database. The GSE73500 dataset includes three liver tissue samples from NAFLD rats and three control liver tissue samples. The GSE89632 dataset includes nineteen liver tissue samples from NAFLD rats and 24 control liver tissue samples.The quality of paired-end reads of raw sequencing data was assessed using the FastQC v0.11.8 software. Preprocessing of raw data was performed by using the Cutadapt v1.18 software: the illumina sequencing adaptors and poly (A) tail sequences were removed. The reads with an N content of over 5% were removed using a Perl script. The reads with a 70% base mass above 20 were extracted using FASTX Toolkit software (v0.0.13). Next, two-end sequences were repaired using BBMap software. Finally, the filtered high-quality reads fragment human reference genome was aligned using HISAT2 software (v0.7.12).p value < 0.05 as the threshold.With normal samples as the control, the R “limma” package (version 3.4.1) was applied for differential analysis to identify the differentially expressed mRNAs with |log2FC| > 1 and NAFLD-related target genes were retrieved from the GeneCards database with “nonalcoholic fatty liver disease” and “fat cell proliferation” as the key words. With Relevance Score  ≥ 1 as the screening criteria, more relevant targets were obtained for subsequent analysis.The Draw Venn Diagram tool was used to perform Venn analysis on the dataset analysis results and the retrieval results of the GeneCards database to identify candidate genes.The Cytoscape (v3.8.2) plug-in Bisogenet and cytoNCA were used for intersection analysis. Bisogenet provides database information, while cytoNCA provides attribute information of nodes after network composition, and is utilized to calculate the degree centrality (DC) of nodes and observe the core genes intuitively. A protein-protein interaction (PPI) network was constructed and topological analysis was performed. Core targets were screened according to the DC and the betweenness centrality (BC).Homo sapiens” as the species, which was then imported into Cytoscape software (v3.8.2) for analysis and ranking. The Degree value and Combine score value were represented by color, and the candidate genes were ranked according to the Degree value.Through the STRING database, the interaction network of E2F1-mediated genes related to fat metabolism, was obtained with the “The crystal structures of seven core proteins E2F1 (PDB ID : 1O9K), MERTK (PDB ID : 2P0C), SOX17 (PDB ID : 2YUI), MMP9 (PDB ID : 1ITV), SULT2A1 (PDB ID : 2QP4), VEGFA (PDB ID : 1BJ1), and BLVRA (PDB ID : 2H63) were downloaded and screened from the Protein Data Bank. The structure of dehydrovomifoliol was downloaded from the PubChem database, converted to a 3D structure using Chem3DUltra 14.0 software, and subjected to energy minimization using the MM2 algorithm. The receptors of the seven target proteins were then subjected to dehydration and organic substance removal using PyMOL software. Target protein receptor molecules were hydrogenated, followed by charge calculation using AutoDockTools 1.5.6 software. Next, compounds and target protein receptors were converted into “PDBQT” files with appropriate box center and box lattice parameters set. Finally, Vina 1.5.6 software was run to evaluate the molecular docking and calculate the docking energy value.p < 0.05 as the threshold. KEGG enrichment analysis was performed to analyze cellular functions and signaling pathways mainly affected by the potential targets and key targets with p < 0.05 as the threshold.The R “ClusterProfiler” package was used for GO enrichment analysis, which consists of biological process (BP), molecular function (MF), and cellular component (CC), with t test. A p value < 0.05 was considered significant.All statistical analyses in this study were performed using SPSS 21.0 software . Measurement data were presented as mean ± standard deviation. Data obeying normal distribution and homogeneity of variance between two groups were compared using unpaired Artemisia frigida willd. This study explored the mechanism of dehydrovomifoliol in NAFLD based on pharmacophore model building and molecular docking methods. The experimental schedule is shown in Dehydrovomifoliol is an active ingredient extracted from the traditional Chinese medicine First, the chemical structure of dehydrovomifoliol was determined using the PubChem database Figures . The PhaSubsequently, the target genes from the PharmMapper database, DEGs from the GSE73500 and GSE89632 datasets, and NAFLD-related genes from the GeneCards database were subjected to Venn analysis, with seven target genes of dehydrovomifoliol in NAFLD treatment obtained , namely When drug molecules interact with receptor targets, it is necessary to generate geometric and energy matched active conformations with targets. Pharmacophore can represent the characteristic elements of pharmacodynamics and their spatial arrangement in the active molecule that play an important role in the activity and can also reflect the active site where the ligand and the receptor interact. Pharmacophore structure analysis of dehydrovomifoliol and the seven receptor targets involved in NAFLD treatment was performed using the PharmMapper database. The results showed that four hydrophobic and one acceptor interact with E2F1; two hydrophobic, one donor, and one acceptor interact with MERTK; six hydrophobic, one donor, and one acceptor interact with SOX17; two hydrophobic, one positive region, two donor, and one acceptor interact with MMP9; three hydrophobic and one acceptor interact with SULT2A1; three hydrophobic, one negative region, and three acceptor interact with VEGFA; four hydrophobic and one acceptor interact with BLVRA Figures . AccordiMolecular docking analysis of candidate proteins and dehydrovomifoliol was performed using AutoDockTools 1.5.6 and Vina 1.5.6 software. When the binding energy is < 0 kJ/mol, the molecular proteins are considered to bind spontaneously and interact with each other; the lower binding energy reflects more stable molecular conformation. The results of docking analysis of candidate proteins with dehydrovomifoliol are shown in Figures Seven candidate genes were imported into Cytoscape software to build a network map, whereupon PPI network analysis was performed using the Cytoscape plug-in Bisogenet. HPRD, BIND, DIP, MINT, INTACT, and BIOGRID databases were selected for analysis, and 238 nodes and 1509 edges were obtained. The DC values of the 238 target proteins were calculated using the plug-in CytoNCA, and the data with DC values > 10 were selected to create a subnetwork, with 103 nodes and 1047 edges determined. The plug-in CytoNCA was then used for topological analysis to calculate the BC of 103 target proteins in the subnetwork, and a total of 14 target proteins of BC ≥ 200 were screened. A PPI network was created, resulting in 14 nodes and 59 edges . Among tGO enrichment analysis revealed that the seven candidate DEGs were mainly enriched in the BP of fat cell proliferation, regulation of fat cell proliferation, and negative regulation of cysteine-type endopeptidase activity involved in apoptotic process entries ; CC of pDuring the development of NAFLD, insulin resistance leads to increased liver lipogenesis and inhibits fat decomposition, resulting in fat storage in adipocytes in the form of triglycerides in the liver and the consequent formation of fatty liver . Among tp value < 1.0e − 16 (β-oxidation, fatty acid catabolic processes, fat cell differentiation, and fatty acid oxidation (The accumulation of fat in liver tissues leads to an increase of free fatty acids and reactive oxygen species, which can cause oxidative stress and endoplasmic reticulum stress, further resulting in damage to hepatocytes and aggravating NAFLD . To expl.0e − 16 . Then, 1.0e − 16 . Further.0e − 16 . Among txidation . EvidencTraditional Chinese medicine have gained widespread attention in recent years, but knowledge of the mechanisms by which they act is still very limited. In this work, we generated a qualitative pharmacophore model and applied the molecular docking technology to elucidate the mechanism underlying dehydrovomifoliol alleviating NAFLD. The results demonstrated that dehydrovomifoliol may alter abnormalities in fat metabolism and consequently alleviate NAFLD by downregulating the E2F1/AKT/mTOR axis.The results of the current study identified seven target genes of dehydrovomifoliol in NAFLD treatment, namely E2F1, MERTK, SOX17, MMP9, SULT2A1, VEGFA, and BLVRA. Among them, MERTK , MMP9 2, and VEGin silico method in drug discovery programs, can predict the binding mode of a given molecule interacting with a specific biological target [It is interesting to note that molecular docking, as a widely applied l target . Subsequl target . Additiol target . The curl target . Nonethevia activation of the PI3K/AKT/mTOR signaling pathway [Further mechanistic investigations suggested that E2F1 may regulate fat metabolism and then participate in the development of NAFLD by mediating the AKT/mTOR signaling pathway. Additionally, the E2F1/DDX11 axis is capable of promoting malignant behaviors of hepatocellular carcinoma cells  pathway . Accumul pathway , 18. Rec pathway . Thus, sin vitro and in vivo experiments are required to substantiate the mechanistic basis and therapeutic effects of dehydrovomifoliol in NAFLD.In summary, the key findings from the present study suggested that dehydrovomifoliol potentially alleviates NAFLD through disruption of the E2F1/AKT/mTOR axis . These r


Background: The Yizhiqinxin formula (YZQX) has been used to treat Alzheimer’s disease (AD) or major depression disorder (MDD). However, its specific underlying mechanisms and therapeutic targets remain unclear.Methods: The ingredients and putative targets of YZQX were screened using the TCMSP and Drugbank databases. Next, the GEO database was used to retrieve relevant differentially expressed genes (DEGs) in AD or MDD and normal tissues. The PPI network was established, merged, and further screened to identify the main ingredients and core targets of YZQX against AD and MDD comorbidities. We performed enrichment analysis of core targets to identify biological processes and pathways. Finally, AutoDock software was used to validate the binding affinity between the crucial targets of direct action and their corresponding ingredients.Results: A total of 43 ingredients were identified from YZQX, of which 43 were screened to yield 504 targets. By establishing the PPI network, 92 targets were regarded as targets of YZQX against AD and MDD comorbidities in the core network. Promising targets  and signaling pathways  were filtered and refined to elucidate the underlying mechanism of YZQX against AD and MDD comorbidities. Molecular docking confirmed the ingredients of YZQX (quercetin and kaempferol) could bind well to multiple crucial targets.Conclusion: The ingredients of YZQX, such as quercetin and kaempferol, might treat AD and MDD comorbidities by acting on multiple targets and pathways. As a complex, multifactorial, progressive neurodegenerative disorder, Alzheimer’s disease (AD) is a major cause of dementia, resulting in the dysregulation of cognitive function, daily behavior, memory, and so on . Accordiin vivo and in vitro  to prevent or treat chronic diseases because of their lower toxicity and more significant curative effects . YZQX foin vitro . It is win vitro . In addiin vitro . HoweverNetwork pharmacology is a novel and powerful tool that could clarify the potential mechanisms of multiple ingredients, targets, and pathways of TCM . Moleculhttps://tcmspw.com/tcmsp.php)  and drug-likeness (DL) are the two main parameters of the absorption, distribution, metabolism, and excretion (ADME) model  that affmsp.php) .https://www.drugbank.ca/) (Homo sapiens” in the Uniprot database (https://www.uniprot.org/) (2017). In addition, we used the Cytoscape 3.7.2 software to construct and present a Herb-ingredient-target network of the YZQX formula. In this network, nodes represent formulas, herbs, ingredients, and targets, while the edges represent their interaction relationships.After screening the active ingredients of the YZQX formula, the next significant process was to identify their corresponding targets. The putative targets of the active ingredients in the YZQX formula were identified and collected using the DrugBank database (ank.ca/) . Furtherhttp://www.ncbi.nlm.nih.gov/geo/) using their microarray platforms, GPL10558 (Illumina HumanHT-12 V4.0 expression bead chip) and GPL570 (Affymetrix Human Gene Expression Array), respectively. Among them, the GSE63061 dataset contained 139 AD samples and 134 normal samples, and the GSE98793 dataset contained 128 MDD samples and 64 normal samples. Thereafter, the probes were converted into gene symbols according to the annotation information of the corresponding platform. The DEGs between disease (AD or MDD) groups and normal groups were identified and screened as implemented in the R software (Version 3.6.0) limma package with a threshold value of p < 0.05. DEGs with p < 0.05, and |log2 fold change (FC)| > 0.1 were then presented visually using volcano plots. Venn diagrams were used to further obtain the overlapping genes from the AD and MDD microarray datasets.The microarray data of GSE63061 and GSE98793 were downloaded from the GEO database , cellular component (CC), and molecular function (MF). The results of GO, sorted from small to large according to the p-value as a parameter, and the top 10 terms of each category were selected using a bubble plot for visualization. For the top five terms, a sub-network was then established in Cytoscape software version 3.7.2 to visualize the relationships between genes and GO terms. Furthermore, based on the genes of the core network of YZQX in AD and MDD treatment, KEGG pathway analysis was performed, and a bubble plot was plotted.ClusterProfiler , a new ohttps://www.rcsb.org/) and saved in PDB format. The MOL2 formats of the bioactive ingredients were downloaded from the ZINC database (http://zinc.docking.org/). Before molecular docking, we used AutoDock software (version 4.2.6) to remove the ligand, hydrogenate, calculate the charge, add protein type for the macromolecules, and saved it in PDBQT format. Subsequently, we ran the AutoDock to perform molecular docking between macromolecules and ligands based on the default parameters. Blinding energy of less than “−5” represented a better binding interaction between macromolecules and small molecules  in-depth, we performed molecular docking based on molecular modeling techniques using AutoDock software . We subsolecules . FinallyA total of 43 ingredients in YZQX were screened from the TCMSP database with the criteria of OB ≥ 30% and DL ≥ 0.18. These were defined as candidate bioactive ingredients, of which seven were from Chuanxiong, 14 from Huanglian, and 22 from Renshen. The detailed characteristics, including molecular ID, molecular name, structure, OB, and DL, of the selected bioactive ingredients are presented in Subsequently, using Cytoscape software, we established a network indicating the relationship among Chinese herbs, ingredients, and putative targets, as shown in p < 0.05, a total of 1,707 DEGs, containing 889 downregulated and 818 upregulated genes, were extracted and analyzed from the microarray data GSE98793. The DEGs with p < 0.05, and |log2 FC| > 0.1 in AD or MDD were visualized in a volcano plot   . As indi < 0.05) .HSP90AA1, ESR1, AKT1, VCAM1, EGFR, CDK1, MAPK1, CDK2, MYC, HSPB1, and HSPA5 are pivotal targets in the core network mentioned above, and these nodes were also overlapping genes of AD and MDD comorbidity. In accordance with key targets, we worked backward to their corresponding bioactive ingredients serving as ligands, thus performing validation of molecular docking. In total, 2 ingredients were docked with AKT1, 2 with CDK1, 3 with CDK2, 6 with ESR1, 11 with HSP90AA1, 1 with EGFR, 1 with HSPA5, 1 with HSPB1, 1 with MAPK1, 1 with MYC, and 1 with VCAM1. The ligand-receptor binding conformation had the lowest binding energy, indicating that this conformation is the most stable, and thus the possibility of interaction is the greatest. The top three docking combinations for the target macromolecules and corresponding ingredients included Fumarine and HSP90AA1, Worenine and ESR1, berberine and HSP90AA1, with binding energies of −6.83, −6.6, and −6.26 kcal/mol, respectively. The other specific docking scores of the ingredients with targets are shown in The PPI network indicated that HSP90AA1, ESR1, AKT1, VCAM1, EGFR, CDK1, MAPK1, CDK2, MYC, HSPB1, and HSPA5 acted directly on these diseases. Further, KEGG pathway analysis revealed that these core targets were mainly related to the PI3K-Akt signalling pathway, ubiquitin-mediated proteolysis, estrogen signalling pathway, MAPK signalling pathway, TGF-beta signalling pathway, etc. The abovementioned evidence suggests the clinically promising potential of YZQX in the treatment of AD comorbidity with depression.Depression is recognized as the most common comorbidity of AD and affects the treatment of AD patients . Hence, in vitro study on rats indicated that kaempferol improved the cognitive function in AD rat models, thereby exerting its neuroprotective effect (In this study, we identified 43 ingredients in YZQX, and molecular docking was performed to compare these crucial targets with the corresponding ingredients. Quercetin and kaempferol could bind well to multiple crucial targets, which served as the main bioactive ingredients. Quercetin is a predominant flavonoid that has multiple beneficial properties, including antioxidative and anti-inflammatory effects . Moreovee effect . Likewise effect . Of notee effect .Moreover, in this study, PPI networks between the ingredient-target of YZQX and comorbidity with AD and MDD were established using Cytoscape, followed by merging and topological parameters. Eventually, 92 targets were identified and enrichment analysis was performed to further clarify the underlying mechanisms of YZQX treatment in patients with comorbid AD and MDD. KEGG analysis indicated that 92 targets were mainly related to the following pathways: PI3K-Akt signalling pathway, ubiquitin-mediated proteolysis, estrogen signalling pathway, MAPK signalling pathway, TGF-beta signalling pathway, etc. Converging reports on the PI3K-Akt signaling pathway have shown that it plays a crucial role in multiple processes of cell reproduction, differentiation, and apoptosis. Further enhancing PI3K/Akt function may prevent the development of AD by alleviating the downstream effects of Aβ . MoreoveESR1 can regulate gene expression and its corresponding function via the interactions between regulatory regions and target genes (ESR1 serves as a key factor of depression, particularly in women (ESR1 can control the activity of serotonin-activated neurons by regulating the quantity and function of serotonin receptors and controlling neurotransmitters (HSP90AA1, a member of the heat shock family, is highly conserved in eukaryotes. Of particular interest was the finding that HSPs are also involved in regulating the stabilization and function of p-tau in AD (Some hub targets predicted by molecular docking in this study have also been reported in previous studies . ESR1, let genes . A studyin women . Interessmitters . HSP90AAau in AD . Apart fau in AD .via action on the PI3K-Akt signalling pathway, ubiquitin-mediated proteolysis, Estrogen signalling pathway, MAPK signalling pathway, and TGF-beta signalling pathway. This study further identified HSP90AA1, ESR1, AKT1, VCAM1, EGFR, CDK1, MAPK1, CDK2, MYC, HSPB1, and HSPA5 as the key targets of YZQX in the treatment of AD with comorbid depression. This study further confirmed that YZQX is a promising therapeutic method for the development of safe and effective multitarget for AD with depression.In the present study, the underlying mechanisms and molecular targets of YZQX for AD with depression were illustrated using integrated network pharmacology and molecular docking approach. YZQX may exert its effects


This research is aimed at identifying the key genes and pathways of cardiac hypertrophy using bioinformatics and at providing a new target for the identification of cardiac hypertrophy.  Microarray data GSE1621 and GSE18801 were acquired from the GEO database. The DEGs of GSE1621 and GSE18801 were analyzed using the online tool GEO2R. “ggplot2” package of R software was utilized to generate the volcano plots. The top and bottom 10 genes were mapped as a heat map. GO functional annotation analysis and KEGG pathway enrichment analysis were performed separately for DEGs using the online software DAVID. Histograms were plotted using the R “ggplot2” package. The DEGs were imported into the STRING online database for constructing PPI networks and analyzing the DEG interaction relationships.  In the present study, 469 DEGs were screened in GSE1621 and a total of 793 DEGs were screened in GSE18801. GO analyses indicate that DEGs were mainly involved in cardiac muscle contraction, regulation of blood circulation, regulation of muscle contraction, muscle contraction, striated muscle contraction, regulation of heart contraction, regulation of striated muscle contraction, and tissue remodeling. KEGG analyses indicate that DEGs were mainly involved in Th17 cell differentiation, Th1 and Th2 cell differentiation, HIF-1 signaling pathway, pathways in cancer, hematopoietic cell lineage, Chagas disease and cell adhesion molecules, viral myocarditis, central carbon metabolism in cancer, acute myeloid leukemia, and JAK-STAT signaling pathway. Eight hub genes were screened, including Akt1, Lox, Timp1, Col1al, Spp1, Ccnd1, Mmp3, and Egfr.  The DEGs associated with cardiac hypertrophy were screened via bioinformatics analysis, and eight hub genes were identified, including Akt1, Lox, Timp1, Col1al, Spp1, Ccnd1, Mmp3, and Egfr, which might be a new target for the identification of cardiac hypertrophy. Cardiac hypertrophy is an adaptive response of the myocardium to increased sustained load and often leads to cardiovascular disease . PatientCardiac hypertrophy is mainly characterized by hypertrophy of cardiomyocytes and alterations in interstitial components . PatholoWith the development of bioinformatics, genomics, transcriptomics, proteomics, and metabolomics have generated big data, which can be analyzed by combining bioinformatics and computer science, providing new methods to study the molecular mechanisms of diseases . With thIn this study, the gene microarray of myocardial hypertrophy in GEO database was used to screen out DEGs, and online databases such as GO and KEGG were utilized to annotate differential gene functions and analyze gene enriched signaling pathways, which provided ideas for identifying new biomarkers of myocardial hypertrophy and revealing potential mechanisms of the disease.Microarray data GSE1621 and GSE18801 were downloaded from the GEO database. GSE1621 included 4 myocardial hypertrophy mice caused by coarctation of aortic arch and 3 sham mice, and the platform was Affymetrix Murine Genome U74A Version 2 Array. GSE18801 included 3 isoproterenol-induced cardiac hypertrophy subjects and 3 normal subjects, and the platform was Affymetrix Mouse Genome 430 2.0 Array.P value < 0.05 and |Log2 fold change (LogFC)| >1.5. “ggplot2” package was utilized to generate the volcano plots. The top and bottom 10 genes were mapped as a heat map.The DEGs of GSE1621 and GSE18801 were analyzed using the online tool GEO2R. The screening criteria for DEGs were adjusted https://david.ncifcrf.gov/tools.jsp). Histograms were plotted using the R “ggplot2” package.GO functional annotation analysis and KEGG pathway enrichment analysis were performed separately for DEGs using the online software DAVID (https://cn.string-db.org/) for analysis. Cytoscape software was used to construct PPI networks and analyze the DEG interaction relationships. Finally, the “MCC” algorithm of “Cytohubba” plugin was used to build the module and screen the candidate hub genes.The DEGs were first imported into the STRING online database  and pathological stimuli  . Patholoα and HIF-1β. HIF-1α expression induced by pressure load will lead to a significant increase in heart mass to body weight ratio and increased BNP expression and promote the occurrence of myocardial hypertrophy [β, mechanical stretch, and tyrosine kinase receptors and is involved in a series of protein kinase phosphorylations which is essential in the progression of myocardial pathological hypertrophy [In the present study, 469 DEGs were screened in GSE1621 and a total of 793 DEGs were screened in GSE18801. GO analyses indicate that DEGs were mainly involved in cardiac muscle contraction, regulation of blood circulation, regulation of muscle contraction, muscle contraction, striated muscle contraction, regulation of heart contraction, regulation of striated muscle contraction, and tissue remodeling. KEGG analyses indicate that DEGs were mainly involved in Th17 cell differentiation, Th1 and Th2 cell differentiation, HIF-1 signaling pathway, pathways in cancer, hematopoietic cell lineage, Chagas disease and cell adhesion molecules, viral myocarditis, central carbon metabolism in cancer, JAK-STAT signaling pathway, and acute myeloid leukemia. HIF-1 is a dimeric transcription factor composed of two subunits, including HIF-1ertrophy . PI3K isertrophy . JAK-STAertrophy . The MAPertrophy . Eight hSerine/threonine kinase (Akt), also known as PKB, is a direct downstream target protein of PI3K . Akt actβ1 upregulated collagen type 1, col1a1, and col3a1. Col1a1 and col3a1 are closely related to the formation of myocardial hypertrophy.The extracellular matrix (ECM), which surrounds the cardiomyocytes, protects the functional and structural integrity of the heart. Alterations in the extracellular matrix, particularly abnormal alterations in type 1 collagen fibers and type 2 collagen fibers, play an important role in myocardial remodeling. Alterations in the extracellular matrix of the myocardium, particularly the deposition of myocardial collagen that develops asynchronously with cardiomyocyte hypertrophy, are important in contributing to the progression of myocardial hypertrophy. Matrix metalloproteinases (MMPs) of myocardial hypertrophy are protein-cleaving enzymes that hydrolyze the extracellular matrix and consist mainly of collagenases . MMPs caIn conclusion, in this study, the DEGs associated with cardiac hypertrophy were screened via bioinformatics analysis, and eight hub genes were identified, including Akt1, Lox, Timp1, Col1al, Spp1, Ccnd1, Mmp3, and Egfr, which might play an important role in the development of cardiac hypertrophy induced by pressure overload, further providing more information for studies on pathogenesis and screening of potential biomarkers of cardiac hypertrophy.