Fenugreek is an ancient herb that has been used for centuries to treat diabetes. However, how the fenugreek‐derived chemical compounds work in treating diabetes remains unclarified. Herein, we integrate molecular docking and network pharmacology to elucidate the active constituents and potential mechanisms of fenugreek against diabetes. First, 19 active compounds from fenugreek and 71 key diabetes‐related targets were identified through network pharmacology analysis. Then, molecular docking and simulations results suggest diosgenin, luteolin and quercetin against diabetes via regulation of the genes ESR1, CAV1, VEGFA, TP53, CAT, AKT1, IL6 and IL1. These compounds and genes may be key factors of fenugreek in treating diabetes. Cells results demonstrate that fenugreek has good biological safety and can effectively improve the glucose consumption of IR‐HepG2 cells. Pathway enrichment analysis revealed that the anti‐diabetic effect of fenugreek was regulated by the AGE‐RAGE and NF‐κB signalling pathways. It is mainly associated with anti‐oxidative stress, anti‐inflammatory response and β‐cell protection. Our study identified the active constituents and potential signalling pathways involved in the anti‐diabetic effect of fenugreek. These findings provide a theoretical basis for understanding the mechanism of the anti‐diabetic effect of fenugreek. Finally, this study may help for developing anti‐diabetic dietary supplements or drugs based on fenugreek. The study workflow is shown in Figure 22.1http://lsp.nwu.edu.cn/tcmsp.php/).http://bindingdb. org/bind/index.jsp), DrugBank (https://go.drugbank.com/), STITCH (http://stitch.embl.de/) and SwissTargetPrediction (http://www.swisstargetprediction.ch/) databases were searched for related targets of active compounds based on their SMILES formulas.A list of compounds in fenugreek was obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (2.2www.genecards. org/) and OMIM (https:// www.omim.org/), using the keywords ‘diabetes’ and ‘hyperlipidemia,’ respectively, with the aim of obtaining the related targets of diseases in Homo sapiens. Of the targets obtained from GeneCards, those with a relevance score > 40 were selected as potential targets. As for the OMIM datasets, all of the targets related to diabetes were involved. Next, an online Venn diagram drawing tool, Venny ver. 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/), was used to visualize the overlapping targets between the selected compounds and diabetes target genes.Searches were conducted of GeneCards . To further investigate the mechanism of action of fenugreek in the treatment of T2DM, the intersection targets were imported into String. The minimum combined score was set to 0.400 and protein‐interaction relationships for Homo sapiens were obtained. Targets without interaction were removed and the data were saved as SIF files. Next, the PPI network was constructed by importing node1, node2 and the combined score into Cytoscape. Finally, the core targets were screened by cluster analysis using the MCODE plug‐in in Cytoscape.Protein‐protein interactions were analysed using String ver. 11.0 (2.3.3p < 0.05) were chosen for analysis. At the same time, KEGG pathway enrichment analysis was performed to investigate the major signalling pathways involved in the anti‐diabetic effects of fenugreek. Finally, the enriched results were analysed using the ‘ggplot2’ package in R.Gene Ontology (GO) enrichment analysis was performed by running the ‘cluster Profiler’ package in R ver. . The Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) modules were selected and significant biological annotations . Then, the progress of dehydration, hydrogenation and charge editing were monitored using AutoDock software 4.2.6, and the modified target proteins and important compounds were exported as PDBQT files. Next, docking progress was monitored separately using AutoDock Vina software 1.2.0. Finally, the docking results were imported into PyMol software for analysis and visualization. Here, we employed Metformin, a commonly used treatment drug for diabetes mellitus, as reference drug.Molecular docking can help to validate key targets in network pharmacology.50, reverse mutation assay (Ames test) and cytotoxicity, were recorded.The SwissADME online server was used to perform machine learning to predict the drug‐like properties. The six important compounds were input into the SwissADME website and the ADMET properties were selected for analysis. The toxicity of the six important compounds was predicted using the free online tool ProTox‐II, and the results, including predicted LD2.5GROMACS (2021.3) with CHARMm36 force field were employed for MD simulations. According to docking results, the top three protein‐ligand complexes in each group were selected for MD simulations. Protein‐inhibitor complex from PDB datebase were employed as the reference systems. Simulate parameters were taken as described.2.65 cells/well and cultured in the cell incubator for 24 h. Then, cells were starved for 24 h by serum‐free medium. The serum‐free medium was then replaced with high‐glucose DMEM (50 μM) with recombinant human insulin (5 × 10−7 M) for 48 h to establish IR model.HepG2 cells were used to establish insulin resistance model. As the method described by Chen et al.6 cells/ml) were added into 96‐well plates. After 24 h pre‐adhesion, the original medium was replaced with fenugreek solution at different concentrations . Cells were cultured for another 24 h and 48 h. Then, CCK‐8 test were performed to evaluate the cell viability.100 μL of HepG2 cells  to estimate the anti‐diabetic effects of fenugreek.33.1Searching the TCMSP database, 14 putative compounds derived from fenugreek, with a threshold value OB ≥30% and DL ≥0.18, were identified by screening. Meanwhile a search of the literature was conducted, and five active constituents were identified. The basic characteristics of all 19 active constituents are presented in Table 3.2After exploring the TCMSP, SwissTargetPrediction, DrugBank and other databases. 277 diabetes‐related targets without duplicate values were obtained. Meanwhile, a total of 462 diabetes targets were identified in the GeneCards and OMIM databases, using diabetes‐related target screening rules. As shown in Figure 3.3To construct the Drug–Compound–Target–Disease network Figure , 71 comm3.4A total of 71 common targets were input to the String database, and the PPI network was exported as a TSV file to Cytoscape, where the PPI network was visualized and analysed. As shown in Figure 3.5p < 0.01) were enriched, including 1553 for BP, 61 for CC and 99 for MF. Top 10 GO terms ranked by p value for BP, CC and MF were plotted in a graph, for a total of 30. In Figure GO functional enrichment analysis was carried out using the Database for Annotation, Visualization and Integrated Discovery. A total of 1713 GO terms  calculated by AutoDock Vina was used to estimate the bonding activity between the docking molecules, with a smaller Vina score indicating stronger bonding activity as well as a higher affinity and more stable structure between the ligand and receptor. Compared to the reference drug, these important compounds showed a stronger affinity for the key targets  in 10 ns of the simulation trajectory. Root mean square fluctuation (RMSF) were used to analyse the fluctuation and flexibility of each complex during simulation. As show in Figure Meanwhile, the binding energies of these complexes were calculated using the GMX‐MM/PBSA method. From Table 3.9Figure 4Previous research has investigated the anti‐diabetic effects of fenugreek.In recent years, molecular docking technology and network pharmacology have been used to screen for active constituents and potential targets of action, to evaluate the degree of compound–target association, and to explain the pharmacological mechanisms of action in traditional Chinese medicines.50 (>150 mg/kg) and no cytotoxicity. In addition, molecular docking and dynamics simulation further screen out that diosgenin, quercetin and luteolin show stronger binding capacity with the corresponding targets. These results suggest that the connection between the selected core active constituents and the core diabetes‐related target genes is stable, indicating that the core active compounds in fenugreek can bind to the key genes involved in diabetes mellitus and induce anti‐diabetic activity. Futhermore, cell culture results demonstrated that fenugreek not only has good biological safety in vitro, but also can effectively improve the glucose consumption of insulin resistance HepG2 cells. Thus, our results further suggest that these core active constituents might be candidate drugs for the treatment of diabetes and its complications.This study systematically investigated the pharmacological mechanism of fenugreek in the treatment of diabetes. First, the active constituents were obtained from the TCMSP, CNKI and PubMed databases. Then, the physical and chemical information of these compounds was analysed, and 19 compounds—mairin, β‐sitosterol, irolone, formononetin, calycosin, kaempferol, diosgenin, gallic acid‐3‐O‐‐glucoside, luteolin, hiyodori lactone A, tigogenone, isovitexin, neotigogenin, quercetin, vicenin‐2, vitexin‐7‐glucoside_qt, isoorientin, vitexin and 4‐hydroxyisoleucine—were determined to be active constituents, which is consistent with the results of a recent study.According to the GO functional enrichment analysis, the anti‐diabetic GO activities of fenugreek affect mainly BP, including response to peptides, response to oxygen levels, cellular response to chemical stress, positive regulation of protein phosphorylation, regulation of the apoptotic signalling pathway and regulation of the circulatory system. Based on the results of KEGG pathway enrichment analysis, in addition to the AGE‐RAGE pathway, which is closely related to diabetes, other related signalling pathways were enriched in terms of diabetes complications, including the cancer signalling pathway, the FoxO signalling pathway, the HIF‐1 signalling pathway, the NF‐κB signalling pathway and lipids and atherosclerosis. A growing number of studies has shown that the AGE‐RAGE signalling pathway plays a key role in the generation of vascular complications related to diabetes, including atherosclerosis and kidney disease.In terms of increasing the levels glucose and other reducing sugars, AGE‐RAGE signalling is strongly amplified, thereby inducing the release of NAD(P)H oxidase and increasing the production of reactive oxygen species. This condition may cause severe oxidative stress as well as vascular endothelial and peripheral nerve dysfunction, which in turn can lead to serious diabetic complications, including diabetic foot, diabetic nephropathy and diabetic myocardial injury.5We used molecular docking, molecular dynamics simulation and network pharmacology to conduct a preliminary study of the active constituents and mechanisms of action of fenugreek in the treatment of diabetes. A total of 19 active constituents were selected, three of which—diosgenin, luteolin and quercetin—were screened as core active constituents. In addition, a total of 71 common target genes were screened, of which ESR1, CAV1, VEGFA, TP53, CAT, AKT1, IL6 and IL1 were predicted to be the core target genes. Furthermore, KEGG pathway enrichment analysis showed that fenugreek exerted anti‐diabetic effects by inhibiting inflammatory signalling pathway, reducing inflammatory factor expression and protecting the vascular endothelium, peripheral nerves and islet cells against inflammatory cytokines. The signalling pathways include the AGE‐RAGE signalling pathway, the FoxO signalling pathway, the HIF‐1 signalling pathway, the NF‐κB signalling pathway and lipids and atherosclerosis. Overall, this study provides profound insights into the biological activity, potential targets of interaction, and mode of action of fenugreek against diabetes. It also provides a theoretical basis for exploring the role and related mechanisms of fenugreek in the treatment of diabetes. Moreover, those selected constituents are biocompatible and antidiabetic. Thus, they could be potential therapeutic agents for diabetes mellitus. Our results may help for developing anti‐diabetic dietary supplements or drugs base on fenugreek. Going forward, we will conduct animal and clinical studies to validate the screening targets based on this predictive analysis, with the aim of providing additional scientific evidence for the clinical application of fenugreek in the treatment of diabetes.Wenfeng Luo: Conceptualization (lead); data curation ; project administration ; resources ; writing – original draft . Jie Deng: Data curation ; formal analysis ; funding acquisition ; software . Jiecheng He: Data curation ; methodology ; software ; visualization . Liang Yin: Data curation ; investigation ; visualization . Rong You: Conceptualization ; methodology ; supervision ; writing – review and editing . Lingkun Zhang: Investigation ; software ; validation . Jian Shen: Resources ; software ; validation . Zeping Han: Conceptualization ; investigation ; software . Fangmei Xie: Data curation ; resources . Jin‐Hua He: Funding acquisition ; visualization ; writing – review and editing . Yanqing Guan: Conceptualization ; funding acquisition ; project administration ; writing – review and editing .This work was supported by the Science and Technology Program of Panyu , the Science and Technology Project of the Guangzhou Health Commission (No. 20211A011114), the Science and Technology Program of Guangzhou (No. 202002020023), the General University Youth Innovative Talent Project of Guangdong Province (No. 2022KQNCX281).The authors have no conflicts of interest to declare.


Background:Macleaya cordata extract (MCE) is effective in the treatment of enteritis, but its mechanism has not been fully elucidated. Therefore, this study combined network pharmacology and molecular docking technologies to investigate the potential pharmacological mechanism of MCE in the treatment of enteritis.Methods: The information of active compounds in MCE was accessed through the literature. Furthermore, PubChem, PharmMapper, UniProt, and GeneCards databases were used to analyze the targets of MCE and enteritis. The intersection of drug and disease targets was imported into the STRING database, and the analysis results were imported into Cytoscape 3.7.1 software to construct a protein–protein interaction (PPI) network and to screen core targets. The Metascape database was used for conducting Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. AutoDock Tools software was used for the molecular docking of active compounds with the core targets.Results: MCE has four active compounds, namely, sanguinarine, chelerythrine, protopine, and allocryptopine, and a total of 269 targets after de-duplication. Furthermore, a total of 1,237 targets were associated with enteritis, 70 of which were obtained by aiding the drug–disease intersection with the aforementioned four active compound targets of MCE. Five core targets including mitogen-activated protein kinase 1 (MAPK1) and AKT serine/threonine kinase 1 (AKT1) were obtained using the PPI network, which are considered the potential targets for the four active compounds of MCE in the treatment of enteritis. The GO enrichment analysis involved 749 biological processes, 47 cellular components, and 64 molecular functions. The KEGG pathway enrichment analysis revealed 142 pathways involved in the treatment of enteritis by the four active compounds of MCE, among which PI3K-Akt and MAPK signaling pathways were the most important pathways. The results of molecular docking showed that the four active compounds demonstrated good binding properties at the five core targets.Conclusion: The pharmacological effects of the four active compounds of MCE in the treatment of enteritis involve acting on signaling pathways such as PI3K-Akt and MAPK through key targets such as AKT1 and MAPK1, thus providing new indications for further research to verify its mechanisms. Macleaya cordata is a perennial, erect, and tall herb of the poppy family, with the advantages of clearing away heat, detoxification, subduing swelling, and eliminating parasites. Macleaya cordata extract (MCE) has anti-inflammatory, antibacterial, and anti-tumor effects  and PubMed (https://pubmed.ncbi.nlm.nih.gov/) were used to review the main active substances of MCE and their active compounds. The 2D structures of the active compounds were consulted and downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Then, the 2D structures were imported into the PharmMapper database (http://www.lilab-ecust.cn/pharmmapper/) to obtain UniPlot (https://www.uniprot.org/) to obtain the targets for the active compounds.Literature search engines such as China National Knowledge Infrastructure ( UniPlot . Furtherhttps://www.genecards.org/) was searched using “enteritis” as the keyword, and the results were exported in an Excel format. The data were de-duplicated and filtered based on the relevance score of >1, and the resulting data were considered enteritis-related targets.The GeneCards database (https://bioinfogp.cnb.csic.es/tools/venny/), respectively, to obtain the drug–disease intersection target Venn diagrams. The intersection site in the diagram was clicked to obtain the intersection target in the result table, which gave the potential target of MCE for the treatment of enteritis.The active compound targets of MCE obtained in Section 2.1 and the targets of enteritis obtained in Section 2.2 were imported into List2 and List1 of the Venny 2.1.0 online website (https://cn.stringdb.org/) and set as “Homo sapiens” with a confidence level of 0.900, hidden unassociated proteins and protein–protein interaction (PPI) data were exported. PPI data were viewed using Cytoscape 3.7.1 software to build the PPI network graphs. The core targets were screened with the topological analysis based on the following criteria: degree ≥2* degree mean, betweenness centrality ≥betweenness centrality mean, and closeness centrality ≥closeness centrality mean. The higher the values of these three criteria, the more important the target was in the network (The intersection targets were imported into the STRING database ( network .https://metascape.org/), and “Homo sapiens” was used as a species for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, which were arranged in the descending order of significance, −LogP. The top 10 enriched biological processes (BP), cellular components (CC), and molecular functions (MF) under the GO classification and the top 20 enriched KEGG pathways were then imported into Bioinformatics (http://www.bioinformatics.com.cn/) for visualization.The PPI data were de-duplicated and imported into the Metascape database , respectively. The SDF files of the active compounds were converted to PDB files using Open Babel GUI software. PyMOL software was used to dehydrate and desolvate the core targets. AutoDock Tools software was used to hydrogenate the active compounds and set them as the ligand and to hydrogenate the core targets and set them as the receptor. Molecular docking of the receptor and ligand was performed using AutoDock Tools software, and the docking result with the lowest binding energy was selected and imported into PyMOL software for visual analysis.The 3D structures of the active compounds and core targets were downloaded from the PubChem and PDB databases , smooth muscle, RET proto-oncogene (RET), endothelin receptor type B (EDNRB), endothelin 3 (EDN3), SRY-box transcription factor 10 (SOX10), nucleotide-binding oligomerization domain-containing 2 (NOD2), glial cell-derived neurotrophic factor (GDNF), interleukin-6 (IL-6), tetratricopeptide repeat domain 37 (TTC37), and transthyretin (TTR) .In total, 269 targets of the four active compounds of MCE and 1,237 targets of enteritis obtained in Section 3.1 and Section 3.2 were imported into Venny 2.1.0 for constructing Venn diagrams. Furthermore, 70 intersection targets were obtained, which were potential targets for the pharmacological effects of the four active compounds of MCE in the treatment of enteritis, including transthyretin (TTR), albumin (ALB), protein kinase C theta type (PRKCQ), interleukin-2 (IL-2), mast/stem cell growth factor receptor kit (KIT), insulin-like growth factor-I (IGF-1), peroxisome proliferator-activated receptor gamma (PPARG), tyrosine-protein kinase (BTK), vitamin D3 receptor (VDR), and retinol-binding protein 4 (RBP4) , which aUsing the STRING database, the highest confidence level (0.900) was selected in the minimum required interaction score column, while other conditions remained unchanged; we exported the Excel table and obtained 49 targets after de-weighting. After importing the 49 targets into Cytoscape 3.7.1 software, a PPI network diagram of enteritis and the four active compounds of targets of MCE  was obtaGO enrichment analysis provided a systematic reflection of the mechanism of the four active compounds of MCE in the treatment of enteritis. A total of 749 BP, 47 CC, and 64 MF were obtained  via MetaThe KEGG pathway analysis allows the prediction of the role of protein interaction networks in different cell activities. In this study, Metascape was used for KEGG pathway analysis, and 142 pathways were obtained , among wTo visualize the potential molecular mechanism of the four active compounds of MCE in the treatment of enteritis, a “drug–active compound–target–pathway–disease” network diagram was built using Cytoscape 3.7.1 software. In the diagram, there are 96 nodes and 410 connections . FurtherTo determine the binding between small molecules of the drug and target macromolecules, binding energy was used: less than −5 kcal/mol indicates good affinity; the lower the binding energy, the stronger the affinity . MoleculThe best binding results were obtained for SAN and PRO with AKT1 and for CHE and ALL with HSP90AA1 . SAN forLactobacillus and decreases those of Escherichia–Shigella, Streptococcus, Proteobacteria, and Actinobacteriota, thus ensuring optimal intestinal health. The four active compounds of MCE primarily act on AKT1 and MAPK1 to activate PI3K-Akt and MAPK signaling pathways for treating enteritis. MCE provides inflammation regulation through PI3K-Akt and MAPK signaling pathways and shows anti-inflammatory effects by inhibiting MAPK activation to alter the synthesis of inflammatory mediators -induced intestinal inflammation via the MAPK signaling pathway. Salmonella typhimurium-induced diarrhea in mice, and reduced intestinal inflammation through the MAPK signaling pathway. Therefore, the MAPK signaling pathway plays a regulatory role in enteritis, a result consistent with our network pharmacological prediction. Thus, it is evident that the four active compounds of MCE possess pharmacological effects in the treatment of enteritis through multiple pathways.GO and KEGG enrichment analyses are helpful in exploring the signaling pathway of the four active compounds of MCE in the treatment of enteritis. According to GO enrichment analysis, the four active compounds of MCE helped in the treatment of enteritis primarily through the involvement of transferase and kinase activity, protein phosphorylation, and cell motility. In addition, cancer-related pathways, PI3K–Akt signaling pathway, MAPK signaling pathway, FoxO signaling pathway, and Rap1 signaling pathway play a vital role in the treatment of enteritis using the four active compounds of MCE. Because the PI3K-Akt signaling pathway enriched five core targets, it could be the main signaling pathway for processing enteritis. The PI3K-Akt signaling pathway is the main pathway in the regulation of inflammation, primarily in the regulation of inflammatory cell recruitment, expression, and activation of inflammatory mediators . This paBased on the aforementioned network pharmacological analysis, four active compounds, SAN, CHE, PRO, and ALL, are stably bound to five core targets, HSP90AA1, MAPK1, HRAS, JAK2, and AKT1, which was validated using molecular docking. Thus, the interaction between the active compounds and the target may be one of the most important pharmacological mechanisms of the four active compounds of MCE in the treatment of enteritis, an outcome that needs to be verified by further experiments.However, the potential pharmacological mechanism of the four active compounds of MCE in the treatment of enteritis was explored using only network pharmacology and molecular docking techniques. In addition, the current network information is not comprehensive, and the real-time data are not available in the databases. Therefore, the findings of this study need to be further validated in animals for better clinical applications.The four main active compounds of MCE involved in the treatment of enteritis are SAN, CHE, PRO, and ALL, and the five core targets are HSP90AA1, MAPK1, HRAS, JAK2, and AKT1. PI3K-Akt and MAPK, obtained through network pharmacology and molecular docking technologies, are the main signaling pathways. These results are of great significance for further exploration of the potential pharmacological mechanism of MCE in the treatment of enteritis.


Saccharomyces cerevisiae undergoes filamentous growth by the filamentous growth (fMAPK) pathway; however, the inducers of the pathway remain unclear, perhaps because pathway activity has been mainly studied in laboratory conditions. To address this knowledge gap, an ecological framework was used, which uncovered new fMAPK pathway inducers, including pectin, a material found in plants, and the metabolic byproduct ethanol. We also show that induction by a known inducer of the pathway, the non-preferred carbon source galactose, required galactose metabolism and induced the pathway differently than glucose limitation or other non-preferred carbon sources. By exploring fMAPK pathway function in fruit, we found that induction of the pathway led to visible digestion of fruit rind through a known target, PGU1, which encodes a pectolytic enzyme. Combinations of inducers  stimulated the pathway to near-maximal levels, which showed dispensability of several fMAPK pathway components , but not others  and required the Ras2-protein kinase A pathway. This included a difference between the transcription factor binding partners for the pathway, as Tec1p, but not Ste12p, was partly dispensable for fMAPK pathway activity. Thus, by exploring ecologically relevant stimuli, new modes of MAPK pathway signaling were uncovered, perhaps revealing how a pathway can respond differently to specific environments.Signaling modules, such as mitogen-activated protein kinase (MAPK) pathways, are evolutionarily conserved drivers of cell differentiation and stress responses. In many fungal species including pathogens, MAPK pathways control filamentous growth, where cells differentiate into an elongated cell type. The convenient model budding yeast Saccharomyces cerevisiae is a convenient model to study MAPK-dependent regulation of filamentous growth, although the inducers of the pathway are not clear. Here, we exposed yeast cells to ecologically relevant compounds , which identified new inducers of the MAPK pathway. In combination, the inducers activated the pathway to near-maximal levels but did not cause detrimental phenotypes associated with previously identified hyperactive alleles. This context allowed us to identify conditional bypass for multiple pathway components. Thus, near-maximal induction of a MAPK pathway by ecologically relevant inducers provides a powerful tool to assess cellular signaling during a fungal differentiation response.Filamentous growth is a cell differentiation response and important aspect of fungal biology. In plant and animal fungal pathogens, filamentous growth contributes to virulence. One signaling pathway that regulates filamentous growth is an evolutionarily conserved MAPK pathway. The yeast  Organisms can sense and respond to signals in the environment. One way this occurs is by signal transduction pathways, such as evolutionarily conserved mitogen-activated protein kinase (MAPK) pathways –3. MAPCandida albicans , presumably as a scavenging response  during Among several MAPK pathways in yeast –59, thS. cerevisiae have mainly been performed in laboratory conditions. However, Saccharomyces yeast are commonly found in wild and domesticated habitats, including fruits, tree sap and bark, insect vectors, leaf litter, soil, and rotten wood  activated the pathway to near-maximal levels, which has not been previously observed in laboratory settings. Maximal activation of the fMAPK pathway partly bypassed the requirement of several core components of the pathway  but not others . We also identified a critical role for the Ras2-protein kinase A (Ras-PKA) pathway, a known regulator of filamentous growth and the fMAPK pathway –129, iFRE-lacZ [The fMAPK pathway regulates pectinase levels ; therefoFRE-lacZ ). To tesPGU1 expression  (pgu1∆), where pectin breakdown does not occur , pectin induced the fMAPK pathway (pgu1∆ mutant shows similar increase between GAL and GAL + P as wild type (WT) in Pectin is a polymer of galacturonic acid and other sugars . Pectin pression , 134; thpression . Therefoig. S1B) , 133. In pathway  (pgu1∆),S. cerevisiae does not break down di-galacturonic acid into galacturonic acid because galacturonic acid inhibits growth  in synthetic medium with 2% pectin as carbon source remained <0.08 after 16 h). Therefore, we focused on testing whether pectin breakdown might improve accessibility to the plant environment.Yeast cells may break down pectin to improve accessibility to plant tissues, and therefore nutrients, as has been suggested 48, 140.pgu1Δ). The reduction in viscosity was also dependent on the fMAPK pathway as seen in cells lacking transcription factors Tec1p or Ste12p (ste12Δ or tec1Δ). Viscosity was strongly reduced when incubated with cells lacking the negative regulator Dig1p, which has elevated fMAPK pathway activity (dig1∆). Therefore, the fMAPK pathway functions to reduce viscosity of pectin-based solutions, and may facilitate accessibility to plant-based environments.Pectin breakdown by Pgu1p has been visualized by a plate-based test that measures enzymatic activity , 141. Toactivity , dig1∆. S. cerevisiae has been isolated on fruit  were required for galactose-dependent induction of the fMAPK pathway (ste11∆). As expected, GAL genes were not required for fMAPK pathway activity in medium with glucose as the carbon source or no carbon source  is induced by activation of the fMAPK pathway and provides an additional readout of fMAPK pathway activity. As previously shown , 64, 145FG1-lacZ  [Fig. S4Yeast has a haploid and diploid stage in their life cycle –167 andig1∆ mutant, which shows very high fMAPK pathway activity seen in laboratory settings  or when all three inducers were combined . These results show that the fMAPK pathway can be activated to near-maximal levels when cells encounter a combination of inducers  showed reduced fMAPK pathway activity compared to wild type in response to the inducers tested .We next tested how different inducers may be sensed and relayed by fMAPK pathway components . In genes tested . Howevermsb2∆, sho1∆, and opy2∆ mutants had a similar reduction in fMAPK pathway activity in glucose . The observation that Opy2p showed a stronger role than Msb2p or Sho1p is in agreement with previous results , whereas the msb2∆ and sho1∆ mutants showed similar reduction in galactose with ethanol. Overall, these findings suggest that the stimuli tested may be sensed in different ways by different proteins.Msb2p, Sho1p, and Opy2p also showed different requirements for signaling compared to each other depending on the environment. For example, the  glucose ; however results , tying O results , 172. WeP-value <0.0004 by Student’s t-test comparing tec1∆ and ste12∆). This was also seen in the diploid strain . Collectively, these data show that the requirement for a subset of fMAPK pathway components changes depending on the condition tested.Similarly, some proteins were required for signaling in all conditions tested  and the The fMAPK pathway regulates differentiation to the filamentous cell type , 29. Celdig1∆ mutant was similar to wild-type cells in the maximally inducing condition  for both fMAPK pathway activity . For example, the plate-washing assay supported the finding that Ste12p plays a more critical role than Tec1p under several conditions . For example, invasive growth  based onMoreover, some fMAPK pathway components made different contributions toward those two phenotypes depending on the environment . Invasivras2∆). This result suggests that the Ras2-PKA pathway is required for fMAPK pathway activity in contexts with ethanol, rather than functioning as an ancillary pathway that augments core pathway activity.Other pathways regulate filamentous growth including a major nutrient-sensing pathway called the Ras2p cAMP-dependent PKA pathway (Ras2-PKA –128), tpk2∆ and pde2∆, GLU) or increase  in fMAPK pathway activity in one environment . New inducers of the fMAPK pathway were identified . FRE-lacZ plasmid  using the sequencing primer CGGAATAGGAACTTCAAAGCG (Yeast strains are listed in Table S1. Experiments were performed in haploid cells unless otherwise noted. The p plasmid  (provide plasmid  (provide plasmid  through  plasmid . Gene de plasmid  as has b plasmid . For CRIescribed . To geneTCAAAGCG . The marTCAAAGCG  as a 140FRE-lacZ plasmid or minus leucine when selecting for pNFG1-lacZ). For some experiments with GLU medium, the dextrose concentration varies as indicated. For media with pectin (+P), a 1% solution was made using citrus pectin from Spectrum catalog #PE100 ; YPD was 1% yeast extract, 2% peptone, 2% dextrose; YPGAL was YPD with 2% galactose instead of dextrose. When solid media were made, 2% agar was used.Synthetic complete medium  included 0.67% yeast nitrogen base without amino acids, amino acids, and one of the following: 2% dextrose, 2% fructose, 2% galactose, 2% sucrose, 2% maltose, or 2% glycerol, respectively (minus uracil when selecting for placZ) assay as previously described (FRE-lacZ or pNFG1-lacZ) as the readout of fMAPK pathway activity. Experiments were done with the FRE-lacZ reporter except where indicated. Cells were grown in indicated medium (without uracil or leucine when maintaining selection for plasmids). Cells were grown to time points indicated and harvested by centrifugation and stored at −80o for at least 30 min. Subtle variation in fMAPK pathway induction or growth by inducers can be noted across some figures. This is due to differences between experiments, as indicated in figure legends, related to different times cells were grown or different volumes of medium. The β-galactosidase (lacZ) assay was performed with at least three biological replicates where the average is reported and error bars represent standard deviation. Differences in values from a previous study .The fMAPK pathway activity was also analyzed by phosphoblot as previously described  by estabImages of cells were taken at 100× magnification by microscopy using differential interference contrast imaging with a Zeiss Axioplan 2 microscope. Digital images were acquired with the Axiocam MRm camera. Some photos were taken by an iPhone 13 (Apple) through the microscope lens. For image analysis, Axiovision 4.4 software was used.For cell differentiation quantification, cells were observed at 100× magnification. At least 85 cells were examined for each strain. Cells that showed elongation and/or cells that budded distally were considered filamentous, whereas cells that were round and budding axially (back toward mother cell) were considered yeast form. The filamentous cells and yeast form cells were expressed as a percentage of total cells.https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z) to calculate (volume/area)/ 10,000  cap side down directly into the test tube, which was placed at a 45o angle. The time for the Eppendorf tube to reach the bottom was recorded for each strain. Viscosity was averaged across three biological replicates and error was determined by standard deviation. The breakdown of pectin in mandarin oranges was tested by growing the indicated strains for 24 h in 200 mL YPGAL liquid cultures shaking at 30o. Cells were spun down by centrifugation and cell supernatants were harvested. Mandarins were purchased commercially from Sunrays (https://sunraysfruits.com/products/mandarins/). The mandarin was sterilely peeled and wedges were separated. One mandarin wedge and one piece of peel was placed in 50 mL supernatants of indicated strains and left at 30o for 24 h. Wedges and peels were removed and placed under a gentle stream of water and washed. Images before and after treatment were captured using an iPhone 13 (Apple).Pectin breakdown was determined by viscosity using a drop assay after cells were grown in 10 mL of indicated medium with 1% pectin for 20 h shaking at 30LYS2::GAL1-HIS3 growth reporter. Positive control of a Cdc42p-Bem4p interaction is described in (RAS2, G19VRAS2, CDC24, STE11, STE50, KSS1, STE7; in reference BEM4, CDC42, BEM1; in reference OPY2-tail; and in reference STE20.Two-hybrid analysis  was perfribed in , 86, 219


Phytohormones exhibit a wide range of chemical structures, though they primarily originate from three key metabolic precursors: amino acids, isoprenoids, and lipids. Specific amino acids, such as tryptophan, methionine, phenylalanine, and arginine, contribute to the production of various phytohormones, including auxins, melatonin, ethylene, salicylic acid, and polyamines. Isoprenoids are the foundation of five phytohormone categories: cytokinins, brassinosteroids, gibberellins, abscisic acid, and strigolactones. Furthermore, lipids, i.e., α-linolenic acid, function as a precursor for jasmonic acid. The biosynthesis routes of these different plant hormones are intricately complex. Understanding of these processes can greatly enhance our knowledge of how these hormones regulate plant growth, development, and physiology. This review focuses on detailing the biosynthetic pathways of phytohormones. They also orchestrate various cellular activities within the plant. Even at minimal concentrations, they are operative in plant cells, tissues, and organs. They are found in all vascular plants and a substantial number of non-vascular species . From thIn addition, peptide hormones regulate many developmental and defense processes, such as meristem maintenance, xylem and phloem differentiation, stomata patterning, pollination, embryo and endosperm development, cell division, nodulation, and systematic response . They arAs such, plants host diverse phytohormone pathways. Significant advancements have been made in the study of phytohormone biology and synthesis over the past decade. An assortment of new tools and methods has been developed, resulting in the discovery of phytohormone substrates, intermediates, and final products. The biosynthetic pathways of plant hormones have been elucidated, and extensive research has been carried out into the genes found in the plant genome, encoding the enzymes that catalyze the various stages of phytohormone synthesis ,2.This review aims to outline the biosynthetic pathways of non-peptide plant hormones .Polyamines (PAs) play a crucial role in plant growth, metabolism, and development. Structurally, these compounds are organic polycationic alkylamines that contain two or more amino groups. PAs such as putrescine (Put), spermidine (Spd), and spermine (Spm) are the most well-studied and recognized PAs found in plants. These three PAs regulate various physiological activities, such as photosynthesis, flower generation, embryogenesis, and organogenesis. They are also accountable for maintaining the stability of nucleic acids, various protein molecules, and the membrane structure. Additionally, they play a substantial role in enhancing the tolerance of many plants to the presence of a variety of abiotic and biotic stress factors, thereby impacting crop yield. Putrescine is a key compound involved in the biosynthesis of PAs. It serves as a common intermediate in the creation of Spd, Spm, and thermospermine. The biosynthesis pathways of Put have been identified in many plants  9,10,1111,12.2 (the ADC pathway) are produced. Subsequently, Agm is used to synthesize N-carbamoyl putrescine (NCPA) and ammonia (NH3), which occurs following the removal of nitrogen. In the succeeding reaction, NCPA amidohydrolase hydrolyzes NCPA, removing its carbamoyl group to produce NH3, CO2, and the first PA—Put. This process is the most prevalent pathway for Put synthesis in plants . T. T122]. f C27-BR . In anotectively . 28-homoectively . 28-homoectively ,127,128.28-BRs -ergostan-4-en-3β-one and campestanol (CN). In the parallel early C-22 oxidation pathway, C-22α hydroxylation of 24-methyleneCR by the enzyme C-22α hydroxylase leads to the biosynthesis of 22-hydroxy-24-methyleneCR . I. I12, wheticulum . KO catareaction . The fir pathway . The rea pathway .ent-kaurenoic acid is converted into GA12 in three steps that involve 7β-hydroxy-ent-kaurenoic acid and GA12-aldehyde. Enzyme KAO is involved in this conversion  that can convert GA12-aldehyde into GA12. These enzymes are present in the development of seeds and vegetative tissues. The pathway branches from GA12. The reaction of 13-hydroxylation to GA53 initiates the formation of 13-hydroxylated GAs, such as GA1. Additionally, a parallel non-13-hydroxylation route from GA12 results in the synthesis of GA4 . G. G19-GAse enzyme . TherefoStriga spp.). SLs are a class of carotenoid-derived terpenoids that are involved in diverse developmental processes, such as seed germination, shoot branching, leaf senescence, and root development, as well as responses to various environmental conditions  ) ) . Thus, 1 process .A. thaliana, tobacco, and tomato , which is characterized by specificity of sn-1 in the structure of fatty acids. Thus, α-LeA oxygenation is the first step in JA biosynthesis. The flower-specific protein DAD1, which is a PLA1, is required for the formation of JA -pentenyl)-cyclopentane-1-octanoic acid (OPC-8) is produced during this step [OPDA is known as a biosynthetic precursor of the phytohormone JA. It is also in-volved in various biological processes, which range from plant defence and stress responses to the regulation of growth and development . OPDA isganelles . OPDA cahis step . In the his step . OPC-8 ihis step . During his step .opr3-3 mutant in which OPR3 activity is completely depleted showed that OPDA and hexadecatrienoic acid-derived dn-OPDA are metabolized to create tetranor-OPDA and 4,5-didehydro-JA. Next, this compound may be reduced to JA by OPR2 after being transported into the cytosol. This process suggests that the OPR2-dependent and OPR3-independent JA biosynthetic pathways may be very ancient in vascular plants and activated under different processes or the influence of certain environmental conditions [Recently, an alternative pathway for JA synthesis has been proposed. Studies of the nditions ,216,217.In this review, the biosynthesis routes of all known plant phytohormones have been explored. Understanding these intricate biosynthetic pathways of plant hormones will provide valuable insights regarding their biological functions.15 farnesyl pyrophosphate and a C40 carotenoid, respectively. Brassinosteroid biosynthesis occurs through three pathways that lead to C27-, C28-, and C29-type BRs, with early steps happening via the MVA or MEP pathways. Cytokinins can be synthesized de novo as low-activity nucleotide mono-, di-, and tri-phosphates. Alternatively, cytokinins can be released from tRNA, creating nucleotide mono-phosphates, with the interconversions of nucleotides, nucleosides, and free bases catalyzed by enzymes involved in adenine metabolism. Gibberellins are formed from GGPP through IPP, which is the C-5 building block of all terpenoid/isoprenoid compounds, including BRs, CKs, ABA, and SLs. Carlactone is the main precursor of SL production in plants. Jasmonic acid synthesis begins with the release of polyunsaturated fatty acid α-linolenic acid from galactolipids present in chloroplast membranes.The biosynthesis of PAs begins with putrescine that leading to the production of other PAs. The ethylene synthesis pathway is more straightforward, with methionine being a precursor. Salicylic acid biosynthesis is believed to occur via two routes: the isochorismate synthase pathway and the phenylalanine ammonia-lyase pathway. Auxin synthesis in plants occurs via two main pathways: the Trp-dependent and independent routes. Four Trp-dependent IAA routes have been proposed: the IAM, IPA, TAM, and IAOX routes. Similarly, melatonin biosynthesis starts with Trp, which is synthesized de novo via the shikimate pathway. Two potential pathways of ABA biosynthesis have been identified in plants. The first pathway is direct, and the second pathway is indirect; in these pathways, ABA is derived from CThe emergence of various phytohormone groups, along with their unique biosynthetic pathways, could be linked to their distinct chemical structures, biosynthetic path-ways, physicochemical properties, plant evolutionary histories, and roles in plants’ adaptation to diverse environmental conditions or developmental programs. Precursors or intermediates involved in one phytohormone pathway may also participate in the biosynthesis of another hormone, thus linking the pathways .


Interstitial fibrosis is involved in the progression of various chronic kidney diseases and renal failure. Diosmin is a naturally occurring flavonoid glycoside that has antioxidant, anti-inflammatory, and antifibrotic activities. However, whether diosmin protects kidneys by inhibiting renal fibrosis is unknown.The molecular formula of diosmin was obtained, targets related to diosmin and renal fibrosis were screened, and interactions among overlapping genes were analyzed. Overlapping genes were used for gene function and KEGG pathway enrichment analysis. TGF-β1 was used to induce fibrosis in HK-2 cells, and diosmin treatment was administered. The expression levels of relevant mRNA were then detected.Network analysis identified 295 potential target genes for diosmin, 6828 for renal fibrosis, and 150 hub genes. Protein–protein interaction network results showed that CASP3, SRC, ANXA5, MMP9, HSP90AA1, IGF1, RHOA, ESR1, EGFR, and CDC42 were identified as key therapeutic targets. GO analysis revealed that these key targets may be involved in the negative regulation of apoptosis and protein phosphorylation. KEGG indicated that pathways in cancer, MAPK signaling pathway, Ras signaling pathway, PI3K-Akt signaling pathway, and HIF-1 signaling pathway were key pathways for renal fibrosis treatment. Molecular docking results showed that CASP3, ANXA5, MMP9, and HSP90AA1 stably bind to diosmin. Diosmin treatment inhibited the protein and mRNA levels of CASP3, MMP9, ANXA5, and HSP90AA1. Network pharmacology analysis and experimental results suggest that diosmin ameliorates renal fibrosis by decreasing the expression of CASP3, ANXA5, MMP9, and HSP90AA1.Diosmin has a potential multi-component, multi-target, and multi-pathway molecular mechanism of action in the treatment of renal fibrosis. CASP3, MMP9, ANXA5, and HSP90AA1 might be the most important direct targets of diosmin. The prevalence of chronic kidney disease (CKD) is estimated to be 8%–16% worldwide . Renal fDiosmin is a glycosylated polyphenolic flavonoid with antioxidant, anti-inflammatory, and anti-apoptotic pharmacological activities . In recehttps://pubchem.ncbi.nlm.nih.gov/) [https://www.lilab-ecust.cn/pharmmapper/) for prediction. This method is based on reverse pharmacophore matching of structural features. The molecular structures of the diosmin components were submitted to the PharmMapper database, and multiple conformations were generated by optimizing the compound structure. The standardized diosmin components of the protein targets were matched with all the human drug targets in the PharmMapper database in UniProt (https://www.uniprot.org/), and protein targets with a parameter matching score (fit) greater than 2 were selected as the component targets.‘Diosmin’ was used as a search term in PubChem database (ih.gov/) . The SDFhttps://omim.org/) [https://www.genecards.org/) [https://www.disgenet.org/) [http://bioinformatics.psb.ugent.be/webtools/Venn/) [Human genes associated with renal fibrosis were gathered from three databases: OMIM (im.org/) , GeneCards.org/) , and Diset.org/) . The seas/Venn/) .https://string-db.org/) [PPIs of the therapeutic targets of diosmin in the treatment of renal fibrosis were gathered using STRING (db.org/) , a databhttp://geneontology.org/) [www.kegg.jp/kegg/kegg1.html) [https://david.ncifcrf.gov/) [P < 0.05 selected, and the results were analyzed by selecting GO Biological Processes (BP), GO Molecular Functions (MF), GO Cellular Components (CC), and KEGG Pathway for GO and KEGG analysis of the obtained results.To further explore the mechanisms of diosmin for renal fibrosis treatment, the intersection of targets of diosmin and the common proteins between renal fibrosis were additionally searched using GO (gy.org/) , enrichmg1.html) –18, and rf.gov/) . The reshttps://pubchem.ncbi.nlm.nih.gov/) [http://www.rcsb.org/pdb/) [The 2D structures of diosmin were downloaded from PubChem (ih.gov/)  and Tradrg/pdb/)  and wererg/pdb/) , 21. Therg/pdb/) .Diosmin (cat. no.: HY-N0178) was purchased from MedChemExpress . Human proximal tubular epithelial (HK-2) cells were obtained from American Type Culture Collection . Fetal bovine serum was obtained from Millipore Sigma . Transforming growth factor-β1 (TGF-β1) was purchased from Proteintech . Reverse transcription kit and SYBR Green PCR Master Mix were purchased from Vazyme .4 cells/well in 96-well plates. After treatment with diosmin for 3 h, they were incubated with 0.5 mg/mL of 3--2,5-diphenyltetrazolium bromide solution (100 μL/well) for 4 h. After washing with 1 × PBS, dimethyl sulfoxide (150 μL/well) was added to dissolve purple crystals. The absorbance of the samples was measured at 570 nm using a microplate reader . Furthermore, diosmin was administered to TGF-β1 (5 ng/mL)-stimulated HK-2 cells. The effects of diosmin on core genes were observed in HK-2 cells.To assess cell viability, HK-2 cells were cultured at 1 × 10Total RNA was extracted from HK-2 cells or mouse kidney tissues using TRIzol® reagent. Complementary DNA was synthesized using Hiscript Q RT SuperMix and used with the qPCR (+ gDNA wiper) reverse transcriptase kit (Vazyme). The sequences of the used primes for Real-time PCR are listed in Table P < 0.05 was designated significant.Values are presented as the mean ± SEM. Quantitative data were tested for normality. Two-tailed unpaired t-test and one-way ANOVA were used to compare differences between two groups and multiple groups, respectively. Prism 9.0  was used for the statistical analyses. 28H32O15 was obtained from PubChem, and the targets related to diosmin were screened using the PharmMapper database and corrected using the UniProKB tool in the UniProt database. A total of 295 potential targets, including alpha1antitrypsin (A1AT), caspase 3 (CASP3), mitogen-activated protein kinase 14 (MAPK14), and matrix metalloprotein (MMP9), were screened after removing targets that were unrelated to humans and those without correspondence or duplication. A total of 266, 570, and 6546 target genes closely related to renal fibrosis were screened in the three commonly used databases of GeneCards, OMIM, and Dis Genet, respectively. A total of 6828 genes were identified after duplicates were eliminated. These results suggest that numerous factors cause renal fibrosis, and the pathogenesis is complex. Gene datasets obtained from the screening of renal fibrosis-related targets and diosmin component-related targets were imported into an online Venn diagram, and a total of 150 intersecting targets were obtained , including cancer, lipids and atherosclerosis, proteoglycans in cancer, fluid shear stress and atherosclerosis, endocrine resistance, and Ras-related protein 1 (Rap1), rat sarcoma (Ras), Phosphoinositide-3 kinase (PI3K)/Akt, chemokine, Mitogen-activated protein kinase (MAPK), Forkhead Box Protein O (FoxO), T-cell receptor, hypoxia-inducible factor-1 (HIF-1), and estrogen signaling pathways  of HK-2 cells did not significantly inhibit the viability Fig. . Cell viRenal fibrosis is a common pathological feature in the terminal stages of various CKDs and is closely related to their prognosis . Early pWe found that core genes are mainly involved in the biological process of apoptosis. The protein encoded by CASP3 is a cysteine-aspartic acid protease that plays a central role in the execution phase of cell apoptosis. Caspase 3 is also a critical upstream regulator in the development of renal fibrosis . CaspaseThe core genes SRC, IGF1, RHOA, ESR1, EGFR and CDC42 may play a key role in the inhibition of the development and progression of renal fibrosis by diosgenin. This SRC gene may play a role in the regulation of embryonic development and cell growth. The protein encoded by this gene is a tyrosine-protein kinase whose activity can be inhibited by phosphorylation by c-SRC kinase. SRC kinase plays an important role in cell proliferation, differentiation, migration, and immune response, and it is a critical mediator of renal fibrosis , 39. Act2 homeostasis [According to the KEGG terms, the therapeutic targets of diosmin against renal fibrosis were mainly associated with the MAPK, Ras, PI3K-Akt, FoxO, and HIF-1 signaling pathways. MAPKs are a group of protein kinases containing three subfamilies: c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 . MAPKs reostasis . Based oeostasis , 58. TheWe explored the potential molecular mechanism of action of diosmin in the treatment of renal fibrosis from a comprehensive and systematic perspective, and our results provide a theoretical basis for further experimental studies. Network pharmacology analysis and molecular docking technology were used to explore the potential mechanism of action of diosmin against renal fibrosis, and the key therapeutic targets of diosmin were identified as CASP3, MMP9, ANXA5, and HSP90AA1. The mechanism of action of diosmin in the treatment of renal fibrosis may be related to the regulation of biological pathways such as cell apoptosis and inflammation. This study provides a theoretical basis for the treatment of diosmin-induced renal fibrosis. A limitation of this study is the lack of additional experimental areas to validate our findings. However, the specific mechanisms involved in this process require further exploration. Future studies should conduct well-designed in vivo and in vitro experiments to further explore the specific mechanisms.Diosmin has a potential multi-component, multi-target, and multi-pathway molecular mechanism of action in the treatment of renal fibrosis. CASP3, MMP9, ANXA5, and HSP90AA1 might be the most important direct targets of diosmin. The mechanism of action may be related to the MAPK, Ras, PI3K-Akt, FoxO, and HIF-1 signaling pathways. This study provides a basis for further studies on diosmin in the treatment of renal fibrosis.


Unsubstituted aromatic hydrocarbons (UAHs) are recalcitrant molecules abundant in crude oil, which is accumulated in subsurface reservoirs and occasionally enters the marine environment through natural seepage or human-caused spillage. The challenging anaerobic degradation of UAHs by microorganisms, in particular under thermophilic conditions, is poorly understood. Here, we established benzene- and naphthalene-degrading cultures under sulfate-reducing conditions at 50°C and 70°C from Guaymas Basin sediments. We investigated the microorganisms in the enrichment cultures and their potential for UAH oxidation through short-read metagenome sequencing and analysis. Dependent on the combination of UAH and temperature, different microorganisms became enriched. A Thermoplasmatota archaeon was abundant in the benzene-degrading culture at 50°C, but catabolic pathways remained elusive, because the archaeon lacked most known genes for benzene degradation. Two novel species of Desulfatiglandales bacteria were strongly enriched in the benzene-degrading culture at 70°C and in the naphthalene-degrading culture at 50°C. Both bacteria encode almost complete pathways for UAH degradation and for downstream degradation. They likely activate benzene via methylation, and naphthalene via direct carboxylation, respectively. The two species constitute the first thermophilic UAH degraders of the Desulfatiglandales. In the naphthalene-degrading culture incubated at 70°C, a Dehalococcoidia bacterium became enriched, which encoded a partial pathway for UAH degradation. Comparison of enriched bacteria with related genomes from environmental samples indicated that pathways for benzene degradation are widely distributed, while thermophily and capacity for naphthalene activation are rare. Our study highlights the capacities of uncultured thermophilic microbes for UAH degradation in petroleum reservoirs and in contaminated environments. Aromatic hydrocarbons (AHs) are a naturally abundant group of hydrocarbons. They are highly hydrophobic and extremely stable molecules because of their planar conformation consisting of one or more six-carbon ring systems stabilized by delocalized π electrons . AHs conThe study of AH biodegradation is of high interest, because AHs are highly toxic and thus their release into the environment is associated with health hazards . MoreovePeptococcaceae bacteria  or tricarboxylic acid (TCA) pathways  by Desulfobacterium strain N47  located in the Gulf of California (Mexico) for this endeavor. In the GB, petroleum-range hydrocarbons, including AHs, are naturally abundant . These hAlvin during RV Atlantis cruise AT42-05 to the GB during dive 4,991 . On the ship, the push cores were transferred to glass bottles, which were sealed with rubber stoppers, purged with argon and stored at 4°C. In the home laboratory, the cores were combined and mixed with anoxic sulfate-reducer medium . Two temperature treatments, 50°C and 70°C, were applied, and bottles were incubated at gentle shaking (40 rpm) in the dark. For each substrate and each temperature, three replicates were prepared.The push cores 4991-13 and 4991-14 used for anoxic cultivations were collected at the “Cathedral Hill” hydrothermal vent site with submersible ium SRM;  in a ratium SRM; . 5 mL ofSulfide production was assessed in bi-weekly intervals via a copper sulfate assay  using ing, 4°C) and the culture medium was discarded. DNA was extracted from the pellets using a modified SDS-protocol (−1) that was produced from the combined cores 4991-13 and 4991-14. The final DNA concentrations were determined in a fluorometric assay. DNA yields were 0.2 μg (B50), 3.4 μg (N50), 4.0 μg (B70), 0.6 μg (N70), and 0.7 μg . Libraries were sequenced as 2 × 150 bp paired-end reads on an Illumina HiSeq3000 platform at the Max-Planck-Genome-Centre . Between 4,142,459 (B70) and 4,247,237 (N70) raw reads were obtained.After around 600 days of cultivation, cultures were sampled and DNA was extracted for metagenome sequencing. By this time, the active cultures had been diluted eight (benzene 50°C-B50), seven (benzene 70°C-B70), ten , and two  times. Of each culture, 40 mL were sampled, centrifuged  Protein and the UniProtKB databases. For putative anaerobic benzene carboxylase (AbcAD), protein sequences were collected from recent publications (bcrACD/bzdNQ) amplified via PCR in the study by pimE) and acetyl-CoA acyltransferase (pimB) were acquired from e-value <1e-10, identity ≥40%, and aligned length ≥ 80%. Proteins containing the heme-binding amino acid motif CxxCH  from the domains ications . The nucif CxxCH  were ideTwo phylogenomic trees were constructed from concatenated alignments of single-copy core genes (SCGs). For the first tree, all 135 publicly available MAGs classified as order Desulfatiglandales in the Genome Taxonomy Database (GTDB) taxonomy treeThe optimal growth temperature (OGT) was predicted for the publicly available MAGs with the OGT_prediction tool (version 1.0.3). Key genes for anaerobic UAH/AH oxidation and downstream degradation were identified in Desulfatiglandales and SZUA-161 MAGs by running the previously built protein databases on the amino acid sequences of the MAGs in the same way and with the same selection criteria as described in the section “Genome annotation.”The raw reads of short-read metagenome sequencing of the original sediment slurry and the four enrichment cultures have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1013425 (accessions SRR25925499-SRR25925503).https://github.com/zehanna/UAH_oxidation.The bash script used for the identification of CxxCH motifs in protein sequences of the MAGs is available under We incubated triplicate batches of an oil-rich sediment slurry from the GB  with UAHAfter more than 1.5 years of cultivation, we retrieved short-read metagenomes from the original sediment used for incubations and from the four active cultures B50, B70, N50, and N70.From the co-assembled metagenomes, we reconstructed 47 MAGs with completeness >50% and redundancy <10%. Cultures that showed high sulfate-dependent substrate turnover  were more enriched in specific taxa, whereas the microbial community of the less active N70 culture remained more diverse . PreviouArchaeoglobus , and the family WUQV01 , and a bacterial MAG  of the Bipolaricaulaceae. The communities of the cultures differed strongly from the ANME-1 dominated sediment slurry, where the enriched MAGs were only present at very low abundances  was classified as species VBQP01 sp008297795 of the archaeal phylum Thermoplasmatota ; Table 1bamY and complete or near complete pathways for reductive dearomatization (RD), ring hydrolysis (RH), lower BCoA pathway, CO-Dehydrogenase/Acetyl-CoA Synthase complex (ACDS), and the H4F methyl branch of the Wood-Ljungdahl (H4F WL) pathway. In addition, both MAGs encode complete DSR pathways. Thus, both organisms could be capable of benzene degradation and can perform DSR. However, because of their low abundance, it is questionable whether they are the main benzene oxidizers in the culture. It is possible that the highly abundant MAG 53 uses a yet unknown mechanism for benzene oxidation. In that case, this archaeon would require partner organisms, potentially the Desulfatiglandales bacteria represented by MAGs 46 and 47, for shuttling electrons into sulfate reduction. This would imply syntrophic interactions within this culture. All three MAGs 46, 47, and 53 encode membrane-bound [NiFe] hydrogenases, which would enable electron exchange via hydrogen. In addition, MAGs 46 and 47 encode archaeal type (FlaB) and bacterial type (PilA) cell appendages , which also affiliates with Thermoplasmatota, lacks acetate kinase for conversion of acetate to acetyl-CoA, but encodes a β-oxidation pathway for utilization of butyrate. In conclusion, multiple symbiotic interactions might co-exist in this culture. More studies are required to elucidate the mechanisms in this complex but highly active culture.In the B50 culture, the most abundant MAG, MAG 53 (Thermoplasmatota), encodes only very few proteins of known pathways for the activation and oxidation of benzene, among others benzoylsuccinyl-CoA thiolase (BbsAB) of the methylation pathway, and benzoate-CoA ligase (BamY) of the carboxylation pathway . We alsopendages , and allpendages . Based opendages . In accot sludge . The infhanogens . Thus, ihanogens . Membershanogens . The autoduction . The CalabcAD is absent from this MAG, and only a single enzyme (HbaA/HcrL) of the hydroxylation pathway is encoded, substrate activation most likely occurs via methylation. MAG 9 encodes a Thauera type RH pathway including BamR, BamQ, and BamA, and an almost complete Rhodopseudomonas type RH pathway. In addition, MAG 9 encodes a major part of the lower BCoA pathway and CODH/ACS complex, and a complete H4F WL pathway for a complete oxidation of benzene to CO2. Encoding a complete DSR pathway, this organism is able to combine benzene oxidation with sulfate reduction in a single cell. MAG 9 also encodes type IV pilin (PilA) and several chemotaxis genes, which could enable it to outcompete other potential benzene degraders and increase its efficiency for benzene degradation, as previously shown for the naphthalene-degrading bacterium Pseudomonas putida G7  encodes an almost complete pathway for benzene activation via methylation, and an almost complete class II BCR for RD . Becauseutida G7 . The estthn operon and lower BCoA pathway facilitates RH and oxidation to acetyl-CoA, followed by complete oxidation to CO2 via the CODH/ACS complex and the remaining H4F WL pathway. MAG 34 encodes a complete DSR pathway, enabling the organism to shuttle electrons from naphthalene oxidation directly into sulfate reduction. The incubation temperature is very close to the estimated OGT of the organism of 55°C. Its high relative abundance and its extensive genomic capacity for naphthalene degradation suggest that the bacterium represented by MAG 34 is the dominant, maybe even the only naphthalene oxidizer in the culture.In the N50 culture , MAG 34 thn operon for RH, among others the putative ring-cleaving hydrolase ThnL , and is therefore probably incapable of sulfate reduction. The next most abundant MAG, MAG 31 , encodes an almost complete DSR pathway, which lacks only the alpha subunit of Apr. Thus, it is possible that naphthalene oxidation in this culture occurs via syntrophic interactions. While membrane-bound hydrogenases are absent in MAG 33, both MAGs 33 and 31 encode formate dehydrogenases, which could facilitate electron transfer from MAG 33 to MAG 31 via formate. Direct transfer via DIET is also conceivable, because MAG 31 encodes both FlaB and PilA, and both MAG 33 and MAG 31 encode several multi-heme cytochromes.In the N70 culture , MAG 33 ase ThnL . FurtherWe aimed to bring the results from our enrichment cultures into a wider ecological context and examined the distribution of AH degradation genes and pathways in the larger taxonomic groups of the microorganisms that we enriched in our study. We refrained from examining the phylogeny of the Thermoplasmatota MAG 53 (M8_bin1702), because the Thermoplasmatota phylogeny was well-resolved in the recent study by Desulfatiglandales currently comprise 135 publicly available MAGs on GDTB, while for SZUA-161 only 26 MAGs are available at the moment. Both Desulfatiglandales and Dehalococcoidia are globally widespread members of marine sediment and subsurface communities . DesulfaThe MAGs of both orders originate from a wide array of environments, including lakes, springs, cold seeps, the seafloor and the subsurface. More than half of publicly available Desulfatiglandales MAGs (75 out of 135) originate from hydrothermal vent sediment, mainly from the GB (70 MAGs) and to a smaller degree (5 MAGs) from the Pescadero Basin, a recently described hydrothermal vent area  located Next, we examined the phylogenomic placement of Desulfatiglandales MAGs and the SZUA-161 MAG from our study in their larger taxonomic groups, and investigated the genomic potential of environmental Desulfatiglandales and SZUA-161 MAGs for anaerobic UAH degradation.Desulfatiglans anilini, which degrades phenol and the aromatic amine aniline , all from hydrothermal vent sediment in the GB and the Pescadero Basin. The clade splits into two groups: (1) MAGs 5, 9, and 46 and GCA_021163815.1 (2) GCA_019306325.1, GCA_003646995.1, MAGs 47, 34, 36, and GCA_019309225.1. Based on ANI, AAI and the tree structure  of the methylation pathway of benzene and the alpha subunit of phenylphosphate synthase (PpsA) of the hydroxylation pathway. Interestingly, more than a third of the Desulfatiglandales MAGs encode AbcA. AbcA, in connection with BamY, could enable many yet uncultured Desulfatiglandales of degrading benzene via the carboxylation pathway. Regarding BCRs, the alustris , is the  evansii . The BamnmsA) for naphthalene degradation after methylation  and SpSt-899 (17 MAGs). This phylogeny is well-resolved in our tree . MAG 33 4F WL pathways, which they could use for oxidation of dearomatized naphthyl-residues to CO2. Surprisingly, two MAGs of the SZUA-161 family encode complete DSR pathways, and the other six MAGs encode partial DSR pathways, insinuating that these organisms are capable of sulfate reduction, a metabolic trait that was previously not associated with members of the Dehalococcoidia.The SZUA-161 family encodes several key pathway genes for benzene degradation. For instance, seven out of eight MAGs encode BssA, all MAGs encode BamY, and six MAGs encode BamA. Interestingly, SZUA-161 seem to rely on ATP-dependent BCRs, since all MAGs encode BzdQ, five MAGs encode BcrA, but only two MAGs encode BamB. Naphthalene activation genes, both for the methylation and carboxylation pathway, are not encoded by members of the SZUA-161 family. Yet, two MAGs encode NCR, three MAGs encode DHNCR, and all MAGs encode one or more subunits of the four-subunit THNCR. Thus, while SZUA-161 likely cannot activate naphthalene directly, they might be able to dearomatize naphthyl-derivatives. All SZUA-161 family MAGs encode complete CODH/ACS complexes and partial or complete HbssA and bamY in two of the MAGs, and bamA in three of the MAGs. Those MAGs also encode several bcrA, bzdQ, and bamB subunits of BCRs, indicating a capacity for RD. MAG 33 is the only MAG of the group encoding a partial pathway for naphthalene degradation . Interestingly, one MAG of this group, GCA_013203045.1, encodes a complete and another MAG, GCA_018657655.1, an almost complete DSR pathway, indicating capacity for DSR in this group. In the remaining members of SpSt-899, genes for both benzene and naphthalene degradation are scarce. Yet, several MAGs contain isolated genes for the degradation of benzene, i.e., bssA, bamY, bamB, and bamA. Curiously, one MAG (GCA_023271155.1) encodes abcA for direct carboxylation of benzene and a copy of UbiD-like naphthalene carboxylase, even though it lacks many genes of the downstream degradation pathway. Most SpSt-899 MAGs encode CODH/ACS and the H4F WL pathway, even though both components are incomplete in some MAGs. All in all, we propose that capacity for UAH/AH degradation is an ancestral trait of the SpSt-899 and was lost in more recent members. However, particularly in several closely related MAGs of the AH-958 group, to which GCA_023271155.1 also belongs, the analysis is impaired by low completeness (52–66%) of the MAGs , GCA_018657655.1, and GCA_013203045.1, contain several genes for the anaerobic degradation of benzene or derivatives, e.g., the MAGs . More hiFerroglobus placidus, which thrives at 85°C  after activation via direct methylation in the Desulfatiglandales. Thus, methylation does not seem to be a frequent mechanism for naphthalene activation, which is in accordance with previous studies  can be found at: BioProject, PRJNA1013425.HZ: Conceptualization, Data curation, Investigation, Methodology, Project administration, Software, Visualization, Writing – original draft, Writing – review & editing, Supervision. CO: Investigation, Methodology, Software, Writing – review & editing. DBM: Investigation, Methodology, Software, Writing – review & editing. GW: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was funded by the Deutsche Forschungsgemeinschaft (DFG) through the Cluster of Excellence EXC 2077 “The Ocean Floor—Earth’s Uncharted Interface” (project no. 390741603) and the Max Planck Society (MPS).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background and Objectives: With the growing incidence and disability associated with myocardial infarction (MI), there is an increasing focus on cardiac rehabilitation post-MI. Kuanxiongzhuyu decoction (KXZY), a traditional Chinese herbal formula, has been used in the rehabilitation of patients after MI. However, the chemical composition, protective effects, and underlying mechanism of KXZY remain unclear. Materials and Methods: In this study, the compounds in KXZY were identified using a high-performance liquid chromatography-mass spectrometry (HPLC-MS) analytical method. Based on the compounds identified in the KXZY, we predictively selected the potential targets of MI and then constructed a protein–protein interaction (PPI) network to identify the key targets. Furthermore, the DAVID database was used for the GO and KEGG analyses, and molecular docking was used to verify the key targets. Finally, the cardioprotective effects and mechanism of KXZY were investigated in post-MI mice. Results: A total of 193 chemical compounds of KXZY were identified by HPLC-MS. In total, 228 potential targets were obtained by the prediction analysis. The functional enrichment studies and PPI network showed that the targets were largely associated with AKT-pathway-related apoptosis. The molecular docking verified that isoguanosine and adenosine exhibited excellent binding to the AKT. In vivo, KXZY significantly alleviated cardiac dysfunction and suppressed AKT phosphorylation. Furthermore, KXZY significantly increased the expression of the antiapoptotic proteins Bcl-2 and Bcl-xl and decreased the expression of the proapoptotic protein BAD. Conclusions: In conclusion, the network pharmacological and experimental evidence suggests that KXZY manifests anti-cardiac dysfunction behavior by alleviating cardiomyocyte apoptosis via the AKT pathway in MI and, thus, holds promising therapeutic potential. Myocardial infarction (MI) is currently a major cause of morbidity and mortality worldwide, posing a significant public health challenge with enormous medical and societal consequences . While tConioselinum Chuanxiong and Angelica sinensis (Oliv.) Diels improve myocardial energy metabolism, promote hematopoiesis, and reduce blood deposition [The use of natural and herbal remedies to treat human problems has been well known for a significant length of time. Because of its holistic and systematic nature, traditional Chinese medicine (TCM) has recently attracted global attention . Thus, hposition ,8. NeverThe approaches of network biology and multitarget drug development have made tremendous strides over the past several years. Thus, some new research concepts, such as network pharmacology, have been created, providing an applicable and efficient research strategy for modern drug research and novel therapy discovery . The priConioselinum Chuanxiong (Chuanxiong), Paeonia lactiflora Pall. (Chishao), Rehmannia glutinosa (Gaertn.) DC. (Dihuang), Angelica sinensis (Oliv.) Diels (Danggui), Prunus persica (L.) Batsch (Taoren), Carthamus tinctorius L. (Honghua), Citrus aurantium L. (Zhike), Bupleurum chinense DC. (Chaihu), Achyranthes bidentata Blume (niuxi), Allium macrostemon Bunge (Xiebai), Trichosanthes kirilowii Maxim. , Ziziphus jujuba Mill. (Hongzao), Hirudo nipponica Whitman. (Shuizhi), Alpinia katsumadai Hayata (Doukou), Platycodon grandiflorus A.DC. (Jiegeng), and Glycyrrhiza uralensis Fisch. ex DC. (Gancao). All of these ingredients were provided by the Hangzhou First People’s Hospital . First, the herbal mixture was soaked in water  for 30 mins and then extracted twice at reflux for 2 times (1 h per cycle). The extracted herbal mixtures were then blended, concentrated, and freeze-dried into a powder for storage.KXZY is composed of sixteen Chinese herbs , namely,v/v formic acid) and B . Using a gradient program, the following profile was created: 0 min , 10 min , 25 min , 30 min  0% B, 40 min , 45 min , 60 min , and 70 min . Q Exactive Orbitrap high-resolution mass spectrometry was used for mass spectrometry (MS) data acquisition, and the detection mode was Full MS-ddMS2 with separate scanning in positive and negative ion modes. The parameter conditions were as follows: mass range: 100–1200 m/z; MS1 resolution: 70,000; MS2 resolution: 17,500; ion source voltage: 3.2 kV; capillary temp: 320 °C; aux gas heater temp: 350 °C; sheath gas flow rate: 40 L/min; aux gas flow rate: 15 L/min.HPLC-MS analysis was conducted on a Q Exactive plus Orbitrap LC-MS/MS  equipped with a Waters ACQUITY UPLC HSS T3 column ; the column temperature was 35 °C. The parameter conditions were as follows: injection volume: 10 L; flow rate: 0.3 mL/min; mobile phase: A  . The preHomo sapiens” was the only species considered.MI-associated targets were compiled by merging the Comparative Toxicogenomics (CTD), Online Mendelian Inheritance in Man (OMIM), and DisGeNET databases ,12, and Potential KXZY targets for treating MI were the targets shared by KXZY component-related targets and MI-related targets.To examine the probable interactions between target-related genes, we imported these genes into the public STRING database . STRING p value of less than 0.05.We used the DAVID V6.8 database to perform GO and KEGG pathway enrichment analyses to show the roles of targets for the active components of TCM in terms of gene function and particular signaling pathways . GO analAutoDockTools 1.5.6 was used to accomplish molecular docking. The critical target’s atomic coordinates were collected from the Protein Data Bank (PDB) and generated in AutoDockTools-1.5.6 by eliminating water molecules, adding charge, and parameterizing. Downloaded from the TCMSP Database, the 3D structures of active components were built in AutoDockTools by calculating atomic partial charges and parameterizing. The docking site was positioned in the middle of the original ligand inside a cuboid box, and a grid map of each atom type within the box was calculated. Molecular docking of possible targets and components was simulated using the AutoDockTools-1.5.6 program. Each compound’s highest-scoring conformer was examined and displayed using AutoDockTools-1.5.6 and PyMOL.Zhejiang Chinese Medical University  approved the procedures for this investigation . Shanghai SLAC Laboratory Animal Co. provided male C57/BL6 mice weighing between 22 and 24 g at the outset . Prior to surgical modeling, experimental mice were acclimated to the laboratory setting for one week. Following surgery, mice were randomly divided into four groups (five to eight mice per group): the sham group (sham), the MI model group (MI), the KXZY group , and the captopril group . The medication dosage was determined using the body surface area. As mentioned earlier, surgery was performed in the Model and KXZY groups by ligating the left anterior descending (LAD) coronary artery. Without ligating the LAD coronary artery, a suture was passed across the LAD of the sham group. All groups received intragastric delivery of water or medication for 28 days. Four weeks after an acute myocardial infarction, the heart tissues of sacrificed mice were swiftly removed. A part of the heart was preserved in 4% paraformaldehyde, and another part was kept at −80 °C for future research.The supine posture was maintained after intraperitoneal injections of 1% pentobarbital sodium rendered the experimental mice unconscious. The left ventricular function in mice was evaluated by transthoracic echocardiography . Under the parasternal short-axis (SAX) ultrasound section, we captured the LVIDd and LVIDs. After this, the EF and FS were separately assessed.The abdominal aorta of mice was used to collect whole blood, which was then processed to isolate serum and stored at −80 °C for further research. The levels of NT-proBNP in serum were measured using a commercial ELISA kit .Cardiac tissues treated with paraformaldehyde were embedded in paraffin and cut into 4 μm thick slices. Subsequently, the sections were processed by hematoxylin-eosin (H&E) and Sirius red staining to evaluate the extent of fibrosis and pathological changes. The reagents were purchased from Abcam .Total protein was extracted from cardiac tissues using radioimmunoprecipitation assay (RIPA) buffer, including a protease inhibitor cocktail. A 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes , which were incubated overnight at 4 °C. Primary antibodies against BAD, AKT, and p-AKT were obtained from Cell Signaling Technology , while Bcl-2, Bcl-xl, and GAPDH were purchased from Abcam . Then, secondary antibodies  were incubated at 25 °C for 1 h. Bands were visualized using enhanced chemiluminescence (ECL) plus Western blotting detection reagents  and subsequently captured by the ChemiDoc Touch Imaging System and Image Lab software .p values less than 0.05 were considered statistically significant.All animal experiment results are reported as the mean ± SD. SPSS 17.0 statistical software was used for the statistical analysis . For numerous comparisons across groups, one-way analysis of variance (ANOVA) was applied in line with tests for normality and homogeneity of variance. Then, the above compounds were input into BATMAN to predict the related targets, and targets with scores > 20 were considered meaningful. After eliminating duplicated genes, a total of 90 vital components with 904 targets were retrieved.In total, 757 genes were identified from the CTD, OMIM, and DisGeNET databases that met the requirements for a connection with MI. After eliminating duplicated genes, 528 MI-related genes were retrieved. As shown in As To analyze potential protein interactions, the predicted targets of KXZY were uploaded into the SPRING database. As shown in KEGG pathway enrichment results indicated that there was significant enrichment in the PI3K/AKT signaling pathway. AKT1 was the top hub target in the structural network. Therefore, we inferred that the PI3K/AKT signaling pathway plays a critical role after MI. Thus, we further mapped the top-ranked hub targets in the PI3K/AKT pathway. As shown in Finally, we screened the key active compounds that precisely hit AKT1, which is the key target protein, based on the above target prediction results . As shown in p < 0.05). Conversely, in mice treated with KXZY or captopril, the EF and FS levels were markedly increased compared with those in the MI group (p < 0.05). The results demonstrated that KXZY could improve cardiac function after MI. In addition, our study indicated that the serum levels of NT-proBNP, which is a cardiac dysfunction marker, were markedly elevated in the model group (p < 0.05). In contrast, KXZY appreciably attenuated the elevation of NT-proBNP levels (p < 0.05).To confirm the cardioprotective benefits of KXZY after MI, KXZY was intragastrically administered to post-MI mice for 4 weeks. Subsequently, we evaluated the function and histopathology of the LV. As shown in Moreover, the histopathological results of cardiac tissue revealed disorganized cardiac muscle fiber arrangements, disruption of cardiac structure, and significant interstitial edema in the MI group. The total cardiac fibrosis area was markedly enlarged in the MI group versus the control group. In the KXZY group, these histopathologic abnormalities were restored. These results provide vital evidence that KXZY can attenuate the extent of myocardial injury in post-MI mice.As shown in TCM’s “holistic perspective” and “syndrome distinction” concepts provide unique advantages in the treatment of diseases. As one of the classic prescriptions for promoting blood circulation and removing blood stasis, KXZY has certain effects on the prevention and treatment of cardiovascular diseases. It is usually added according to clinical syndrome differentiation and disease, such as in post-MI patients with chest distress, shortness of breath, heart palpitations, and tiredness. Wang et al.’s research shows that supplementing qi and activating blood therapy were effective and safe in the treatment of coronary heart disease . In addiWe first identified 193 substances using the HPLC-MS technique. Among these, quercetin, liquiritigenin, and naringin have been reported to have antiapoptotic, antihypertensive, anti-inflammatory, and anti-cardiovascular injury properties ,24,25. TCardiomyocyte apoptosis plays an important role during chronic cardiac remodeling and heart failure. Especially in the acute and subacute phases of MI, apoptosis is a prevalent pathogenic characteristic . StudiesThe AKT protein is one of the serine/threonine protein kinase families that regulates several biological activities, including cell growth, metabolism, and survival . The AKTTherefore, based on the above results, further investigation was conducted to explore the potential anti-apoptotic mechanism of KXZY. To assess the potential cardioprotective and anti-apoptotic effects of KXZY, a post-MI mouse model was utilized in this study. Our study shows that post-MI mice exhibit better EF and FS values with the treatment of KXZY. Our research suggested that KXZY could improve left ventricular systolic function in post-MI mice. Furthermore, we assessed the AKT-related anti-apoptotic effects of KXZY after MI damage. AKT phosphorylation was significantly inhibited, and BAD expression was markedly reduced. There was an increase in the expression of Bcl-2 and Bcl-xl. We therefore infer that KXZY may exert its anti-apoptotic effects by regulating the phosphorylation and activation of AKT1, which in turn inhibits the pro-apoptotic protein BAD, allowing Bcl-2 and bcl-xl to inhibit apoptosis and maintain cell survival. These findings indicated that AKT-related antiapoptotic mechanisms might be the main mechanism of KXZY in post-MI damage.Finally, molecular docking was used to verify the mode of interaction between compounds of KXZY and AKT1 protein molecules. Eight compounds of KXZY were predicted to bind to AKT1: isoguanosine, naringenin, liquiritigenin, isosakuranetin, hesperetin, dehydrodiisoeugenol, glabridin, and adenosine. Our study indicated that isoguanosine exhibits high binding energy through molecular docking. Thus, we inferred that isoguanosine might improve cardiac dysfunction after MI by inhibiting AKT-related apoptosis.This study identified 193 chemical compounds of KXZY by HPLC-MS. In addition, 228 potential targets were predicted, and a PPI network was constructed for further network pharmacology research. The findings of functional enrichment analysis suggest that the apoptotic process is strongly associated with AKT1. Additionally, we used a post-MI mouse model to verify the anti-apoptotic and anti-cardiac dysfunction behaviors of KXZY, and isoguanosine and adenosine were found to be two important pharmacodynamic compounds. Consequently, the herbal method provides a wide repository for the development of novel therapeutic agents for the treatment of post-MI cardiac damage, and KXZY might serve as an important supplementary substance.