In order to explore and further understand the efficacy of donepezil (DNP) in the treatment of Alzheimer's disease (AD), this research was conducted based on network pharmacology and molecular docking.Compounds of DNP and its effective targets were collected using the TCMSP Chinese medicine system pharmacology database. Disease targets were screened and selected utilizing GeneCards, TTD, DrugBank, CTD, and other online databases. Then, Venn diagrams were generated to identify the intersections. A diseases-drug-active ingredient-key target protein interaction (PPI) network was constructed using the STING database. GO and KEGG enrichment analyses were conducted to predict the function and mechanism of DNP, which were visualized by graphs and bubble charts. After the screening, the top five interacting targets in the PPI network and the compound containing the most active target were selected for molecular docking.The study received 110 potential targeting genes and 155 signaling pathways. A strong association between DNP and modulation of chemical synaptic transmission and the regulation of trans-synaptic signaling is noted. Signaling pathways related to the proliferation, differentiation, and survival of cells are also found positively relative. The results revealed that the mechanism of its therapeutic effect is multi-component, multi-target, and multi-pathway, laying a foundation for the follow-up in-depth study of the mechanism of DNP in the treatment of AD.This research provides a superior prediction that AD could be treated using DNP which targets the key proteins and essential pathways associated with the recovery of AD. Alzheimer's disease (AD) is a neurodegenerative disorder in the central nervous system, prevalently observed among the elderly and near-elderly, characterized by progressive cognitive impairment, and memory degeneration . Despite the effectiveness of the existing therapies in treating the symptoms of AD, no significant influence has been found in curing or preventing the disease  stimulations . With the structure acquired, data of the donepezil targeted sites were collected from four separate databases: swisstarget (http://www.swisstargetprediction.ch/), pharmmapper (http://lilab-ecust.cn/pharmmapper/), batman-tcm (http://bionet.ncpsb.org.cn/batman-tcm/), and stitch (http://stitch.embl.de/), each with a different standard for data selection. For swisstarget, data with Probability*>0 was included. For batman-tcm, the inclusion standard was set Score cutoff≥ 20. No standard was set for screening in stitch or pharmmapper. The corresponding genes of the donepezil target sites were collected through Uniprot (http://www.uniprot.org/www.uniprot.org/), utilizing data downloaded in pharmmapper.First, the structural formula of donepezil was downloaded from PubChem  from three aspects, Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), with p-value cutoff and q-value cutoff both as 0.05. The results from each aspect were visualized with a graph and bubble chart.With the selected genes, KEGG pathway enrichment analysis was conducted to predict its mechanism of action using DOSE, clusterProfiler, and pathview packages (Bioconductor), with p-value cutoff and q-value cutoff both as 0.05. Columns and bubble charts were generated to visualize the result.*>0, score cutoff≥20 was screened and downloaded from the swisstarget and batman-tcm databases. The structure was also searched in the stitch and pharmmapper databases to further verify the results. The structure was visualized using PubChem  was utilized to visualize the intersection result , including 10 BP, 10CC, and 10 MF. Therefore, it can be concluded that the mechanism of the therapeutic effect of donepezill to AD could be associated with biological processes such as positive regulation of RNA polymerase II promoter, signal transduction, and positive regulation of DNA template transcription. At the same time, a variety of substances such as plasma membrane, cytosol, and extracellular space are involved, such as protein binding, enzyme binding, transcription factor activity, sequence-specific DNA binding, same protein binding, and other molecular functions.Genes for potential target sites were selected using the R package from “org.Hs.eg.db” database, and GO enrichment analysis was conducted using the DOSE, clusterProfiler, and pathview packages (Bioconductor) from three aspects, Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), with p-value cutoff and q-value cutoff both as 0.05. The results from each aspect were visualized with a bubble and bar chart . 30 GO lP < 0.05, FDR < 0.05), and the top 20 are listed by Count number (In KEGG pathway enrichment analysis, 155 signaling pathways are screened (t number . The KEGt number .The development of drugs with clinical value has met with extreme obstacles (Becker et al., DNP is a kind of AChEI, which functions as an inhibitor to the cholinesterase enzyme and sustains the activity of acetylcholine at cholinergic synapses. DNP has been shown to significantly improve cognition and daily function and some behavior manifestations of patients with AD (Massoud and Gauthier, The study received 110 potential targeting genes and 155 signaling pathways in total. The diversity of the associated genes and pathways demonstrate that the mechanism of DNP in the treatment of AD is multifactorial. A strong association between DNP and modulation of chemical synaptic transmission and the regulation of trans-synaptic signaling is noted, further confirming the ability of DNP in sustaining the activity of synapses. In addition, signaling pathway related to the proliferation, differentiation, and survival of cells are also found positively relative, such as Ras and MAPK signaling pathways, suggesting a potential of promoting the growth and proliferation of neurons using DNP.The cholinergic system plays an indispensable role in neuronal function in memory, learning, and other essential aspects of cognition and plays a wider role in the promotion of neuronal plasticity (Drachman and Leavitt, in vivo or in vitro, and deficiencies remain noticeable. However, this study can provide new ideas and directions for further exploration of relevant experiments.In this study, due to the incomplete information in the database and unclear interaction between DNP and other factors, the prediction results have certain limitations. This study did not verify the possible molecular mechanism of DNP in the treatment of AD syndromes either The simulation experiment data used to support the findings of this study are available from the corresponding author upon request.LL and PF prepared the figures. JY and YZ finished the paper. All authors contributed to the article and approved the submitted version.This work was supported in part by the Science and Technology Commission of Shanghai Municipality, General Program, No.20ZR1456400.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.


The possible mechanism of Xiyanping injection treatment COVID-19 is discussed through the network pharmacology.  Obtaining the chemical structure of Xiyanping injection through the patent application and obtaining control compounds I, II, III, IV, V, Yanhuning injection , Chuanhuning injection , 10 compounds were analyzed by D3Targets-2019-nCoV. The human anti-COVID-19 gene in COVID-19 DisGeNET was intersected with the CTD Andrographolide target gene and then combined with D3Targets-2019-nCoV, resulting in 93 genes, using the Venny 2.1 platform. The PPI network was constructed by the String platform and Cytoscape 3.8.2 platform. The GO, KEGG, and tissue of the target were analyzed using the Metascape platform and DAVID platform. The gene expression in the respiratory system was analyzed using the ePlant platform. The CB-Dock is used for the docking verification and degree values of the first 20 genes.  Finally, 1599 GO and 291 KEGG results were obtained. GO is mostly associated with the cell stress response to chemicals, the cell response to oxidative stress, and the cell response to reactive oxygen species. In total, 218 KEGG pathway concentrations were related to infection and other diseases and 73 signaling pathways mostly related to inflammation and immune pathways, such as TNF signaling pathway and MAPK signaling pathway. The molecular docking results show that Xiyanping injection, compound III, has a good docking relationship with 20 target proteins such as HSP90AA1. Tissue has 22 genes that are pooled in the lungs.  Xiyanping injection may inhibit the release of various inflammatory factors by inhibiting intracellular pathways such as MAPK and TNF. It acts on protein targets such as HSP90AA1 and plays a potential therapeutic role in COVID-19. Thus, compound III may be treated as a potential new drug for the treatment of COVID-19 and the Xiyanping injection may treat patients with COVID-19 infection. It mainly has anti-inflammatory, antibacterial, anticough, and other effects and enhances the body's immunity, [According to expert consensus, Xiyanping injection belongs to Chinese medicine injections, primarily composed of Coronaviruses (CoVs) consist of a single positive-chain RNA virus that belongs to the coronavirus group and can develop into respiratory, intestinal, nervous, reproductive, and other diseases in infected animals and humans . In 2019Recently, network pharmacology has been widely used in the study of Chinese medicine anti-SARS-CoV-2 . The emeBased on clinical data, this article uses network pharmacology to explore the possible mechanism of Xiyanping injection anti-SARS-CoV-2, while andrographolide, compound III, can be used as a potential new anti-SARS-CoV-2 drug .https://go.drugbank.com/): Yanhuning injection compounds: VI and VII; Chuanhuning injection compounds: VIII and IX. Ten compounds are drawn and saved in SDF format via ChemDraw 20.0 software [We obtained the chemical structures of six andrographolide salts, including Xiyanping injection and other andrographolide salts synthesized in process production, through the contents in the patent book of pharmaceutical companies. These five compounds were derived from the production process of Xiyanping injection, but they were not produced in the market as the objects of subsequent pharmacodynamic development, and there were no related clinical studies: I, II, III, IV, and V. We obtained similar andrographolide Chinese medicine injections via the DrugBank  platformsoftware .https://www.d3pharma.com/D3Targets-2019-nCoV/index.php), the Xiyanping injection anti-COVID-19-related target genes were docked. We obtained human gene targets related to andrographolide based on the CTD platform [https://ctdbase.org/). The CTD platform [https://ctdbase.org/) takes the target intersection collection of these two gene sets through the Venny 2.1 platform (https://bioinfogp.cnb.csic.es/tools/venny/). The collection is then merged with the human anti-COVID-19 target obtained by the D3Targets-2019-nCoV platform [Based on the D3Targets-2019-nCoV platform  , and the target protein interaction network (PPI network) is built through the String platform [https://www.string-db.org/). Finally, a compound-target PPI network is built using the Cytoscape 3.8.2 software platform [The target protein is converted into the corresponding gene name through the UniProt platform  (https:/platform  (https:/platform .https://metascape.org/); the species is limited to “H. sapiens,” the threshold P < 0.05, and its GO biofunction , molecular function (MF), and cell composition (CC)) enrichment analysis is carried out. At the same time, KEGG signal path enrichment analysis and tissue structural analyses are carried out through the DAVID platform [https://david.ncifcrf.gov/), and Enrichment Plot is drawn through the image GP platform (http://www.ehbio.com/).To further analyze the function of the target, the target is uploaded to the Metascape platform  (https:/platform  (https:/https://www.rcsb.org/): 20 target macromolecules, hydrogenation, removal of water molecules, removal of ligands, etc., through Discovery Studio 2019 Client software [https://clab.labshare.cn/cb-dock/php/) for molecular docking to bind the energy ≤ −5.0 kcal/mol as the basis for molecular-target interactions [Download the Human Target Protein  in the Tsoftware . The tarsoftware  . The modern injection has adverse reactions because it directly enters the internal circulation without passing through the liver metabolism of the digestive system. The adverse reactions of andrographolide injections—Yanhuning injection and Chuanhuning injections—are more serious; therefore, they should be used with caution in children. China's National Medical Products Administration has reported security issues with Yanhuning injection/Chuanhuning injection and noted that Yanhuning injection/Chuanhuning injection was used cautiously for children [The main component of Xiyanping injection is andrographolide, which is the main effective component of the natural plant tic drug . In clinchildren . In the κB and other pathways in the nucleus through various pathways, inhibits the release of various inflammatory factors, and plays a role in the treatment of COVID-19.Excessive TNF can cause tissue damage, which is one of the core cytokines of an “inflammatory storm” caused by infection. TNF can induce cells to release cytokines, but in some cases, it can also induce apoptosis or programmed cell necrosis . After XκB (RELA) and STAT3 transcription procedures, including proinflammatory cytokines IL-6 and IL-8 [Target gene HSP90AA1: HSP90AA1 is an important regulator of inflammation because HSP90AA1 is currently understood as an extracellular agent secreted in wound healing and inflammation. Extracellular HSP90 + induces inflammation by activating NF-and IL-8 . In addiTarget gene CAT: CAT gene occurs in almost all aerobic breathing organisms, which helps protect cells from the toxic effects of hydrogen peroxide. It promotes cell growth, including T cells, B cells, bone marrow leukemia cells, melanoma cells, breast cells, and normal and transformed fibroblasts ; XiyanpiTarget gene TNF: TNF gene induces VEGF production by cooperating with IL1B and IL6 , plays aTarget gene FOS: FOS is considered to play an important role in signal transduction, cell proliferation, and cell differentiation. In growth cells, phospholipid synthesis may be activated by CDS1 and PI4K2A . This acFrom the molecular docking Vina data, it was found that the overall score of compound III was better than that of Xiyanping injection. This also indicates the possibility of a potential new drug, with compound III having better water solubility than Xiyanping injection.κB and other pathways in the nucleus, thus playing a role in the treatment of COVID-19. Compound III is a potential new drug for the treatment of COVID-19. Xiyanping injection can achieve a therapeutic effect in patients with infectious pneumonia, which is in line with the “Chinese Association of Traditional Chinese Medicine Expert Consensus (literature reports)” [In this study, through network pharmacology, molecular docking, target analysis, and tissue analysis, it can be concluded that Xiyanping injection and andrographolide sulfonates such as compound III in the treatment of COVID-19 mainly act on HSP90AA1, CAT, TNF, FOS, and other targets. At the same time, Xiyanping injection inhibits the release of a variety of inflammatory factors by inhibiting intracellular MAPK, TNF, and other pathways and inhibiting NF-


Rhodococcus sp. AD45 two decades ago, the isoprene metabolic pathway still remains largely undefined. Limited understanding of the functions of isoG, isoJ and aldH and uncertainty in the route of isoprene‐derived carbon into central metabolism have hindered our understanding of isoprene metabolism. These previously uncharacterised iso genes are essential in Variovorax sp. WS11, determined by targeted mutagenesis. Using combined ‘omics‐based approaches, we propose the complete isoprene metabolic pathway. Isoprene is converted to propionyl‐CoA, which is assimilated by the chromosomally encoded methylmalonyl‐CoA pathway, requiring biotin and vitamin B12, with the plasmid‐encoded methylcitrate pathway potentially providing robustness against limitations in these vitamins. Key components of this pathway were induced by both isoprene and its initial oxidation product, epoxyisoprene, the principal inducer of isoprene metabolism in both Variovorax sp. WS11 and Rhodococcus sp. AD45. Analysis of the genomes of distinct isoprene‐degrading bacteria indicated that all of the genetic components of the methylcitrate and methylmalonyl‐CoA pathways are not always present in isoprene degraders, although incorporation of isoprene‐derived carbon via propionyl‐CoA and acetyl‐CoA is universally indicated.Bacteria that inhabit soils and the leaves of trees partially mitigate the release of the abundant volatile organic compound, isoprene . While the initial steps of isoprene metabolism were identified in  Anthropn et al.  and the 1 Greve, . Studies1 Greve, , 2012. I1 Greve, , and can−1 in early estimates  were abundant components of isoprene‐degrading communities, including from soil from a tyre dump, soils taken from the vicinity of willow and oil palm trees, and the phyllospheres of willow and poplar trees  catalyses the initial oxidation of isoprene to epoxyisoprene , then from GMB to 2‐glutathionyl‐2‐methyl‐3‐butenoic acid (GMBA). The structures of these intermediate metabolites are shown in Figure Rhodococcus sp. AD45, van Hylckama Vlieg et al.  and aldH (a putative dehydrogenase), also lack functional characterizations. A possible role of AldH was suggested during a study of the styrene metabolic pathway of G. rubripertincta CWB2, wherein AldH may perform the same second oxidation reaction as StyH, which catalyses the oxidation of glutathione to (1‐phenyl‐2‐acetic acid)glutathione  and two phylogenetically distinct aldH genes. However, genes involved in the methylcitrate cycle and methylmalonyl‐CoA pathway, which were predicted to be involved if isoprene were incorporated by a similar mechanism as odd‐chain fatty acids . These cultures were switched to one of four conditions (1% (v/v) isoprene, 0.01% (w/v) epoxyisoprene, 10 mM succinate, no substrate), and 10 ml aliquots were harvested at specific intervals and stored for RNA extraction , and switched onto one of two growth conditions (10 mM succinate or 1% (v/v) isoprene). The 200 ml aliquots were removed at Timepoint 0, after 6, 24, or 30 h and the total protein content was extracted. Fractionation, Tandem Mass Tagging, and proteome analysis were run at the Proteomics Facility  using nanoLC‐MS/MS on an Orbitrap Eclipse™ Tribrid™ mass spectrometer, coupled to an UltiMate® 3000 RSLCnano LC system . Data processing was run using Proteome Discoverer 2.4.1.14 (Thermo). A total of 5364 proteins were identified, of which 4350 were quantified with at least two unique peptides. Hypothesis testing was run using the background‐based t‐test  was PCR‐amplified from pCM351 to include the 5′ and 3′ loxP sites  isoprene to an OD540 of 0.6, using three biological replicates for each condition. Cells were pelleted by centrifugation at 4000g for 10 min at 4°C. Total RNA was extracted using TRIzol reagent  according to the manufacturer's instructions. Contaminating DNA was removed by a single treatment with RNase‐free DNase , then DNase‐treated samples were purified using RNeasy spin columns . RNA concentration was determined using a Qubit RNA Broad Range assay kit (ThermoFisher Scientific), and a lack of contaminating DNA was verified by a negative PCR assay using 16S rRNA primers. cDNA was generated from approximately 900 ng RNA in 20 μl reactions using the Superscript III reverse transcriptase kit (ThermoFisher Scientific) with random hexamers (Invitrogen), including controls for each condition without reverse transcriptase for reference. Primers were designed to amplify garB_1, garB_2, and garB_3, using rpoB as an internal reference gene  isoprene, 10 mM pyruvate, or a combination of 10 mM pyruvate and 1% (v/v) isoprene. Cells were pelleted by centrifugation at 4000g for 10 min at 4°C, then resuspended to an OD540 of 10.0 in 1 ml of ice cold 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) . Cell suspensions were sealed in 30 ml vials and pre‐incubated for 3 min in a water bath at 30°C with shaking at 160 rpm, then spiked with 1 ml of headspace from a 1% isoprene stock. After a further 1 min, 50 μl headspace samples were taken using a gas‐tight syringe  and injected into a Fast Isoprene Sensor. Further samples were taken every 3 min for 1 h.Isoprene uptake assays were conducted as described by Dawson et al. . VariovoTranslated amino acid sequences were used as query sequences against a specific subject genome sequence using tBLASTn .All experiments were conducted using three biological replicates for each condition, with the exception of the proteome study, which was run using two biological replicates per condition. Statistical differences between two experimental conditions were calculated using the Student's Variovorax sp. WS11 transcriptome over a range of timepoints (see Variovorax sp. WS11. Sequences at the 5′ end of the iso gene cluster were more abundant in the RNAseq data than at the 3′ end at Timepoint 1 (20 min), possibly due to the fact that termination of transcription often occurs at the 5′ end of the sequence. Transcription of the iso genes , increasing to 451‐ to 694‐fold at Timepoint 4 (24 h), relative to Timepoint 0 (0 h)  exhibited a weak response to isoprene   Figure , althougepoint 1 0 min, poe Figure . The morepoint 1 0 min, poh Figure , rather Variovorax sp. WS11 to isoprene, the corresponding Iso proteins were not significantly expressed above Timepoint 0 (0 h) levels after 6 h of growth on isoprene, with the exception of IsoI (p ≤ 0.01) , largely failed to exceed 1.0, with the greatest positive increase demonstrated by IsoI with a ratio of 2.5 after 24 h of growth on succinate . The transcription of aldH increased significantly from a fold‐change of 143 after 20 min to 514 after 6 h in the presence of isoprene, relative to Timepoint 0 (0 h) . An AldH homologue in the isoprene‐degrading Rhodococcus opacus PD630, which shares the same genetic context with AldH1 from Rhodococcus sp. AD45 and shares 90% amino acid identity with the latter, was characterized as a NAD(P)+‐dependent glyceraldehyde 3‐phosphate dehydrogenase   catalyses the reductive cleavage of the disulfide bond of oxidized glutathione‐disulfide (GSSG) to form reduced glutathione (GSH) in order to restore its redox function , suggesting that the functional prediction as a glutathione‐disulfide reductase is correct. The putative LysR‐type transcriptional regulator (LTTR) dmlR_4 has consistently been identified upstream of isoG in the iso metabolic gene clusters from known Gram negative isoprene degraders   and 3,4‐dichloro‐1‐nitrobenzene (DCNB) , isoJ (ΔisoJ) or aldH  were unable to grow on isoprene, although Variovorax sp. WS11 ΔisoJ and ΔaldH mutants consumed a small amount of isoprene from the headspace of the respective vials , which consumed all detectable isoprene within 72 h. The Variovorax sp. WS11 ΔaldH mutant consumed significantly less isoprene than mutants WS11 ΔisoG and WS11 ΔisoJ after 120 h (p ≤ 0.0005)  and ΔaldH (p ≤ 0.01). Each wild‐type copy of the deleted genes was expressed from the de‐repressed lac promoter of pBBR1MCS‐2  was a succinyl‐CoA:d‐citramalate CoA transferase from Chloroflexus aurantiacus. The deduced IsoG amino acid sequence from Variovorax sp. WS11 encodes conserved domains which indicated CoA‐transferase activity, as determined by BLASTp analysis. If isoprene is incorporated into central metabolism via β‐oxidation, it stands to reason that a CoA group would be incorporated into the isoprene‐derived carbon chain. A GMBA‐CoA conjugate (2‐glutathionyl‐2‐methyl‐3‐butenyl‐CoA) is the most likely intermediate compound, since the alternative, removal of glutathione by glutathione‐S‐transferase IsoJ before CoA addition, is not supported due to the lack of activity towards GMBA by IsoJ reported by van Hylckama Vlieg et al. , two additional copies of garB  were detected on the chromosome with 45.3% and 42.9% identity to garB_3 from the isoprene cluster, respectively, at the translated amino acid level. It was clear that neither garB_1 or garB_2 was involved in isoprene metabolism by the wild‐type strain, since neither transcripts nor translated gene products were detected by either ‘omics experiment , but there was no change between the succinate‐grown and isoprene‐grown conditions in the mutant strain. The continued growth of Variovorax sp. WS11 ΔgarB on isoprene may have been supported by de novo glutathione biosynthesis or possibly due to the activity of an alternative thiol recycling mechanism. Escherichia coli was resilient to the loss of glutathione‐disulfide reductase, presumed to be due to redundancy provided by the thioredoxin system, although a glutathione‐disulfide reductase‐deficient and thioredoxin‐deficient double‐mutant was similarly able to maintain intracellular reduced glutathione pools , but not in succinate‐grown cells, suggesting the role of this protein in isoprene metabolism. Enoyl‐CoA hydratase (ECH) catalyses the hydration of an alkene group in a CoA‐thioester to form a sub‐terminal hydroxyl group, but this typically occurs at the 2–3 carbon position rather than the 3–4 carbon position as in GMBA‐CoA. However, some enoyl‐CoA hydratase/isomerase family proteins may catalyse both the hydration and isomerase reactions    , indicating that the enzymatic functions may differ. Additionally, the expression of NDZ14590.1 was downregulated during growth on isoprene   of the approximately 30 genes , while the genome of Variovorax paradoxus B4 (GCA_000463015.1) contains genes encoding 2‐methylcitrate synthase (prpC) and 2‐methylcitrate dehydratase (prpD), with the prpD gene annotated as a bi‐functional 2‐methylcitrate dehydratase/aconitate hydratase which shared no identity with methylcitrate dehydratase (acnD) from Variovorax sp. WS11 when compared by BLASTp. Instead, prpD from V. paradoxus B4 shared 84% predicted amino acid identity with a non‐isoprene‐induced chromosomal copy of prpD  in Variovorax sp. WS11, which was adjacent to a second copy of methylcitrate synthase (prpC_2 ‐ NDZ16657.1) which had a relative expression of 91.4 after 30 hours of growth on isoprene (Supplementary Data sheet prpC_2 and prpD genes was not upregulated in the transcriptome of isoprene‐grown cells (Supplementary Data sheet Both the Variovorax sp. WS11 (S1b) with predicted roles in fatty acid metabolism and biotin synthesis, including acetyl‐/propionyl‐CoA carboxylase α‐subunit (NDZ12928.1) and β‐subunit (NDZ12936.1), acyl‐CoA dehydrogenase (NDZ12938.1), and propionate‐CoA ligase (NDZ12937.1)  measuring approximately 20 kbp was located on the chromosome of ) Figure , L33‐48.) Figure , L50‐52,Variovorax sp. OPL2.2 , with the closest relative at the nucleotide level found in Acidovorax avenae KL3 (GCA_003029905.1) , although this bacterium contains no isoprene gene cluster. Ramlibacter sp. WS9 (GCA_003797765.1), a Gram negative isoprene degrader, and Rhodococcus sp. AD45 (GCA_000949305.1), a Gram positive isoprene degrader, each contained the methylcitrate cycle‐encoding genes except for methylaconitate isomerase, indicating that they may lack the ability to convert mixed 2‐methyl‐cis/trans‐aconitate into 2‐methyl‐cis‐aconitate  and Sphingopyxis sp. OPL5 (GCA_003797775.2) do not encode complete methylcitrate pathways, suggesting a reliance on incorporating carbon from propionyl‐CoA via methylmalonyl‐CoA. The presence of an alternative metabolic pathway for the incorporation of carbon from isoprene cannot be discounted in these extant isoprene degraders. Overall, comparative genomics analysis suggested that the metabolic pathway predicted in Variovorax sp. WS11 is available to distinct isoprene degraders, albeit in varying levels of completeness, but the need for additional proteomics and metabolomics studies is clearly indicated.The genomes of cultivated isoprene‐degrading bacteria were studied for the presence of genes involved in the predicted isoprene metabolic pathway Table . Propioniso genes  likely to contribute to the assimilation of carbon from isoprene. While essential roles have been confirmed for previously uncharacterized iso genes, specific enzyme activity assays must now be developed for IsoG, IsoJ and AldH with a view to confirming the enzymatic steps predicted in this study. The incorporation of isoprene‐derived carbon is likely to vary according to the vitamins available to Variovorax sp. WS11 during growth, such as biotin and vitamin B12, permitting the conversion of propionyl‐CoA to methylmalonyl‐CoA or methylcitrate. The significant upregulation of the megaplasmid‐encoded methylcitrate gene cluster strongly indicated that Variovorax sp. WS11 had acquired an additional mechanism of incorporating carbon from isoprene, one that was not available to all extant isoprene degraders. Metabolomics analysis of isoprene‐grown Variovorax sp. WS11 is now required to verify the metabolic predictions made in this study. Additional proteomics studies are also required to assess the use of the proposed pathway by a greater diversity of isoprene degrading bacteria.Using a combination of proteomics and transcriptomics, a complete isoprene metabolic pathway has been proposed for the first time. Gene expression during growth on isoprene strongly indicated a β‐oxidative pathway to generate propionyl‐CoA from GMBA, with confirmed roles for essential The work on this project was funded through a European Research Council (ERC) Advanced Grant to J. C. Murrell (694578‐IsoMet), a Natural Environment Research Council (NERC) grant to J. C. Murrell (NE/J009725/1), a Leverhulme Trust Early Career Fellowship (ECF‐2016‐626) to A. T. Crombie and the Earth and Life Systems Alliance (ELSA) at the University of East Anglia.The authors declare no conflict of interest that could influence this work.Figure S1 Confirmed steps of isoprene metabolism, adapted from van Hylckama Vlieg et al. (2000). Steps which are catalysed by enzymes are marked by an asterisk. IsoABCDEF: isoprene monooxygenase. IsoI: glutathione S‐transferase. IsoH: dehydrogenase.Click here for additional data file.Figure S2 (A) iso metabolic gene cluster encoded on Megaplasmid 1 of Variovorax sp. WS11 . (B) Putative isoprene‐induced gene cluster with predicted roles in β‐oxidation. acdA: acyl‐CoA dehydrogenase (NDZ12938.1), fadD: propionyl‐CoA ligase (NDZ12937.1), pccB: propionyl‐CoA carboxylase, β‐chain (NDZ12936.1), hypoth: hypothetical protein , dpgD: enoyl‐CoA hydratase (NDZ12934.1), bioA: adenosylmethionine‐8‐amino‐7‐oxononanoate aminotransferase (NDZ12932.1), bioF: 8‐amino‐7‐oxononanoate synthase (NDZ12931.1), bioD: ATP‐dependent dethiobiotin synthetase (NDZ12930.1), bioB: biotin synthase (NDZ12929.1), accA: acetyl‐/propionyl‐CoA carboxylase α‐chain (NDZ12928.1). (C) Putative methylcitrate pathway gene cluster. prpC: 2‐methylcitrate synthase (NDZ17600.1), acnD: 2‐methylisocitrate dehydratase (NDZ17599.1), prpF: 2‐methylaconitate isomerase (NDZ17598.1), dml: 2,3‐dimethylmalate lyase (NDZ17597.1).Click here for additional data file.Figure S3iso metabolic gene transcripts  as a percentage of all detected transcripts during growth on isoprene or incubation with epoxyisoprene, measured over time.Click here for additional data file.Figure S4 Growth of Variovorax sp. WS11 ΔisoG on 1% (v/v) isoprene, compared with growth of Variovorax sp. WS11 ΔisoG transformed with pBBR1:isoG. Error bars represent the standard deviation about the mean (n = 3). An asterisk (*) denotes a statistically significant difference between the indicated data (p ≤ 0.05), determined by student's t‐test.Click here for additional data file.Figure S5 Growth of Variovorax sp. WS11 ΔisoJ on 1% (v/v) isoprene, compared with the growth of Variovorax sp. WS11 ΔisoJ transformed with pBBR1:isoJ. Error bars represent the standard deviation about the mean (n = 3). An asterisk denotes a statistically significant difference between the indicated conditions .Click here for additional data file.Figure S6 Growth of Variovorax sp. WS11 ΔaldH on 1% (v/v) isoprene, compared with the growth of Variovorax sp. WS11 ΔaldH transformed with pBBR1:aldH. Error bars represent the standard deviation about the mean (n = 3). An asterisk denotes a statistically significant difference .Click here for additional data file.Figure S7 Isoprene oxidation by Variovorax sp. WS11 grown in the presence of 1% (v/v) isoprene, 10 mM pyruvate, or a combination of 1% (v/v) isoprene and 10 mM pyruvate, measured as nmol isoprene consumed minute−1 mg dry weight−1. Error bars represent the standard deviation about the mean (n = 3). An asterisk denotes a statistically significant difference (p ≤ 0.05) between the indicated conditions, determined by students t‐test.Click here for additional data file.Figure S8 Growth of Variovorax sp. WS11 ΔgarB on 1% (v/v) isoprene, compared to wild‐type Variovorax sp. WS11. Error bars represent the standard deviation about the mean (n = 3). An asterisk denotes a statistically significant difference between the indicated conditions (p ≤ 0.01).Click here for additional data file.Figure S9 Fold‐change in expression of garB_1, garB_2, and garB_3 by Variovorax sp. WS11 (wild‐type) and Variovorax sp. WS11 ΔgarB, during growth on isoprene compared to growth on succinate, determined by RT‐qPCR, relative to the expression of rpoB. The fold‐change in expression of garB_3 determined by RNA‐seq was calculated relative to timepoint 0. Error bars represent the standard deviation about the mean (n = 3). An asterisk (*) denotes a statistically significant difference between the indicated conditions (p ≤ 0.05).Click here for additional data file.Figure S10 Isoprene‐induced changes in the transcription of typical β‐oxidation genes (bars), and the relative protein abundance of the respective gene products (lines).Click here for additional data file.Figure S11 Alignment of the deduced amino acid sequence of NDZ12934.1 (ECH) with published ECH and ECI sequences, using the MAFFT online multiple sequence alignment tool with default parameters . Conserved residues  are highlighted in red. Glu144 has been substituted with a glycine residue in Variovorax sp. WS11. Where available, protein databank identifiers have been included in brackets.Click here for additional data file.Figure S12 Isoprene‐induced changes in the expression of transcribed genes (bars) and translated gene products (lines) with predicted roles in the isoprene metabolic pathway , subsequent to GMBA formation by IsoH.Click here for additional data file.Figure S13 Growth of Variovorax sp. WS11 on 1% (v/v) isoprene in the presence of a complete vitamin solution, in the absence of biotin (−biotin), in the absence of vitamin B12 (‐B12), or in the absence of both biotin and vitamin B12 (‐Biotin ‐B12). Error bars represent the standard deviation about the mean (n = 3). An asterisk denotes a statistically significant difference between the indicated conditions .Click here for additional data file.Figure S14 Growth of Variovorax sp. WS11 on 10 mM propionate in the presence of all vitamins, in the absence of biotin (−biotin), in the absence of vitamin B12 (‐B12), or in the absence of both biotin and vitamin B12 (‐Biotin ‐B12). Error bars represent the standard deviation about the mean (n = 3).Click here for additional data file.Appendix S1 Supporting Information.Click here for additional data file.Appendix S2 Supporting Information.Click here for additional data file.Appendix S3 Supporting Information.Click here for additional data file.Appendix S4 Supporting Information.Click here for additional data file.Table S1 Top 30 most abundant transcripts and polypeptides after 24 hours' growth on isoprene, calculated as the fold‐change in transcripts and ratio of abundance of polypeptides, compared to the respective timepoint 0 samples.Click here for additional data file.Table S2 Top 30 most upregulated transcripts and polypeptides after 24 hours' growth on isoprene, calculated as the fold‐change in transcripts and the ratio of abundance of polypeptides, compared to the respective timepoint 0 samples.Click here for additional data file.Table S3 Presence (✓) or absence (X) of genes involved in the methylcitrate pathway and propionyl‐CoA assimilation pathways in isoprene degrading bacteria.Click here for additional data file.Table S4 Vectors used in this study.Click here for additional data file.Table S5 Primers used in this study.Click here for additional data file.


It can be considered as a meansto generate metabolite biomarkers, although it is also important tonote the cogent criticisms of the environmental biomarker approachthat have been made within ecotoxicology: briefly, that biomarkersare surrogates for macro phenotypes  that have population-level effects and that it is generallymore straightforward and meaningful to measure these end points directly.4 ORA uses the intuitive approach of identifying metabolite biomarker“hits” and comparing them to the numbers of metabolitesin specific pathways, to determine if there are either more or fewerhits than one would expect by chance. It therefore has the twin advantagesof being simple to calculate and simple to understand. It does, though,have disadvantages. One potential limitation is shared with all methodsthat rely on predetermined pathway definitions: traditional pathwaysare, generally, subjective and heuristic approaches to imposing orderon a biochemical network.5 While this isan important point, we will simply note it here and pass on, and bearin mind that “pathways” are, at least to some extent,arbitrary definitions. The problem is exacerbated for nonmodel organisms,in that accurate metabolic pathway definitions may not be available.It should also be noted that metabolites may contribute to many differentpathways: for example, glucose is present in 23 of 263 pathways , and ATP is present in 880 of 1669 pathways .Just because a metabolite may be part of a particular pathway, then,does not mean that changes in that metabolite necessarily mean changesin that pathway. Particular care must be taken with environmentalorganisms not to misinterpret changes with respect to examples fromhuman medicine. A second obvious limitation of ORA is that the criteriafor defining significant metabolites are also arbitrary, usually,but not necessarily, based on selecting a threshold for P values from null hypothesis significance testing.So far, so good, but there is a problem: there isno simple, universallyaccepted way of reverse engineering mechanistic understanding frommetabolomic data, even for model organisms, and the problem is evenmore complicated for nonmodel species. The closest thing to a standardapproach is pathway analysis (PA), i.e., making use of existing biochemicalknowledge. There are multiple approaches to PA, but we will focuson just one, over-representation analysis (ORA).  It should clearly be understood, though,that ORA is certainly not the only approach to analyzing metabolomicsdata. It is beyond the scope of this work to review the options available,but we direct the interested reader to recent reviews.6 These have become justly popular, as they arefree to use, available online, integrated with data processing andbiostatistical modules, and updated to ensure they remain current.They also provide some opportunities to set parameters that affectthe results, opening up the possibility of inadvertently misusingthe tools.  We recently publisheda study of the sensitivity of ORA of metabolomics data to some ofthe different parameters that can be chosen.7 A wide range of different factors affect the results  is critically important: if the background is not takeninto consideration, the results tend to be very overoptimistic—the P values obtained are much more significant than they shouldbe.It is alsopossible to draw incorrect conclusions from ORA. Forinstance, the online Metaboanalyst web server provides a suite oftools for metabolomic analysis, including, but not limited to, ORA. results 1. First,Table S1) and checked to see what form of PA, if any, was used. Two-thirdsof the studies (20 of 30) employed PA ;all of these (20 of 20) used ORA, although this generally was notspecified by name. Two of them used simultaneous enrichment of transcriptomicand metabolomic data, although full details were not given. Fourteenof the studies used Metaboanalyst; one used the R package Mummichog,and seven failed to specify which software was used. With the exceptionof the study that had used Mummichog, they generally failed in reportingkey parameters that affect the outcome. No studies specified exactlywhich pathway database was used for the analysis, including for whichorganism; eight mentioned KEGG pathways but with no more detail given.No studies reported if they corrected for multiple testing in thesoftware output ; several used plots including an uncorrected P value scale with no additional information given. No studiesmade any mention of a reference or background metabolome set. Somestudies set ad hoc thresholds based on the “pathway impact”statistic provided by Metaboanalyst. It is clear that ORA is beingunintentionally misused in environmental metabolomics research, ina fashion that is likely to lead to misleading results.We decided to survey the literature to get an idea of the currentpractice in environmental metabolomics. We searched for environmentalmetabolomics papers  and identified988 recent papers. We randomly selected 30 papers from this list .(1) Accurately report the analyses carried out.The specific softwarepackage/online tool used should be reported, along with all of theparameters, even if they were left as defaults, including the databaseversion and organism used for pathways. Specify what (2) Always upload areference metabolome, or “backgroundset”. In other words, the list of all metabolites that havebeen identified in that specific study. If this is not done, the resultsshould be treated with extreme caution, as they may inaccurately identifypathways as significantly enriched.(3) Avoid definitive statementsabout which pathways have beenimpacted in a particular study. This type of pathway-based approachis, ideally, used to help generate hypotheses that can then be validatedby independent experiments; even if further experiments are not feasible,the limitations should be appreciated.We hope these simplerecommendations should help researchers avoidsome of the common errors that currently plague environmental metabolomicsresearch.


Osteoporosis is an important health problem worldwide. Liuwei Dihuang Decoction (LDD) and its main ingredients may have a good clinical effect on osteoporosis. Meanwhile, its mechanism for treating osteoporosis needs to be further revealed in order to provide a basis for future drug development.  A systematic biological methodology was utilized to construct and analyze the LDD-osteoporosis network. After that, the human transcription data of LDD intervention in patients with osteoporosis and protein arrays data of LDD intervention in osteoporosis rats were collected. The human transcription data analysis, protein arrays data analysis, and molecular docking were performed to validate the findings of the prediction network (LDD-osteoporosis PPI network). Finally, animal experiments were conducted to verify the prediction results of systematic pharmacology. β-catenin mRNA expression (P < 0.05). (4) Molecular docking results showed that alisol A, dioscin, loganin, oleanolic acid, pachymic acid, and ursolic acid may stably bind to JAK2, ESR1, and CTNNB1.  (1) LDD-osteoporosis PPI network shows the potential compounds, potential targets , clusters, biological processes , and signaling and Reactome pathways  of LDD intervention in osteoporosis. (2) Human transcriptomics data and protein arrays data validated the findings of the LDD-osteoporosis PPI network. (3) The animal experiments showed that LDD can improve bone mineral density (BMD), increase serum estradiol (E2) and alkaline phosphatase (ALP) levels, and upregulate Wnt3a and  LDD may have a therapeutic effect on osteoporosis through regulating the targets , biological processes , and pathways  found in this research. Osteoporosis is a common systemic metabolic bone disease with a decrease in bone density and bone quality and bone microstructural damage caused by various reasons . The serRehmannia glutinosa (Gaertn.) DC , Cornus Officinalis Sieb. et Zucc. (Shanzhuyu), Paeonia × suffruticosa Andrews , Dioscorea oppositifolia L. , Poria cocos (Schw.) Wolf. (Fuling), and Alisma plantago-aquatica L.  Juz., Zexie). Current clinical studies showed that LDD alone or in combination with other antiosteoporosis drugs  for osteoporosis has a certain effect: it can effectively improve the patient's bone density  and the clinical efficacy rate  and were raised in the Experimental Animal Center of Hunan University of Chinese Medicine. The animals were weighed, numbered, and divided into 4 groups according to the random sequence method: sham operation group (10 rats), model group (10 rats), LDD group (10 rats), and positive control group (10 rats). Animal experiments have been approved by the Animal Ethics Committee of Hunan University of Chinese Medicine  and performed in accordance with the guidelines for the care and use of experimental animals.After 1 week of adaptive feeding, rats underwent bilateral ovarian resection under anesthesia  to create postmenopausal osteoporosis (PMOP) model. On the 7th day after the operation, the rats began to administer the medicine by gavage daily. The sham operation group and the model group were intragastrically administered with 10 mL/kg of normal saline. The rats in the LDD group were gavaged with LDD 6.75 g crude drug/kg. The rats in the positive control group were gavaged with alendronate sodium 0.1 mg/mL.Under anesthesia with 3% pentobarbital sodium 30 mg/kg, blood was collected from the abdominal aorta. After the blood was placed at room temperature for 1 hour, the blood was centrifuged at 3 500 r/min for 15 minutes. The serum was separated and stored in a refrigerator at −80°C. The right femur of the rat was stored at −20°C for bone density detection and Quantitative Real-time PCR (qRT-PCR). The left femoral head of the rat was fixed with 4% paraformaldehyde for HE staining.Serum E2 and ALP were detected by ELISA. BMD was measured by a dual-energy X-ray bone densitometer.The left femoral head was fixed with a volume fraction of 4% paraformaldehyde for 72 h and then decalcified with 10% EDTA decalcification solution, and the decalcification solution was changed every 3 days. After decalcification was completed, it was rinsed with PBS, dehydrated with gradient ethanol, cleared with xylene, embedded in paraffin, and sliced for HE staining. The histopathological changes in the femoral head were observed under a light microscope.μM) 2 μL; PCR reverse primer (2 μM) 2 μL; 2X All-in-One qPCR Mix 10 μL; Template 2 μL; 50X Rox Reference Dye 0.4 μL; ddH20 3.6 μL; total 20 μL. The reaction conditions were set as follows: predenaturation: 95°C 10 min, 1 cycle; amplification reaction: 95°C 10 S, 62°C 20 s, 72°C 15 S, 45 cycles; draw melting curve: 95°C 10 S, 25°C 30 s. Each sample was repeated 3 times. For data analysis, the 2−ΔCt method was used to calculate the difference in Wnt3a and β-catenin gene mRNA transcription levels. The gene sequence was searched in the NCBI database, and primers were designed, as shown in Blood RNA was extracted and reverse-transcribed into cDNA for qRT-PCR detection. Reaction system was as follows: PCR forward primer (2 x ± s), and the comparison between groups adopts multisample mean comparison and analysis of variance. The test level adopts α = 0.05; P < 0.05 indicates that the difference is statistically significant.SPSS22.0 software was used for statistical analysis. The measurement data are expressed by mean ± standard deviation (β-catenin (CTNNB1), and JAK2 were selected as receptors. The “sdf” format file of the top 6 components was input into ChemBioDraw for energy minimization and saved as “mol2” format. The PDB database (https://www.rcsb.org/) was used to retrieve the 3D structure of ESR1 (PDB ID: 1QKT), CTNNB1 (PDB ID: 1JDH), and JAK2 (PDB ID: 3E62) [The top 6 components in the compound-compound target network of LDD were selected as ligand, and ESR1, D: 3E62) . DiscoveD: 3E62) –39.Two thousand and nine hundred and eighty-three osteoporosis genes were obtained from GeneCards and OMIM database. The osteoporosis genes with a relevance score ≥5.0 were selected to construct the LDD-osteoporosis PPI network see . A totalThe target shared by LDD potential target and osteoporosis genes is LDD-osteoporosis targets. The LDD-osteoporosis PPI network contains 743 nodes  and 8081 edges . After aThe Cytoscape's plug-in MCODE was utilized to analyze the LDD-osteoporosis PPI network, and 13 clusters were obtained . The tarκB, TGF-beta/SMAD, BMP, and Wnt signaling pathway). Cluster 3 is associated with bone resorption and reconstruction, mesenchymal cell differentiation, osteoblast differentiation, osteogenic matrix, and osteoporosis-related signaling pathways . Cluster 4 is mainly involved in endothelial cell proliferation in bone remodeling and differentiation of osteoblasts and chondrocytes. Cluster 6 is mainly related to steroid metabolism. Cluster 10 is related to steroid metabolism. Cluster 15 failed to return any human biological processes. Clusters 5, 7, 8, 9, 11, 13, 14, 16 and 17 do not return any osteoporosis-related biological processes. The details of each cluster and biological processes are described in Cluster 1 is mainly involved in several signaling pathways in osteoporosis  and positive regulation of calmodulin 1-monooxygenase activity, estrogen metabolism, endothelial cell proliferation, and neovascularization during bone remodeling. Cluster 2 is mainly involved in endothelial cell proliferation in bone remodeling, differentiation of osteoblasts and chondrocytes, bone resorption, and some signaling pathways , while those of Rhizoma Dioscoreae regulate 216 targets. The compounds of Rhizoma Dioscoreae and Rhizoma Dioscoreae regulate 206 targets, respectively. This suggests that Cornus officinalis Sieb. et Zucc. and Rhizoma Dioscoreae play a major role in LDD  are as follows: Cytokine Signaling in Immune system, Signaling by Receptor Tyrosine Kinases, Negative regulation of the PI3K/AKT network, PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling, Interleukin-12 family signaling, SUMOylation of intracellular receptors, Interleukin-12 signaling, Extracellular matrix organization, and Degradation of the extracellular matrix. The top 30 Reactome pathways are shown in Table S5The LDD targets and osteoporosis in the LDD-osteoporosis PPI network were input into Reactome Pathway Database to collect the Reactome pathways. These osteoporosis-related Reactome pathways were arranged according to the β-catenin signaling pathway [α gene promoter and increase the expression of ERα mRNA and protein and promote the proliferation and differentiation of osteoblasts by AMPK and β-catenin signaling mediated by this pathway [At present, it was found that LDD has obvious antiosteoporosis effect in ovariectomized rats, which is mainly achieved by activating the Wnt/ pathway . Ge et a pathway , 41. Xu  pathway . In addi pathway . Moreoveβ-catenin pathway [κB pathways [κB transcriptional activity and nuclear transport [For example, Luo et al. found that Rehmanniae Radix Praeparata extract has osteoprotective effects, the mechanism of which is related to the IGF-1/PI3K/mTOR pathway . Lai et  pathway . Ferulic pathway , 48. Fer pathway . Paeonolpathways . Furtherpathways . Kaempfepathways –56. Evidpathways , 58. Quepathways , 60. In pathways . Paeonifpathways , 63. (+)ransport , 65. Furransport . Dioscinransport , 68. Dioransport –72. Ursoransport –75. Olearansport . Loganinransport –79.Through the study of small molecule compounds, we determined the synergy between the main active compounds of LDD in the treatment of osteoporosis. Meanwhile, through the construction and analysis of the network, we obtained important targets for LDD and the potential biological processes and signaling pathways for the treatment of osteoporosis . However, since the above processes are all completed by computer simulation, experiments are still needed for further verification. Therefore, we used the human transcriptomics data in the GEO database (GSE57273) and protein arrays data in reference  for furtP-value <0.05 were thought to be differentially expressed genes  were selected. After the selection, a total of 4031 genes were obtained. Finally, the top 3000 genes were selected for enrichment analysis.To further analyze the transcriptomics data, the identified genes with log2FC ≥2 or ≤−2 and After the enrichment analysis, several biological processes, Reactome pathways, and signaling pathways are obtained. The results of GO enrichment are mainly related to insulin-like growth factor and its receptor signaling pathway (GO:0048009), extracellular matrix (GO: 0098609), endothelial cell formation (GO:0061028), skeletal system morphogenesis (GO: 0048705), several osteoporosis-related signaling , and so on .The results returned by Reactome pathways enrichment show that LDD is able to regulate Cell-extracellular matrix interactions, Interleukin-7 signaling, SUMOylation of transcription cofactors, SUMOylation of intracellular receptors, Downregulation of TGF-beta receptor signaling, MET activates RAP1 and RAC1, Insulin-like Growth Factor-2 mRNA Binding Proteins (IGF2BPs/IMPs/VICKZs) bind RNA, Interleukin-6 signaling, Cohesin Loading onto Chromatin, Interleukin-12 family signaling, and so on .The results of signaling pathway enrichment are protein processing in the endoplasmic reticulum, TGF-beta signaling pathway, Phosphatidylinositol signaling system, Neurotrophin signaling pathway, Thyroid hormone signaling pathway, Focal adhesion, Signaling pathways regulating pluripotency of stem cells, FoxO signaling pathway, Estrogen signaling pathway, Insulin signaling pathway, and NF-kappa B signaling pathway . The detThe osteoporosis-related protein arrays data comes from . The netProtein arrays data were input into DAVID and Reactome for enrichment analysis and returned several biological processes, Reactome pathways, and signaling pathways. The results of GO enrichment are shown in Figures The results of GO enrichment are mainly related to immune response (GO:0060334), cell proliferation , and other aspects  .The results of signaling pathway enrichment are related to bone homeostasis  and other pathways (such as hsa04060:Cytokine-cytokine receptor interaction) .The results returned by Reactome pathways enrichment are associated with immunomodulatory , cell growth, proliferation, and cell cycle (such as R-HSA-9617828 and R-HSA-452723), transcription factor and coactivated genes that interact with STAT proteins (R-HSA-2173796 and R-HSA-9006936), and other transcription factors and their coactivated genes (R-HSA-2173789 and R-HSA-76002) . The detIn summary, we found that the mechanism of LDD in the treatment of osteoporosis is related to immunity, cell growth and proliferation, endocrine hormones and cytokines, and differentiation of osteoclasts and osteoblasts. These pathways work together to regulate the activity of osteoclasts and balance the process of bone turnover through the interaction of crucial proteins. Next, animal experiments were conducted to verify the mechanism of LDD on osteoporosis.P < 0.01). The BMD of the proximal femur in the LDD group and the positive control group was higher than that in the model group (P < 0.01). Compared with the positive control group, the BMD of the proximal femur in the LDD group was not statistically significant (P > 0.05)  .P < 0.05), suggesting that LDD can increase serum E2 and ALP levels in osteoporotic rats . Compared with the model group, Wnt3a and β-catenin were significantly increased in the LDD group and the positive control group (P < 0.05), similar to the sham operation group. It is suggested that LDD can improve osteoporosis in rats by regulating the expression of Wnt3a and β-catenin , and osteoprotegerin (OPG) [κB pathway, MAPK pathway, PI3K/AKt pathway, and CN/NFAT pathway [The skeletal structure undergoes subtle changes throughout life, requiring the involvement of osteoblasts and osteoclasts . The twoin (OPG) , 85, of in (OPG) . Bone biin (OPG) . The regin (OPG) –90: (1) in (OPG) . For exain (OPG) . The sig pathway . Hence,  pathway .β), interferon (INF), and insulin-like growth factor (IGF) are also associated with the activation of osteoclasts [MCSF is produced by various cells in the bone microenvironment and acts on the osteoclasts in a paracrine manner, resulting in increased expression of genes involved in osteoclast differentiation and increased osteoclast activity . In addieoclasts .α of osteoclasts, stimulate OPG expression, inhibit the activation of osteoclast-associated cytokine expression, and ultimately reduce the rate of osteoclastogenesis through the RANKL signaling pathway [Hormones also play an important role in the process of bone regulation, especially the regulation of osteoclast activity . For exa pathway . For thi pathway . Other h pathway . The hor pathway . The syn pathway .β, INF, IGF, and calmodulin 1-monooxygenase, which are closely associated with the activation of osteoclasts. [Through the construction and enrichment analysis of the predicted target network of small molecule compounds in LDD, we can find that the small molecule compounds of LDD can regulate some biological processes. For example, they can regulate endothelial cell proliferation and neovascularization, bone formation, bone resorption and reconstruction, differentiation of mesenchymal stem cells, formation of extracellular matrix and collagen of bone formation, differentiation of osteoblasts, and osteoclasts, and so on. These biological processes play an important role in the development of osteoporosis. Meanwhile, this study suggests that LDD can regulate some cytokines related to osteoporosis, such as TGF-oclasts. , 102. Thoclasts. . The resoclasts. , and ultoclasts. , romosozoclasts. , and rapoclasts. .κB signaling, MAPK signaling, PI3K/AKt signaling, and CN/NFAT signaling [γ [β-catenin [β/SMAD signaling pathway [The most important signaling pathways associated with osteoporosis are the OPG/RANKL/RANK signaling pathway, which includes NF-ignaling , 96. Theignaling . In addialing [γ , Wnt/β-c-catenin , Hedgeho-catenin , BMP [11-catenin , Notch [-catenin , JAK/STA-catenin , and TGF pathway .In conclusion, although this study explored the molecular mechanism of LDD in the treatment of osteoporosis through bioinformatics and systems pharmacology and transcriptomics and verified some related molecular mechanisms, its molecular mechanism still needs to be further elucidated in order to promote the thorough disclosure and elucidation of the molecular network mechanism of LDD in the treatment of osteoporosis.LDD may have a therapeutic effect on osteoporosis through regulating the targets , biological processes , and pathways  found in this research.


Polycystic ovary syndrome (PCOS) is a common reproductive metabolic disorder, normally accompanied by insulin resistance (IR). The specific pathogenesis of this disease remains unclear. To identify the underlying pathogenesis of PCOS with IR and explore the potential efficacy and mechanism of Jiawei Huanglian-Wendan decoction (JHWD) by a network pharmacology approach. The effective components and the potential drug and disease-related targets are retrieved. Drug-disease overlapped targets are being obtained by Venny analysis. The construction of protein-protein interaction network relied on Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING), after uploading drug-disease overlapped targets. The drug-component-target-disease interaction network map was displayed , after importing their data into Cytoscape 3.7.2 software. Bioinformatics analyses are being performed by Metascape and Kyoto Encyclopedia of Genes and Genomes databases, respectively. Further, molecular docking analysis was carried out using AutoDock software. Finally, the influence of JHWD is verified by means of traditional Chinese medicine syndrome score, the rate of resumption of normal menstrual cycles and regular ovulation, the blood lipid levels, the blood glucose and insulin levels, and the inflammatory cytokines in PCOS with IR patients. Four primary interaction networks of JHWD are constructed. The enrichment analysis of PCOS-IR-related targets demonstrated that the top enriched pathways in the development of PCOS with IR are pathways in cancer, metabolic, phosphoinositide-3-kinase-protein kinase B signaling, lipid and atherosclerosis, and mitogen-activated protein kinase signaling pathways. Molecular docking analysis revealed strong binding interactions of the key targets with the active components. Further confirmations showed that the active components of JHWD exhibited significant clinical efficacy in improving the clinical syndromes, menstrual cyclicity and ovulatory function, and significantly reducing the blood lipid levels, blood glucose and insulin levels, and inflammatory cytokines in PCOS with IR patients. The combination of the network pharmacological analysis and clinical validation stated that the active compounds in JHWD could regulate glycolipid metabolism, reduce IR, and exert anti-inflammatory effects in the treatment of PCOS with IR, promoting Chinese classical formulations. IR can further aggravate chronic inflammation by activating the inflammatory response pathway. Therefore, ameliorating low grade chronic inflammation may be an effective therapy for PCOS with IR. However, the therapeutic options for PCOS with IR are minimal, as most of these drugs result in severe adverse effects and unclear long-term risk-benefit ratio.Polycystic ovary syndrome (PCOS), a heterogeneous disease marked by insulin resistance (IR), has become a challenging pathological condition in gynecology and endocrinology due to unclear pathogenesis. In this context, chronic low-grade inflammation has been implicated as a key pathogenic mechanism of PCOS and consequent IR. significantly improving the holistic functional status of patients. From the perspective of TCM, phlegm-dampness stagnation is the primary etiology and pathology of PCOS with IR, making disorder of qi movement of the body, disharmony of qi and blood, as well as stagnation of skin and meridian. Consequently, these pathological conditions lead to the typical symptoms of PCOS, such as menstrual irregularity, infertility, obesity, hirsutism, and acne.To this end, traditional Chinese medicine (TCM) offers unique advantages in clinical practice via the synergism of multiple targets, links, and pathways as well as fewer side effects compared to chemotherapeutic agents,liu yin tiao bian written by Lu Tingzhen in the Qing Dynasty. Studies confirmed that Huanglian-Wendan decoction could improve liver lipid accumulation and enhance insulin sensitivity in IR rats. Moreover, the Huanglian-Wendan decoction could decrease the area of aortic plaques and prevent the progression of atherosclerosis. It was demonstrated that Huanglian-Wendan decoction could ameliorate dysregulated glucose and lipid metabolism and regulate pancreatic β-cells. In this framework, Jiawei Huanglian-Wendan decoction (JHWD), a modified compound prescription, is often used to clear heat and dry dampness, resolve phlegm, regulate qi, disperse stasis, obviate retention, strengthen spleen, and replenish kidney. Notably, the JHWD is comprised of several TCM molecules: Coptidis Rhizoma (Huanglian), Scutellariae Radix (Huangqin), Caulis Bambusae in Taeniam (Zhuru), Aurantii Fructus Immaturus (Zhishi), Poria Cocos (Schw.) Wolf. (Fuling), Citrus Reticulata (Chenpi), Cyperi Rhizoma (Xiangfu), Arum Ternatum Thunb (Banxia), Radix Salviae (Danshen), Angelicae Sinensis Radix (Danggui), Cuscutae Semen (Tusizi), and Atractylodes Macrocephala Koidz. (Baizhu). Our previous studies have confirmed that JHWD had achieved high efficacy in treating PCOS with IR.,7 However, the potential mechanism of JHWD for PCOS with IR is not well defined.Huanglian-Wendan decoction is a classic traditional Chinese prescription recorded in  Therefore, this study aims to identify the underlying pathogenesis of PCOS with IR and the potential mechanistic action of JHWD in treating PCOS with IR using the network pharmacology method. Further, a clinical experiment is performed to verify the efficacy of JHWD in treating PCOS with IR and explore insight into the underlying mechanisms , Chemistry database (http://www.organchem.csdb.cn/), and the Encyclopedia of Traditional Chinese Medicine , using the Chinese pinyin “Huanglian, Huangqin, Zhuru, Zhishi, Fuling, Chenpi, Xiangfu, Banxia, Danshen, Danggui, Tusizi, and Baizhu” as keywords. Further, we screened eligible components based on ADME . Then, in this study, we identified active components under standard conditions of oral bioavailability (OB) of ≥30% and drug-likeness (DL) of ≥0.18. The target names of each herb were screened from the TCMSP database.The components of 12 herbs in JHWD were acquired from the TCM System Pharmacology database , (2) Drug Bank (https://www.drugbank.ca/), (3) National Center for Biotechnology Information , (4) Disgenet (https:www.genecards.org/), and (5) Online Mendelian Inheritance in Man . The search results from each database were combined, and duplicates were eliminated. Then, we converted the drug-related and disease-related target protein names into gene names by the UniProt Knowledgebase , with the species limited to “Homo sapiens.”With “polycystic ovary syndrome” “insulin resistance” as keywords, disease-related targets were searched and screened from the following 5 existing databases: (1) GeneCards (https://bioinfogp.cnb.csic.es/tools/venny/), and the overlapped targets between drug and disease were gained using Venny online platform.The collected drug-related and disease-related targets were input into Venny 2.1 (https://string-db.org/) and Cytoscape 3.7.2 software.To illustrate the potential mechanisms of JHWD in PCOS with IR treatment, some major networks were constructed, including key-target protein-protein interaction (PPI) network, drug-component-target-disease network, and target-pathway network, using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://metascape.org/) and KEGG database (https://www.kegg.jp/). GO categories included various criteria of biological process (BP), cellular component (CC), and molecular function (MF). Further, the enrichment analysis results were screened, using the screening criteria of P < .01.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using Metascape database (https://pubchem.ncbi.nlm.nih.gov) in .sdf format. The 3-dimensional (3D) structures of key target proteins were downloaded from RCSB PDB database (https://www.rcsb.org/) in .pdb format. Then, the 2-dimensional (2D) ligands were optimized using ChemBio 3-dimensional (3D) software (version 20.0) in .mol2 format. AutoDock Tools (ADT version 1.5.7) was used for protein and ligand preparation, and AutoDock Vina (version 1.1.2) was used for molecular docking. Finally, the further visualization of the docking results was performed in PYMOL software (version 2.5.4).The 2-dimensional (2D) ligand structures of active components were downloaded from PubChem  who were treated at Shandong Provincial Hospital were randomly distributed into 2 groups: experimental and control groups, in which each group was allocated with 40 subjects.The clinical diagnosis of PCOS was performed by following the Chinese Guidelines for PCOS based on revised Rotterdam Consensus criteria.The homeostasis model assessment of insulin resistance (HOMA-IR) value of ≥1.66 was investigated for IR.TCM syndrome elements diagnosis standard: phlegm-dampness syndrome refers to the Guidelines for Diagnosis and Treatment of Common Diseases of Gynecology in Traditional Chinese Medicine.Patients with conditions of diabetes mellitus, hyperprolactinemia, hypothyroidism, hyperthyroidism, non-classic congenital adrenal hyperplasia, Cushing syndrome, pelvic organ pathologies, severe hepatic or renal disease, malignant tumors, psychosis, and digestive tract disease; patients on antibiotics, or other medications affecting the sex hormones in the past 3 months; and patients with hypersensitivity to metformin.All the patients were received with metformin (500 mg thrice a day) and Diane-35  for 3 menstrual cycles.Moreover, apart from the control group, the experimental group was provided with JHWD deprived from the following herbs: 6g Coptidis Rhizoma, 10g Scutellariae Radix, 15g Caulis Bambusae in Taeniam, 12g Aurantii Fructus Immaturus, 15g Poria Cocos (Schw.) Wolf., 12g Citrus Reticulata, 12g Cyperi Rhizoma, 10g Arum Ternatum Thunb, 12g Radix Salviae, 12g Angelicae Sinensis Radix, 24g Cuscutae Semen, and 15g Atractylodes Macrocephala Koidz.. These Chinese herbal pieces for decoction were provided by TCM pharmacy of Shandong Provincial Hospital, Affiliated to Shandong First Medical University. Herbs were boiled in water for 30 minutes and condensed into 300mL decoction. Patients were administrated with 150mL twice a day after meal for 3 menstrual cycles.The Ethics Committee of the Shandong Provincial Hospital Affiliated to Shandong First Medical University had approved the designed study protocol (BF201902011-02). In accordance with the guidelines, all the subjects who participated in the study have signed the written informed consent prior to conducting the study.Phlegm-dampness syndrome score: The clinical efficacy is determined by symptomatic improvement upon treatment, according to the Guiding Principles for Clinical Research of New Chinese Medicines .The rate of resumption of normal menstrual cycles and regular ovulation.Blood sample collection and testing. All blood samples were collected after 8 hours of fasting. Initially, the fasting blood glucose (FBG) and the fasting insulin (FINS) were recorded to enumerate the insulin resistance index (HOMA-IRI = (FBG × FINS)/22.5). Further, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) in serum were measured by flow cytometry . Finally, the concentrations of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were enumerated using an automatic biochemical analyzer .t test for comparisons between 2 groups; otherwise, the nonparametric test was performed. The counting data were presented as numbers or percentages, and comparison between groups was conducted by χ2 test. P < .05 was considered as statistically significant, and P < .01 as statistically highly significant.The statistical analyses were performed using the SPSS software 20.0. Data were expressed as mean ± standard deviation (x ± s). The measured data conforming to a normal distribution were analyzed using the A total of 180 active components were collected, including 11 from Coptidis Rhizoma, 32 from Scutellariae Radix, 12 from Arum Ternatum Thunb., 4 from Atractylodes Macrocephala Koidz., 5 from Citrus Reticulata, 2 from Angelicae Sinensis Radix, 59 from Radix Salviae, 6 from Poria Cocos (Schw.) Wolf., 10 from Cuscutae Semen, 16 from Cyperi Rhizoma, 19 from Aurantii Fructus Immaturus, and 4 from Caulis Bambusae in Taeniam. The active components of Coptidis Rhizoma, the main herb of JHWD, are presented in Table https://bioinfogp.cnb.csic.es/tools/venny/) to acquire 197 key targets, as specified in Figure After eliminating duplicates, a total of 294 compound-related targets, as well as 3126 PCOS-related and IR-related targets, were retrieved from the databases mentioned above. These retrieved targets were then submitted into the Venny platform , these significant targets were predominantly related to various attributes of influence of inorganic substance, response to xenobiotic stimulus, lipopolysaccharide, radiation, organic, and nitrogen-based compounds, circulatory system process, apoptotic signaling pathway, as well as blood vessel development; In terms of cell composition, the main targets were associated with membrane raft, receptor complex, vesicle, and endoplasmic reticulum lumen, and protein kinase complex, among others; In terms of molecular functionalities, the targets were predominantly relevant to protein domain-specific binding, protein kinase-specific binding, deoxyribose nucleic acid (DNA)-binding transcription factor binding, and protein homo-dimerization as well as nuclear receptor activities, among others.On the other end, the experimental results of KEGG pathway analysis demonstrated that the key targets of JHWD and PCOS-IR were predominantly associated with the pathways of cancer, lipid and atherosclerosis, PI3K-Akt signaling, hepatitis, human cytomegalovirus infection, advanced glycation endproducts-receptor for advanced glycation endproducts (AGE-RAGE) signaling in diabetic complications, chemical carcinogenesis-receptor activation, kaposi sarcoma-associated herpesvirus infection, chemical carcinogenesis-receptor activation, and MAPK signaling pathway, among others. The relationships between these notified main targets and pathways were submitted in the Cytoscape 3.7.2 software to establish the pathways-target network Fig. . FurtherThe results of molecular docking showed that wogonin and quercetin docked well into RAC-alpha serine/threonine-protein kinase (AKT1), MAPK1, MAPK3, signal transducer and activator of transcription 3 (STAT3) and tumor protein 53 (TP53), while nobiletin, luteolin and kaempferol had strong binding affinity only with TP53. The results were shown in Table Before treatment, there were no significant differences in the TCM syndrome scores between the 2 groups. The phlegm-dampness syndrome of the experimental group showed significant improvement after treatment, compared with control group Table .P < .05). There was significantly difference in the resumption of regular ovulation between the 2 groups (P < .05)  after therapy, in which these indicators of the experimental group were remarkably lower than those of the control group (P < .01). Together, it was evident that JHWD could regulate the levels of lipid, glucose, insulin, and inflammatory cytokines in the blood samples of PCOS with IR patients.The effects of JHWD on the levels of lipid , glucose and insulin , and inflammatory cytokines  in the blood samples of PCOS with IR patients were observed Table . It shou MAPK1 and MAPK3 play important roles in ovulation of PCOS involved in transmitting signals. It has been reported that the expression of MAPK1 and MAPK3 genotypes were differed significantly between PCOS patients and controls, and they were related to the levels of body mass index (BMI), HOMA-IR, and C-reactive protein (CRP). STAT3 activation is closely associated with PCOS-IR. It has been shown that the expression of phosphor-STAT3 and IL-6 were increased in the ovarian tissues of rat models of PCOS-IR, which suggested that STAT3 signaling pathway was activated mediated by IL-6 in the pathology of PCOS-IR. TP53 protein is mainly involved in promoting apoptosis and inhibiting proliferation. A previous study in India showed that there was a significant increase of TP53 genotype frequency in PCOS patients compared to controls, indicating that TP53 might contribute to genetic susceptibility to PCOS. Therefore, analysis of the predicted targets may represent the therapeutic targets for JHWD in the treatment of PCOS-IR.In the current investigation, we are intended to reveal the possible mechanism of pathogenesis behind the PCOS with IR and the potential therapeutic effects and mechanistic exploration of JHWD through the network pharmacology approach along with preliminary validation through clinical experiments. In this context, a total of 197 overlapped targets and 20 predicted key targets may be the potential therapeutic targets for JHWD in the treatment of PCOS-IR. AKT1 has previously been shown to be localized in both granulosa cells and oocytes and involved in inflammation, cellular growth, survival and metabolism, and AKT1-deficient leads to reduced fertility. Briefly, the PI3K-AKT signaling often regulates glucose and lipid metabolism.To further identify the related signaling pathways of JHWD on PCOS-IR, the enrichment analyses of PCOS-IR-related targets were performed to confirm that the chief enriched pathways in the progress of PCOS with IR were related to the pathways in cancer, metabolic, PI3K-Akt signaling, lipid and atherosclerosis, as well as MAPK signaling, indicating their importance in the pathogenesis of PCOS with IR. Moreover, the abnormalities in insulin receptor and insulin-signaling pathways could contribute to the development of IR in PCOS patients, which could be indicated by the suppression of the PI3K-Akt signaling and the extreme instigation of the MAPK signaling pathways. In this context, several reports indicated that the dominant insulin pathways in regulating glucose and lipid metabolism included the PI3K-Akt and MAPK signaling pathways.,30 In addition, the PI3K-Akt/NF-κB pathway is an important signaling pathway in the chronic low-grade inflammation of PCOS. In this case, it was demonstrated that blocking the MAPK signaling pathway could substantially suppress the proliferation of KGN cells (human ovarian granulosa cell line) of PCOS. Lipid and atherosclerosis are the metabolic, inflammatory reactions that may contribute to dysfunction of the vascular endothelial cells and lipid metabolism disorder of PCOS with IR. In this framework, several reports demonstrated that the generated inflammatory response was the leading contributor to the pathogenesis of IR in women with PCOS conditions. Thus, a chronic inflammation-mediated metabolic dysfunction IR might be the underlying pathogenesis of PCOS, which could be demonstrated by relying on these crucial pathways.In contrast, the inhibition of PI3K-AKT signaling can block the proliferation efficacy of various cancer cells, promoting their apoptosis and eventually death. In a case, quercetin exerted anti-inflammatory effects by inhibiting the NF-κB and MAPK signaling pathways. In another case, luteolin significantly reduced the fasting insulin and blood glucose, as well as serum lipid levels in hyperlipidemic rats. Moreover, quercetin and luteolin synergistically decreased the generation of inflammatory cytokines .In this study, it was observed that the analysis of the active compounds of JHWD resulted in several notable active compounds, such as quercetin, luteolin, kaempferol, wogonin, and nobiletin, among others. Moreover, the primary enriched pathways included cancer, lipid and atherosclerosis, PI3K-Akt and MAPK signaling pathways, advanced glycation endproducts-receptor for advanced glycation endproducts (AGE-RAGE) signaling in diabetes-associated complications, human cytomegalovirus, and hepatitis B, confirmed by enrichment analysis of overlapping targets of JHWD and PCOS-IR. These pathways were closely correlated with multiple biologic processes, including response to hormone and inorganic substance, as well as a cellular response to lipid, cytokine, and xenobiotic stimulus, positive regulation of cell migration, as well as inflammatory responses. Previous studies indicated that quercetin could substantially increase insulin sensitivity and alleviate IR by acting on the MAPK signaling pathway. In another case, it was demonstrated that kaempferol could play an anti-inflammatory role via the NF-κB and MAPK pathways. Wogonin suppressed IL-6, TNF-α, and (cyclooxygenase-2) COX-2 production in peritoneal macrophages induced apoptosis by modulating the PI3K-AKT pathway. Consequently, based on the above network pharmacology approach and previous literature reports, it could be concluded that the participation of active components of JHWD in the regulation of lipid and atherosclerosis, PI3K-Akt, and MAPK signaling pathways might be the underlying mechanisms of JHWD for PCOS-IR.Interestingly, it was observed that kaempferol could ameliorate atherosclerosis significantly through lowering lipids and alleviating vascular inflammation.Meanwhile, the molecular docking analysis was carried out on the key targets to improve the accuracy of network pharmacological prediction. The docking result of the 5 active compounds and the 5 key target proteins showed that wogonin and quercetin had good affinity with AKT1, MAPK1, MAPK3, STAT3, and TP53, while nobiletin, luteolin and kaempferol had strong binding affinity with TP53.Together, these results strongly suggested that the main active components in JHWD could effectively treat PCOS-IR by acting on these key target proteins, involving multiple signaling pathways. Moreover, the clinical validation of this study explored that JHWD exhibited significant clinical efficacy in improving the clinical syndromes, menstrual cyclicity and ovulatory function, and remarkably reduced the lipid levels , the glucose and insulin levels , and the inflammatory cytokines  in the blood samples of PCOS with IR patients. Therefore, JHWD showed exceptional therapeutic events towards regulating the glycolipid metabolism, reducing IR, and exerting anti-inflammatory effects.In summary, we employed the network pharmacology approach, molecular docking analysis and a clinical study to demonstrate the significant clinical potential of multiple components in JHWD for the treatment of PCOS-IR. It was observed that a chronic inflammation-mediated metabolic dysfunction predominantly associated with lipid and atherosclerosis, PI3K-Akt, and MAPK signaling pathways might be the underlying pathogenesis of PCOS with IR. However, JHWD dramatically exerted functions of anti-inflammatory and improved glycolipid metabolism. We firmly believe that JHWD will offer a potential value to exploring the pathogenesis of PCOS and the promotion of Chinese classical prescriptions, indicating their ability towards eradicating various metabolic diseases.NS designed the study, analyzed the data, and wrote the article. YZ collected and analyzed parts of the data. HM supervised the study and revised the article. All authors read the final version and approved submission.Conceptualization: Na Shi.Data curation: Yuhe Zhou.Formal analysis: Na Shi.Funding acquisition: Hongbo Ma.Investigation: Na Shi.Methodology: Na Shi.Project administration: Na Shi.Resources: Na Shi, Yuhe Zhou.Software: Na Shi.Supervision: Hongbo Ma.Validation: Na Shi.Visualization: Na Shi.Writing – original draft: Na Shi.Writing – review & editing: Na Shi, Hongbo Ma.


Pumpkin is a well-known multifunctional ingredient in the diet, full of nutrients, and has opened new vistas for scientists during the past years. The fruit of pumpkin including the flesh, seed, and peel are a rich source of primary and secondary metabolites, including proteins, carbohydrates, monounsaturated fatty acids, polyunsaturated fatty acids, carotenoids, tocopherols, tryptophan, delta-7-sterols, and many other phytochemicals. This climber is traditionally used in many countries, such as Austria, Hungary, Mexico, Slovenia, China, Spain, and several Asian and African countries as a functional food and provides health promising properties. Other benefits of pumpkin, such as improving spermatogenesis, wound healing, antimicrobial, anti-inflammatory, antioxidative, anti-ulcerative properties, and treatment of benign prostatic hyperplasia have also been confirmed by researchers. For better drug delivery, nanoemulsions and niosomes made from pumpkin seeds have also been reported as a health promising tool, but further research is still required in this field. This review mainly focuses on compiling and summarizing the most relevant literature to highlight the nutritional value, phytochemical potential, and therapeutic benefits of pumpkin. Products from natural sources have been used for centuries as functional and nutraceutical foods ,3,4,5,6.Cucurbita pepo L., Cucurbita maxima Duchesne, and Cucurbita moschata Duchesne are harvested worldwide due to their economical and environmentally friendly properties [Pumpkin has attracted increasing attention from scientists due to its nutritional profile. It is a nutritious and economical product and belongs to the Cucurbitaceae family. operties . In manyoperties . Worldwioperties .For decades, several research studies have been conducted on the active ingredients of pumpkin peel, flesh, and seeds to provide a thumbnail sketch of their health-related impacts, which have demonstrated its anti-inflammatory , antibacThis review article focuses on compiling all of the research related to the nutritional composition of pumpkin peel, flesh, and seeds as well as their health-related benefits, involving scavenging activities against many chronic diseases. Safety concerns in regard to the use of pumpkins in some situations and in vulnerable groups are also discussed. In addition, recommendations for future research are proposed at the end of this paper.C. maxima, C. pepo, C. moschata). Moreover, research has demonstrated that pumpkin seeds and flesh are significant sources of proteins, carotenoids, tocopherols, and antioxidants and are low-caloric [All foodstuffs are regarded as food supplements, although the term nutraceutical is typically used to describe goods that have not been properly evaluated for their health-related benefits on the market. Considering that pumpkin is a good source of nutrients, it may be regarded as nutraceutical . The aut-caloric .In 2006, a study found that pumpkin peel contains alcohol insoluble polysaccharides (AIP), which tend to attenuate bile acid and support the growth of gut microbiota . MoreovePumpkin fruit and its derivative products have become more popular in agriculture, medicines, and food processing due to their nutritious and health-promoting properties. Of note, the chemical composition of pumpkin varies across species and cultivars grown in various regions . Pulp anProteins, carotene, mineral salts, vitamins, polysaccharides, such as pectin, as well as other elements, such as phenolic compounds and terpenoids are found in pumpkin pulp, which have both nutritional and health-protective values.Minerals required by the human body are obtained through the regular consumption of food. Minerals play vital roles in a variety of bodily processes. Several minerals essential to human health may be found in pumpkins. Phytosterols and phytonutrients are found in abundance in pumpkin pulp ,29. CaroBioactive chemicals found in pumpkin seeds, which are typically regarded as agricultural waste, have fascinating nutraceutical qualities . The eleA detailed view of the nutritional composition, bioactive profile, and health benefits of pumpkin bioactive compounds is shown in Hyperglycemia, which may result from the absence of insulin (DM-type 1) or due to a low response to insulin (DM-type 2), is one of the world’s emerging problems. Chronic hyperglycemia leads to severe complications, such as damage to the eyes (retinopathy), brain (neuropathy), and kidneys (nephropathy) . DiabeteIn Mexico and China, herbal extracts are used to treat hyperglycemia as they usually contain pumpkin ,58. In rThe breakdown of complex carbohydrates usually occurs in the small intestine by a-glycosidase, which is present in epithelial mucosa. A-glycosidase is responsible for the breakdown of glycosidic bonds present within complex carbs, and thus increases the blood glucose level . It is oDiabetic rats are one of the most commonly used tools to study the antidiabetic effect of any therapeutic agent. Alloxan is a toxin that affects the B-cells of pancreas, and thus leads to diabetes in rats . A case-Numerous researches established the diabetic-friendly effects of pumpkin . One stuHypoglycemic properties of germinated seed oil(s) and seed protein(s) have also been reported. However, ungerminated seeds do not show hypoglycemic properties .In addition to the in vivo studies, several clinical studies have been conducted to understand the efficacy of pumpkin in treating and controlling DM-type 2. It has been reported that fasting blood glucose in people with diabetes is due to the use of juices of pumpkin fruits. Similarly, pumpkin polysaccharide granules have also been proven to control postprandial sugar levels in clinical studies ,70. PumpA diet rich in oxidants and antioxidants has a significant association with cancer. Diet tends to make the condition worse or better, as cancer is associated with oxidative stress . In 2017Prevention of cancer is possible using a proper diet and dietary ingredients. Additionally, only 5 to 10% of all cancers are inherited, while the others are due to lifestyle . PumpkinIn vivo and in vitro studies have demonstrated the inhibitory effects of pumpkin against prostate cancer. Moreover, a study on Sprague Dawley rats reported the inhibition of testosterone-induced hyperplasia using pumpkin seed oil, suggesting the beneficial use of pumpkinseed oil against benign prostatic hyperplasia . FurtherPumpkin seeds are a major source of phytoestrogen, such as lignans and isoflavones. Phytoestrogen tends to bind with estrogen receptors (ER) in the female body . A studyMalnutrition is a common issue worldwide, affecting children with limited calorie and protein(s) intake. Malnutrition usually results in behavioral disorders . It is rOrganic compounds known as aflatoxins have substantial toxic effects, such as carcinogenic, mutagenic, and hepatotoxic. In addition, they contribute to lipid peroxidation, thus affecting the brain . In 2013Depression is the most common brain disorder and is specified as a state in which one loses interest, pleasure, sleep, and eating patterns are disturbed. Similarly, in mood swings, the sufferers may feel guilty and ashamed. In addition, they have poor interest/concentration in daily tasks. According to the 2015 World Health Organization statistics, people suffering from depression were more than 300 million . In a stFurthermore, a recent study highlighted that several nutritional deficiencies result in the progression of diabetes, such as vitamin B, amino acids, and omega 3-fatty acids . TryptopIn the past, several researchers reported the liver-protective effects of pumpkin. In 2005, a study was conducted on male Sprague Dawley rats, which were administered a low protein diet for 5 days to produce liver dysfunction. Thereafter, they were administered CCl4 injections, which resulted in significantly higher levels of four liver enzymes; aspartate transaminase (AST), alkaline phosphatase (ALP), alanine transaminase (ALT), and lactate dehydrogenase (LD). One group was administered with pumpkin seed protein isolate, which lowered the level of both aforementioned enzymes, suggesting the beneficial role of pumpkin in treating liver dysregulation . In a siNon-alcoholic fatty liver disease (NAFLD) further leads to atherosclerosis, and CVD is a major health issue worldwide that leads to mortality and morbidity . To cureIn 2015, a study was conducted on APOE*-3Leiden (E3L) mice, which is the most suitable model to study NAFLD . Cocoa bAspartame, which is often used in more than 60 countries worldwide as a sweetener, has also been found to be associated with hepatotoxicity, thus affecting liver functioning . A studyAlthough the death rate associated with CVD has been controlled during the past years, in the US, heart disease is 1st, while stroke remains the 5th leading cause of death . Of noteSeveral researches have demonstrated an inverse relation between the Mediterranean diet and CVD ,126. CucPumpkin affects serum lipid levels directly and can also affect them indirectly. A research was conducted on Lohmann Brown Lite hens, and they were administered with a diet based on flaxseed and pumpkin oil. It was reported that eggs laid by the hens with a pumpkin diet had the highest MUFA content, lower PUFA, lower myristic acid, and other saturated fatty acids content . HoweverA similar study was conducted in 2015, where pumpkin cake was prepared with pumpkin extract  and wheat flour (72%). Pumpkin cake was high in fiber (8%), carotenoid (41 mg/100 g), and ash (6.45%) compared with the control cake, which was made of wheat. On a biological basis of analysis, it was reported that pumpkin meal lowers the cholesterol and LDL level and increases the HDL level in a dose-dependent manner . Other rWhen the body is in hypercholesterolemic situation, dysfunctioning of the endothelial layer occurs, leading to the enhanced level of vascular cell adhesion molecule (VCAM). This occurs due to the activation of reactive oxygen species (ROS), which reduces nitric oxide formation and stops LDL oxidation. Due to the lower levels of nitric oxide (NO), oxidization of LDL will occur, which is responsible for all this VCAM enhancement mechanism . PumpkinIn addition to all of the aforementioned properties, pumpkin has been reported to play an important role in inflammatory diseases, such as arthritis due to its anti-inflammatory properties ,140,141.The worldwide male population above 50 years mostly suffers through an outgrowth of stromal and epithelial cells, usually in the periurethral area and in the transition zone ,143. Manv/v), pumpkin seed oil has the tendency to treat Staphylococcus aureus, Salmonella enterica, Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Candida albicans, and Serratia marcescens [Currently, although antimicrobial drugs are available, the focus of scientists is to develop dietary strategies to treat these infections as the use of these antimicrobes can lead to drug resistance. At a concentration of 2%  are rich sources of micro and macro nutrients, including carbohydrates, fiber, amino acids, MUFA, PUFA, tocopherol, and carotenoids. The presence of various bioactive phytochemical compounds in pumpkin showed health-promising properties and may be used as an ingredient of choice in functional foods and pharmaceutical products. All of these components have an essential role in the body’s normal mechanism, and thus can aid in treating several diseases as functional therapeutic agents. Previous studies confirmed the significant role of pumpkins in managing and treating diabetes, cancer, liver disorders, cardiovascular diseases, and depression. Additionally, the use of cucurbits species as antioxidant, antimicrobial, anti-inflammatory, and anti-ulcerative properties has also been reported by researchers. Moreover, these bioactivities can be improved using environmental stimulators, such as atmospheric COAlthough previous studies demonstrate the therapeutic uses of pumpkins in major chronic diseases, further research is still required. The effect of pumpkin on a-amylase and the pathway of this climber to exhibit hypoglycemic properties have not yet been reported. Data on the role of pumpkin in prostate and breast cancer are not sufficient, and minimal investigations are conducted on rectal, stomach, and lung cancer. Moreover, pumpkin in spermatogenesis can be used as an effective strategy after chemotherapy, where males suffer from reproduction disorders. In addition to all of these therapeutic effects, pumpkin meals can be used in poultry to produce healthy eggs due to the high MUFA and low saturated fatty acid content of pumpkin. Furthermore, an appropriate collaboration of researchers and industrialists is required to produce pumpkin-based useful products. Finally, awareness among the masses should also be emphasized in regard to the nutritional and therapeutic uses of pumpkin as an ingredient in the daily diet.