This study aimed to use network pharmacology to predict the therapeutic mechanism of oroxyli semen (OS) on triple-negative breast cancer (TNBC) and validate it through in vitro experiments.https://www.bioinformatics.com.cn/) to perform gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses to evaluate biological processes, molecular functions, and cellular components and generate simulated signal pathways. Additionally, molecular docking was used to evaluate the binding ability of small molecule drugs and signaling pathway targets. CCK8 assay was conducted to detect the effect of small molecule drugs on TNBC cell viability, and Western Blot was utilized to verify the expression of AKT, VEGF, and hypoxia-inducible factor 1-alpha (HIF-1α) proteins.The active ingredients and target proteins of OS were retrieved from the Traditional Chinese Medicine Systems Pharmacology database, and the TNBC-related target genes were obtained from the GeneCards database. The overlapping genes were used to construct a protein–protein interaction (PPI) network via the String database. Furthermore, we employed an online bioinformatics analysis platform (P < .05), increased the cleaved PARP.Fifteen active ingredients and 166 therapeutic targets of OS were obtained from the Traditional Chinese Medicine Systems Pharmacology database. The Venn diagram revealed that 163 targets were related to TNBC. The protein–protein interaction network analysis identified AKT1, IL-6, JUN, vascular endothelial growth factor A (VEGFA), CASP3, and HIF-1α as potential core targets through which OS may treat TNBC. Furthermore, the molecular docking results indicated that the active ingredient chryseriol in OS had good binding ability with VEGFA, and HIF-1α. CCK8 assay results indicated that chryseriol inhibited the viability of MDA-MB-231 and BT-20 cells. Western Blot demonstrated that chryseriol intervention led to a decrease in VEGFA, and HIF-1α protein expression compared with the control group (OS may exert its therapeutic effects on TNBC through multiple cellular signaling pathways. Chryseriol, the active component of OS, can enhance the apoptosis of TNBC cells by targeting VEGFA/HIF-1α pathway. This study provided new insights into the potential therapeutic mechanism of OS for TNBC and may aid in the development of novel therapeutic approaches for TNBC. Its high metastatic potential and chemoresistance make it a challenging type of breast cancer to treat. Currently, research on TNBC mainly focuses on the molecular biology mechanisms of the disease and targeted therapies. Many studies use in vitro and in vivo models to evaluate the therapeutic effects of existing drugs and new compounds on TNBC. In addition, an increasing number of studies are focused on screening potential therapeutic drugs from traditional Chinese medicine and natural products, and predicting and validating their mechanisms of action using bioinformatics methods. Future research needs to continue to explore the molecular mechanisms of TNBC and find more effective treatment methods.Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2.Oroxyli semen (OS) is a common traditional Chinese medicinal herb with a history spanning thousands of years. It contains various bioactive compounds, such as apigenin, luteolin, and quercetin, and exhibits diverse pharmacological effects including anti-inflammatory, antibacterial, lipid-lowering, and anticancer activities. Among its bioactive compounds, apigenin is one of the most active and has been shown to inhibit the proliferation and migration of multiple tumor cells and induce tumor cell apoptosis. Although considerable progress has been made in OS research, due to its complex composition and diverse pharmacological mechanisms, further research is still needed to gain a deeper understanding of its impact on human health and to develop more effective treatment methods and applications.Current research on OS primarily focuses on the extraction, isolation, and purification of its bioactive compounds, as well as investigating its pharmacological and anticancer effects.The active constituents of drugs and corresponding therapeutic targets were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. The targets for TNBC were sourced from the GeneCards database. The cellular pathways were enriched by employing the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The 2D structure of the small molecule drug chryseriol, which was utilized for molecular docking analysis, was obtained from the TCMSP database. The protein structures for vascular endothelial growth factor A (VEGFA) and hypoxia-inducible factor 1-alpha (HIF-1α) were sourced from the protein data bank database.https://tcmspw.com/tcmsp.php) was used to retrieve information on the drug components and targets of OS. The aim was to screen for components with good oral bioavailability (OB ≥ 30%) and drug-likeness (DL ≥ 0.18) and predict their components and targets.The TCMSP database (https://www.genecards.org/) using the keyword “TNBC” to obtain relevant targets information. Targets with a relevance score of at least 50 were selected to obtain more relevant disease targets.A search was performed on the GeneCards database . We selected the default settings and set the species as “Homo sapiens.” The results obtained from the STRING database were imported into Cytoscape 3.6.0, and then NetworkAnalyzer was used for topological analysis based on degree, betweenness centrality, average shortest path length, and closeness centrality. We sorted the genes by their degree and selected the top 20 genes with scores higher than the average score as key targets. Ultimately, we identified 163 key targets and plotted them as a graph based on their respective values.We constructed a protein–protein interaction (PPI) network for the shared target genes of OS and breast cancer using the STRING database  was used to list the top 5 gene enrichments for biological processes, molecular functions, and cellular components.We conducted GO enrichment analysis on the shared targets of OS and triple-negative breast cancer, using the GO database to analyze biological processes, molecular functions, and cellular components. We filtered the enriched pathways based on a corrected P-value of ≤.05. Subsequently, we annotated the pathways in which each gene was involved using a bioinformatics online analysis platform to further investigate the association between these pathways and the diseases.We conducted KEGG pathway enrichment analysis on the common targets of OS and triple-negative breast cancer using the David database. We filtered out enriched pathways based on a corrected To better elucidate the complex interaction between OS components and breast cancer and their corresponding target genes, we constructed an OS component-TNBC target network based on the corresponding components and disease target genes included in this study. The network was visualized using Cytoscape 3.6.0. The components were sorted based on their degree values, with higher values indicating greater importance.https://www.rcsb.org/). To meet the requirements of AutoDock, we converted the pdb files into pdbqt format files and added hydrogen atoms and calculated charges. Next, we configured the AutoDock parameters according to our needs, including defining the grid box, selecting the search algorithm, setting the search space, and energy function. Defining the grid box is an important step that determines the size of the search space and the position of the ligand molecule. Selecting the appropriate search algorithm and energy function also have a significant impact on the accuracy of the results. After the configuration is complete, AutoDock scans the ligand molecule, searches all possible positions, and calculates the interaction energy at each position. After docking, we can analyze and visualize the results, such as calculating the interaction energy between the ligand and protein, and drawing energy contour maps. These results can provide important references for subsequent drug design and optimization.We used AutoDock software for molecular docking. Firstly, we obtained the 3D structures of the required protein and ligand molecules from the protein data bank database (2 and the next step was performed when the cells reached approximately 70% to 80% confluency.MDA-MB-231 cells were cultured in DMEM medium (with high glucose) and 10% fetal bovine serum (FBS). BT-20 cells were cultured in RPMI 1640 medium and 10% FBS. 1% penicillin–streptomycin (PPS) was added to avoid bacterial infection during cell culture. Cells were cultured at 37 °C and 5% CO3 cells per well) were seeded into a 96-well plate. Different concentrations of OS  were added to the treatment and control groups, followed by incubation for 48 hours at 37 °C and 5% CO2. Cell viability was measured using the Cell Counting Kit-8 . CCK-8 reagent was added to each well and incubated for 1 to 4 hours in a cell culture incubator. Absorbance at 450 nm was measured using a microplate reader  to assess cell viability.MDA-MB-231 and BT-20 breast cancer cells  and the experimental group with 20 μmol/L OS added. After 48 hours of incubation, the culture medium was removed and the cells were washed 3 times with sterile PBS buffer. Then, pre-cooled protein lysis buffer containing 10% phosphatase inhibitor was added to the cells, and the cells were scraped with a cell scraper and collected into a 1.5 mL EP tube. The tube was placed on ice and the cells were lysed for 20 minutes. After lysis, an appropriate amount of loading buffer was added, and the sample was heated at 95 °C for 10 minutes and stored for later use. The proteins were separated by 10% polyacrylamide gel electrophoresis and transferred onto a PVDF membrane  when the bromophenol blue was close to the bottom of the gel. The membrane was then blocked with 10% skim milk at room temperature for 30 minutes, followed by overnight incubation with the primary antibody at 4 °C. The next day, the membrane was washed 3 times with TBST buffer and incubated with a goat anti-rabbit or anti-mouse secondary antibody labeled with horseradish peroxidase at 37 °C for 1 hour.P-value was <0.05.The data from all samples were presented as mean ± standard deviation (SD) after being measured 3 times. Single-factor analysis of variance (ANOVA) was conducted using Graphpad Prism 9.0 software to compare the differences among different groups. A difference was considered statistically significant when the In this study, we applied specific screening criteria for drug components in the TCMSP database, selecting components with drug-likeness (DL) and oral bioavailability (OB) and only retaining those with OB >30% for further analysis. Following these criteria, we identified 15 effective components and 166 targets. We then associated these targets with breast cancer-related genes, resulting in 15,960 genes associated with TNBC. By intersecting the drug target genes with TNBC-related target genes, we ultimately obtained 163 common targets. We used Venny 2.1 software to draw a Venn diagram  using the GO database, we found that the core biological processes of OS treatment for TNBC are mainly related to xenobiotic stimulus, nutrient levels, cellular response to nitrogen compounds, response to hormones, and response to inorganic substances. These processes are primarily involved in cellular components, such as the membrane raft, perinuclear region of the cytoplasm, transcription regulator complex, and plasma membrane protein complex. The core biological processes are completed through molecular functions, including protein domain-specific binding, protein homodimerization activity, polymerase II-specific DNA-binding transcription factor binding, and protein kinase binding Fig. A.The KEGG enrichment analysis revealed that the intersection genes between OS and TNBC are primarily enriched in processes related to cancer, signal transduction, endocrine system, and cardiovascular disease Fig. B. Based To investigate whether the biological activity of chryseriol is related to the VEGFA and HIF-1α signaling pathways, we used molecular docking to evaluate its binding to the key targets in these pathways. The research results showed that hydrogen bonding is the main mechanism for the interaction between the targets and chryseriol, and a binding energy <0 indicates better docking efficiency. Specifically, we found that chryseriol can bind to VEGFA and HIF-1α, with binding energies of −7.0 kcal/mol and −7.4 kcal/mol, respectively. This research result suggests that chryseriol may exert its anticancer effects through the VEGFA/HIF-1α signaling pathway Fig. .P < .05)  significantly reduced the survival rate of the 2 triple-negative breast cancer cells when compared to the control group treated with 0 μmol/L chryseriol (05) Fig. A.P < .05) and increased the expression of the cleaved PARP  for 48 hours led to a decrease in the protein levels of VEGFA and HIF-1α , inducing the accumulation of regulatory T cells while concurrently suppressing T cell functionality. Utilizing antiangiogenic agents that target VEGFA/VEGFR may assist in restricting immunosuppression induced by tumors. HIF-α, a subunit of the HIF-1 transcription factor, is encoded by the HIF1α gene. It contributes to cancer development by facilitating the shift from oxygen-reliant ATP generation to glycolysis, and is linked to genetic instability, inflammation that promotes tumor growth, and evasion of the immune system. The molecular docking results demonstrate that chryseriol has a good binding ability with proteins VEGFA and HIF-α. The CCK8 assay confirmed that chryseriol indeed exerts an inhibitory effect on the viability of TNBC cells. Meanwhile, Western Blot experiments demonstrated that in MDA-MB-231 and BT-20 cells, the expression of VEGFA and HIF-1α proteins was significantly suppressed at a chryseriol concentration of 20 μmol/L, while the expression of Cleaved PRAP increased. The results suggest that chryseriol may induce TNBC cell apoptosis by inhibiting the VEGFA/HIF-1α pathway. However, additional experiments are necessary to determine its potential cytotoxicity and to validate its efficacy in vivo.In the present research, the KEGG database was employed for pathway enrichment analysis. It was observed that within the simulated pathway, VEGFA and HIF-1α serve as crucial nodes in combating TNBC cell pathways. A multitude of studies suggest that VEGFA also functions as an immunosuppressive factor, playing a vital role in the process of tumor angiogenesis.OS has a strong therapeutic effect in combating TNBC, with most of its therapeutic targets directed towards TNBC. Its main active ingredient, chryseriol, may promote TNBC cell apoptosis by inhibiting the VEGFA/HIF-1α pathway. This article proposes a relatively new view on the therapeutic mechanism of the Chinese herbal medicine OS, providing further evidence for its clinical use.Conceptualization: Wei Jiang, Kehui Hu.Data curation: Lulu Chen, Aishen Yang.Investigation: Yangan Li, Xin Liu.Software: Yangan Li, Xin Liu.Visualization: Yangan Li, Xin Liu.Writing – original draft: Lulu Chen, Aishen Yang.Writing – review & editing: Wei Jiang, Kehui Hu.


Aldh2, Aldh3a2, and Aldh9a1 in carnitine biosynthesis and Acsl5 in carnitine shuttle system were up-regulated by increased CR, which are consistent with our previous work on metabolome of the same individuals. Overall, the patterns of protein expression were more consistent with a “clean cupboards” than a “disposable soma” interpretation.Calorie restriction (CR) extends life span by modulating the mechanisms involved in aging. We quantified the hepatic proteome of male C57BL/6 mice exposed to graded levels of CR (0%–40% CR) for 3 months, and evaluated which signaling pathways were most affected. The metabolic pathways most significantly stimulated by the increase in CR, included the glycolysis/gluconeogenesis pathway, the pentose phosphate pathway, the fatty acid degradation pathway, the valine, leucine, and isoleucine degradation pathway, and the lysine degradation pathway. The metabolism of xenobiotics by cytochrome P450 pathway was activated and feminized by increased CR, while production in major urinary proteins (Mups) was strongly reduced, consistent with a reduced investment in reproduction as predicted by the disposable soma hypothesis. However, we found no evidence of increased somatic protection, and none of the 4 main pathways implied to be linked to the impact of CR on life span  as well as pathways in cancer, were significantly changed at the protein level in relation to the increase in CR level. This was despite previous work at the transcriptome level in the same individuals indicating such changes. On the other hand, we found  Aging is accompanied by many metabolic changes. Obesity, insulin resistance, inflammation, and high blood pressure are known as the metabolic syndrome of aging . One hunThe liver plays a central role in energy metabolism and glucose homeostasis. Previous work suggested that 40% CR changed lipid metabolism in the liver through reduced lipogenesis and increased lipolysis and ketogenesis , as wellCR was also reported to regulate several canonical aging-related pathways, including insulin/insulin-like growth factor (IGF-1) signaling ,11, mTORIncreased CR in both male and female mice and rats are roughly linearly related to the increase in life span ,26 up top < .05). Almost three-quarters (99) of the 134 correlations were positive, meaning the protein expression of a majority of significantly altered genes was up-regulated by the increase in CR level. Forty-nine of the 134 genes remained significant after correction for multiple testing using the Benjamini-Hochberg procedure, with 32 of these positively correlated  and the largest number (77) of enriched genes. The majority (63) of the genes enriched in the “metabolic pathways” were up-regulated , 126 out of which overlapped with the correlation results. After adjusting the p values for multiple testing, only 45 regressions were statistically significant (adjusted p < .05), 44 out of which overlapped with the adjusted correlation results. There were consequently very few differences between the results of the correlation analysis and the linear regression modeling, and therefore the following analyses are based on the correlation results to maintain consistency with the hepatic transcriptome paper we published previously relating to the same individual animals , and 132 regressions were statistically significant  and 2 (Idh2), and malate dehydrogenase 2 (Mdh2) were significantly up-regulated as the CR level increased . IsocitrThe pentose phosphate pathway was stimulated by increase in the CR level . AlthougGlyoxalase 1 (Glo1) and Glyoxalase 2 (Glo2) play essential roles in the detoxification of methylglyoxal, and the product of the detoxification, glycolate, is a substrate for glycolate oxidase to produce glyoxylate. There was no significant change in protein expressions of Glo1 and Glo2 with the increased CR level, and in the glyoxylate and dicarboxylate metabolism pathways, only Mdh2 and serine hydroxymethyltransferase 1 (soluble) (Shmt1) proteins were significantly upregulated by the increase in CR .r = −0.03692, p = .8307) showed no significant correlation to the increased CR level, which indicated that it was the β-oxidation process, aka the degradation pathway, that was stimulated rather than the elongation pathway. Sterol carrier protein 2, liver (Scp2), a nonspecific lipid-transfer protein was significantly inhibited by the increase in CR , carnitine palmitoyltransferase 2 (Cpt2), solute carrier family 25 , member 20 (Slc25a20), and acyl-CoA synthetase long-chain family member 1(Acsl1), showed no significant changes in protein expression along with the increase in CR levels, except for Acsl5, which was up-regulated. The carnitine biosynthesis pathway starts from trimethyllysine (obtained from the diet or synthesized from L-lysine) and is part of the pathway of lysine degradation. Aldehyde dehydrogenase 2, mitochondrial (Aldh2), aldehyde dehydrogenase family 3, subfamily A2 (Aldh3a2), and aldehyde dehydrogenase 9, subfamily A1 (Aldh9a1) are included in the pathway of carnitine biosynthesis and their protein expression were significantly up-regulated by the increase in CR . Sphingor = −0.3436, p = .01681) was down-regulated (Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by fatty acids and their derivatives. However, no protein expression of PPARs was detected from the liver proteome. Protein expression of fatty acid transporter Slc27a5/FATP of the downstream PPAR signaling pathway was significantly up-regulated by the increase in CR level . Of PPARegulated . Among oegulated .Increasing CR level stimulated amino acid biosynthesis pathways mostly because it stimulated the protein expression of genes  overlapping with the glycolysis, TCA cycle, and pentose phosphate pathways, which provide activated precursors for biosynthesis of various amino acids, such as histidine. However, cystathionase (cystathionine gamma-lyase) (Cth), enzyme participating in cysteine biosynthesis, was also significantly up-regulated . ArginasSerine dehydratase (Sds), the enzyme mediating the conversion from serine to pyruvate, was up-regulated; Methionine adenosyltransferase I, alpha (Mat1a), a liver-specific enzyme that catalyzes the formation of S-adenosylmethionine, the principal biological methyl donor, was also up-regulated; Serine hydroxymethyltransferase 1 (soluble) (Shmt1), an enzyme which plays an important role in cellular one-carbon pathways by catalyzing the conversions of L-serine to glycine and 5,6,7,8-tetrahydrofolate to 5,10-methylenetetrahydrofolate, was also up-regulated .Degradation of various amino acids were also activated by the increased CR level. Expressions of aldehyde dehydrogenase 9, subfamily A1 (Aldh9a1), aldehyde dehydrogenase family 3, subfamily A2 (Aldh3a2), aldehyde dehydrogenase 2, mitochondrial (Aldh2), Hmgcs2, Ehhadh, Hadh, and Acadm, enzymes mediating degradation of the branched-chain amino acids  were significantly up-regulated . Expressr = −0.3061, p = .03437) by the increased CR.In de novo biosynthesis pathways, NAD is generated from tryptophan via 2-amino-3-carboxymuconic-6-semialdehyde and quinolinic acid, which is part of tryptophan metabolism, and genes mapped to the NAD biosynthesis, including tryptophan 2,3-dioxygenase (Tdo2), indoleamine 2, 3-dioxygenase 2 (Ido2), arylformamidase (Afmid), kynurenine 3-monooxygenase (Kmo), and amino carboxymuconate semialdehyde decarboxylase (Acmsd), showed no significant changes in protein levels, except for kynureninase (Kynu), which converts N′-formylkynurenine into kynurenine, down-regulated  and sulfotransferase family 2A, Dehydroepiandrosterone-preferring (DHEA-preferring), member 8  were down-regulated by the increased CR level  by the increase in CR. Gstp1 and Mgst1 were also down-regulated, while Gstm1, Gsta4, Gstt1, and Gstt3 were significantly up-regulated. Those are the glutathione S-transferase enzymes, which are the phase II enzymes of the biotransformation enzyme group responsible for detoxification of xenobiotics and protection against cancers.Pathways in Cancer obtained from KEGG included the Wnt, Hedgehog, Notch, HIF-1, cAMP, mTOR, PI3K-Akt, Jak-STAT, MAPK, Calcium, TGF-β, VEGF, p53, and PPAR signaling pathways, and none of these pathways showed significant changes as the CR level was increased . Howeverp = .01). Yet in the present analysis, protein expression of genes in those pathways was almost unaffected by the CR levels , flavin containing monooxygenase 3  and Fmo1 , sulfotransferase family 1D, member 1  and Sult1a1 , and Gsta4. Expression of these genes has previously been shown to be more highly expressed in females in the mouse liver because of the suppressive effects of both androgens and male-pattern growth hormone (GH) secretion , Ugt2b1, and Gstp1 were down-regulated by the increased CR, which normally have a greater expression in males because of the inducing effect of male-pattern GH secretion , Mup20 , Mup3 , Mup2 , Mup14 , and Mup10 . These changes suggested an increasing feminization in relation to the level of restriction.On the other hand, carboxylesterase 3A  with the increased CR level, there was a lack of significant correlation between the protein expression and level of CR (mean p = .60) and a lack of significant correlation between the protein and mRNA expression. These data suggest that mRNA levels are only a good indicator of altered protein expression when the relationship to a given treatment variable is very strong. This is in agreement with a previous study which found that in the livers of mice treated for different periods of time with 3 different peroxisome proliferative-activated receptor agonists, the differential expression of mRNA (up or down) captured at most 40% of the variation of protein expression  to the increase in CR level. In contrast, when the mRNA expression levels did not respond significantly to the increase in CR, there was only one out of 30 genes showed a significant correlation between the mRNA transcript levels and protein levels. Even though the mRNA expression of genes in the 4 main longevity-regulating related pathways were significantly correlated ” . The DSHRecently, a novel idea called the “clean cupboards hypothesis” was proposed which suggests that when animals are under CR, they do not strategically reallocate resources between reproduction and somatic protection, but rather their aim is only to make an immediate energy balance . ConsequThe metabolic pathways, including carbohydrate, lipid, and amino acid metabolism pathways were the most significantly stimulated by the increased CR level. There was the significant feminization of these male mice and reduced investment in major urinary proteins. We found no upregulation of pathways that might be considered diagnostic of somatic protection. Moreover, the lipid metabolism modulating pathways carnitine shuttle system and biosynthesis were up-regulated, which is consistent with our previous metabolome work. Several key pathways previously suggested to play an important role in CR’s effect to life span  were virtually unchanged at the protein level, despite previous transcriptomics work in the same individual animals showing significant changes.Mus musculus) purchased from Charles River  were individually housed with free access to water. Mice were exposed to 12-hour dark/light cycles (lights on at 0630 hours) and body mass and food intake were recorded daily, immediately prior to nocturnal feeding. At 20 weeks of age (resembling early adulthood in humans), mice were randomly allocated into 6 different treatment groups: . Mice in 24AL and 12AL groups were fed AL for 24 hours or 12 hours during the dark period, respectively. 10%CR, 20%CR, 30%CR, and 40%CR indicate 10%, 20%, 30%, and 40% lower calories, respectively, than their own individual intakes measured over a baseline period of 14 days prior to introducing CR.All procedures were approved by the University of Aberdeen ethical approval committee and carried out under the Animals (Scientific Procedures) Act 1986 Home Office license (PPL 60/3706 held by J.R.S.). Forty-eight male C57BL/6 mice  may overfeed and become obese. CR-associated changes compared to 24AL are, therefore, most likely to reflect the anti-obesity effect of CR ,47. In a2 overdose, the liver was removed as part of the overall dissection, weighed, and divided into 7 pieces which were immediately snap frozen in liquid nitrogen and stored at −80°C until one piece was used for proteome isolation. The proteins were digested using the filter-aided proteome sample preparation method by sequencing grade trypsin . The resultant tryptic peptides from each sample were then labeled with the 8-plex iTraq-reagents . The labeled samples were mixed together with an equal molar ratio. After prefrcationed by off-line reversed phase-high-performance liquid chromatography, the peptides were analyzed by a TripleTOF 5600 mass spectrometer (AB SCIEX) coupled online to an Eksigent nanoLC Ultra in Information Dependent Mode and with iTRAQ reagent collision energy adjustment on. Data are available via ProteomeXchange with identifier PXD033436.After culling by a terminal COIn this work, we analyzed the liver proteome as one of the series of studies on the role of graded CR levels ,10,29,48https://www.R-project.org/) mainly using the Hmisc package and the for a loop. The adjusted p value of the correlation test was obtained using the Benjamini–Hochberg method. DAVID: Functional Annotation Tools (https://david.ncifcrf.gov/tools.jsp) was used for enrichment analyses, both based on G.O. and KEGG pathways (https://www.kegg.jp/kegg/tool/map_pathway2.html) was used for pathway mapping , which was transferred into a numeric vector , by Pearson correlation method in the statistical environment R (version 4.1.1; pathways ,50, and  mapping .We validated the top 30 genes correlated either negatively or positively and the 30 least correlated genes of the transcriptome  with theglad017_suppl_Supplementary_MaterialClick here for additional data file.


Periodontitis is a chronic oral disease prevalent worldwide, and natural products are recommended as adjunctive therapy due to their minor side effects. Curcumin, a widely used ancient compound, has been reported to possess therapeutic effects in periodontitis. However, the exact mechanism underlying its activity remains unclear. In this context, the present study aimed to conduct computational simulations to uncover the potential mechanism of action of Curcumin in the treatment of periodontitis.Single-cell analysis was conducted using a dataset  curated from the Gene Expression Omnibus (GEO) database through an R package "Seurat package." Bulk RNA sequencing data were curated from GSE10334 and GSE16134 and processed by R package "Limma." Then, the marker genes in the single-cell transcriptome and differentially expressed genes (DEGs) in the bulk transcriptome were integrated. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were also carried out to reveal their functionalities. Key targets were mined from their protein–protein interaction (PPI) network topologically. Afterward, molecular docking was performed. The top-ranked pose was subjected to molecular dynamics simulations to investigate the stability of the docking result.FOS, CXCL1, CXCL8, and IL1B, were filtered after a series of selected processes. The results of molecular modeling suggested that except for IL1B, the Vena Scores of the rest exceeded -5 kcal/mol. Furthermore, the molecular dynamic simulation indicated that the binding of the CXCL8-Curcumin complex was stable over the entire 100 ns simulation.The present study unlocked the binding modes of CXCL1, FOS, and CXCL8 with the Curcumin molecule, which were relatively stable, especially for CXCL8, hindering its promising potential to serve as the critical targets of Curcumin in periodontitis treatment.The online version contains supplementary material available at 10.1186/s12903-023-03181-x. Periodontal disorders encompass a broad spectrum of inflammatory problems that damage the supporting components of teeth  . PeriodoMoreover, as antimicrobial resistance is a significant threat to public health globally, the excessive use of antibiotics in dentistry is under debate . Host mohttp://ctdbase.org/). R software (version 3.6.1) was implemented for data processing. Unless specifically emphasized, a P-value < 0.05 indicated statistical significance.Bulk transcriptome data were acquired from the GSE10334 and GSE16134 datasets, of which the GSE57338 dataset had 247 samples; GSE16134 had 310 samples. The single-cell transcriptome data were curated from GSM5177042, GSM5177043, GSM5177044, and GSM5005052. References-based curcumin targets were obtained from the CTD (Comparative Toxicogenomics Database) database  to generate the object and exclude samples with poor quality –13. We fP < 0.05 and | Log2  |> 1 as thresholds, and R package "ggplot2" (version 3.3.5) was used to draw volcano plots to showcase the level of DEGs' expression [We screened the DEGs between periodontitis samples and normal samples from the 2 GSE datasets with the R package "limma" (version 3.16) by setting pression .Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment were carried out , 16. Thehttps://genemania.org/) system to construct a PPI network for the candidate genes which was imported into the Cytoscape software (version 3.16) to produce a more esthetic visualization [We used the GeneMANIA  and docked them on the CB-dock platform  [https://vina.scripps.edu/) [To determine the best interactive pose between the curcumin molecule and the targets of interest, we downloaded the 3D structures of the hub targets from the AlphaFold Protein Structure Database (st_load) –22. Afteps.edu/) –29.As molecular and condensed-phase systems are inherently dynamic, it is important to understand the fundamental physicochemical phenomena by analyzing their behaviors at the atomic level. From this point of view, we assessed the conformational stability of the protein–ligand complexes of interest over 100 ns. The Desmond module of the Schrödinger software package  implemented in Maestro was exploited to conduct MD simulation studies , 31. TheP < 0.05 and | Log2FC |> 1, 108 DEGs were identified from the GSE10334 dataset between periodontitis and normal samples in which 76 genes were up-regulated and 32 genes were down-regulated , which once again supported the fact that our extraction is reasonable  was performed for a more in-depth exploration of the possible targets of curcumin. We first attempted molecular docking for the curcumin molecule and FOS, CXCL1, CXCL8, and IL1B, respectively, discovering that except for IL1B, they all showed relatively high affinities. Then, as CXCL8 exhibited the most stable binding mode, we continued with a molecular dynamics simulation to verify this interaction. The curcumin-CXCL8 complex was quite reliable during the entire 100 ns simulation, which supports the idea that CXCL8 may serve as a direct target for curcumin’s function in the treatment of periodontitis. The inhibition of CXCL8 binding to its receptor on proinflammatory immune cells in the context of chronic lesions such as those found in periodontitis might limit the recruitment of these cells and lead to the end of the proinflammatory cycle in these lesions, ultimately leading to their resolution without the use of antibiotics or more drastic, surgical interventions.In conclusion, in the present study, through a comprehensive series of omics data cooperation, network analysis, and molecular modeling, CXCL8 was found as a promising potential direct target for the curcumin molecule in treating periodontitis. Although certain limitation exists in the present study, namely, the lack of in vitro experimental data to validate these exciting results, we sincerely hope our findings can act as a theoretical base for either old drug reproposal or new drug development in the future.Additional file 1.


Agrobacterium tumefaciens in PureWheat, a combination of scanning electron microscopy (SEM), complete transcriptome analysis, and metabolome analysis was conducted to understand the progress. The results of the SEM analysis revealed that Agrobacterium tumefaciens were deposited under the damaged cortex of immature embryos as a result of pretreatment and contacted the receptor cells to improve the TE. Transcriptome analysis indicated that the differentially expressed genes were mainly enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, plant–pathogen interaction, plant hormone signal transduction, and the MAPK (Mitogen-activated protein kinase) signaling pathway. By analyzing the correlation between differentially expressed genes and metabolites, the expression of many genes and the accumulation of metabolites were changed in glucose metabolism and the TCA cycle (Citrate cycle), as well as the amino acid metabolism; this suggests that the infection of wheat embryos with Agrobacterium is an energy-demanding process. The shikimate pathway may act as a hub between glucose metabolism and phenylpropanoid metabolism during Agrobacterium infection. The downregulation of the F5H gene and upregulation of the CCR gene led to the accumulation of lignin precursors through phenylpropanoid metabolism. In addition, several metabolic pathways and oxidases were found to be involved in the infection treatment, including melatonin biosynthesis, benzoxazinoid biosynthesis, betaine biosynthesis, superoxide dismutase, and peroxidase, suggesting that wheat embryos may be under the stress of Agrobacterium and, thus, undergo an oxidative stress response. These findings explore the physiological and molecular changes of immature embryos during the co-culture stage of the PureWheat technique and provide insights for Agrobacterium-mediated transgenic wheat experiments.The transformation efficiency (TE) was improved by a series of special chemical and physical methods using immature embryos from the cultivar Fielder, with the PureWheat technique. To analyze the reaction of immature embryos infected, which seemed to provide the necessary by  Agrobacterium-mediated genetic transformation and biolistic transformation are commonly used in plant systems, among the many methods of delivering DNA into cells [Agrobacterium-mediated transformation has some unique advantages, including low-copy-number integration, low cost, high stability, and a simple procedure [Triticum aestivum L.) plant was obtained by biolistic particle bombardment in 1992. Agrobacterium-mediated transgenic wheat plants were first developed in 1997, and the protocol required about 3 months to obtain stable transgenic plants [Agrobacterium infection, greatly improving the transformation efficiency, reaching 50–90% [Agrobacterium infection, recovery after cutting embryo axes, and selection of positive plants. The PureWheat technique sheds light on the genetic engineering of wheat.to cells . Comparerocedure ,3. For ac plants . After tAgrobacterium and plant receptors, which occurs after Agrobacterium infects plant receptors, and generally lasts 1–4 days [Agrobacterium-infected plants. However, how the molecular mechanism and endogenous substances change at this stage—which further affects the transformation efficiency—is unknown.Co-cultivation is an important step in the direct contact between 1–4 days . Several1–4 days ,7. For e1–4 days ,9. In coToday, high-throughput omics approaches—including genomics, transcriptomics, proteomics, and metabolomics—have been widely used by researchers to study plants’ life processes and different abiotic stresses, deepening the understanding of multiple biological pathways . CombineAgrobacterium and immature embryos is mysterious, including changes in the internal molecular mechanisms caused by the introduction of exogenous genes. In this investigation, we referred to the protocol used in PureWheat and performed an integrated analysis of the transcriptomes and metabolomes of immature embryos from cv. Fielder in the co-culture stage to analyze the changes in gene expression and metabolites during Agrobacterium treatment. This helped us to understand the physiological and molecular changes of immature embryos in this regimen after infection by Agrobacterium tumefaciens, and it can further explain the mysterious T-DNA insertion of Agrobacterium tumefaciens in response to the recipient plant.Common wheat is a staple food crop for more than one-third of the world’s population, providing approximately 20% of the daily human calorie consumption. The increasing nutritional demands posed by the growing world population and environmental stresses present major challenges for wheat research and breeders ,27,28. IAgrobacterium infection. Firstly, we observed through scanning electron microscopy that the upper epidermis of immature embryos would break and create gaps after the pretreatment , an increase in proline, and an increase in Malondialdehyde (MDA), indicating that some stress response occurred in immature embryos.Subsequently, some physiological indicators related to oxidative stress were measured . The resAgrobacterium tumefaciens, whole-genome transcriptomic analysis was employed to analyze the expression of transcriptome genes. Three biological replicates of cDNA libraries were prepared, for a total of four samples of control and treatment after Agrobacterium infection of immature embryos. At first, the four cDNA libraries were prepared in Illumina sequencing from three biological replicates per sample, which generated 73,174,653, 75,342,269, 71,580,265, and 72,900,690 sequences with 10.98, 11.3, 10.74, and 10.93 G from CK1, CK2, T1, and T2 respectively  suggested close clustering of replicates within the samples and, hence, excellent sample data reproducibility . A largeA total of 1570 DEGs  were identified in the comparison of CK1 vs. T1 ; GO Gen, and theAs shown in Agrobacterium tumefaciens were selected for qRT-PCR  with a screening criterion of FC > 2/q < 0.05 and compared the metabolite numbers between the two control conditions and the two treatment levels to identify DAMs following ie chart c. These ie chart . A totalie chart . In addiie chart d,e.Agrobacterium tumefaciens showed significant differences in metabolites and significant enrichment pathways for CK1 and T1 , including purine metabolism, phosphonate and phosphinate metabolism, histidine metabolism, arginine and proline metabolism, tryptophan metabolism, phenylpropanoid biosynthesis, phenylalanine metabolism, alanine, aspartate and glutamate metabolism, and vitamin B6 metabolism. Pathways significantly enriched in CK2 and T2  included phenylpropanoid biosynthesis, purine metabolism, tryptophan metabolism, the pentose phosphate pathway, phosphonate and phosphinate metabolism, glutathione metabolism, phenylalanine metabolism, tyrosine metabolism, monoterpenoid biosynthesis, and glycine, serine, and threonine metabolism.Comparative analysis of immature embryos treated with Agrobacterium infects immature embryos, differentially expressed genes and differentially accumulated metabolites were mapped to the KEGG pathway map simultaneously. The results showed that the same pathway of DEGs and DAMs was enriched in glycolysis, the TCA cycle, amino acid biosynthesis, phenylpropionin, lignin, and other related pathways As shown in Agrobacterium-infected immature embryos through glycolysis metabolism, TCA cycle metabolism, and their related pathways; the upregulated genes included PFK (TraesCS3D02G109600), gapN (TraesCS2D02G197300), gpmI (TraesCS4B02G172700), pyk (TraesCS2D02G561700), RAFS (TraesCS3B02G133400), tktA (TraesCS2D02G073900), IDH1 (TraesCS2A02G205900), GLT1 , and POP2 (TraesCS2A02G421400), and the downregulated genes included PDHA (TraesCS6B02G342000), PDHB (TraesCS5B02G290600), DLAT (TraesCS4A02G481800), DLD (TraesCS1D02G109600), ACO (TraesCS6B02G342000), LSC1 (TraesCS5B02G290600), LSC2 (TraesCS7D02G011600), SDHA (TraesCS1D02G109600), fumC (TraesCS2A02G336500), MDH2 (TraesCS1A02G412900), and GSS . The upregulation of PFK, gapN, and gpmI promoted the accumulation of 2PGA (2-Phospho-D-glycerate), which promoted the accumulation of quinate and 5-o-(1-carboxyvinyl)-3-phosphoshikimate in the downstream shikimate pathway. Pyk was upregulated 2-fold in immature embryos treated with Agrobacterium tumefaciens, which is a key enzyme that catalyzes the conversion of PEP (Phosphoenolpyruvate) to pyruvate and then promotes the biosynthesis of the important downstream osmotic substance betaine. Acetyl-CoA (Acetyl coenzyme A) is a hub metabolite linking the glycolytic pathway and the TCA cycle. Several regulated genes were downregulated, including PDHA, PDHB, DLAT, and DLD, suggesting that Agrobacterium infection activates the glycolytic pathway of immature embryos, and several metabolites enter the shikimic acid pathway through PEP. Six genes encoding five enzymes in the TCA cycle pathway were downregulated in Agrobacterium-infected embryos. GSH is an important regulatory metabolite and affects the metabolic process in cells; the GSH-encoding enzyme GSS gene was downregulated, and the accumulation of its downstream metabolites in immature embryos was increased. The biosynthesis of GSH was mediated by succinyl-CoA and 2-oxoglutarate in the TCA cycle. Meanwhile, we also detected an accumulation of intermediate metabolites, including γ-glutamylcysteine, glutamate, GABA, and succinate semiphosphate. The results indicated that the TCA cycle pathway participated in the response of immature embryos to Agrobacterium and, finally, participated in the GSH biosynthesis pathway through intermediate metabolites. In addition, we also detected changes in some amino acid metabolites, including D-glucuronide, D-serine, L-histidine, isoleucine, D-aspartate, β-alanine, tyrosine, etc.To better understand the relationships between genes and metabolites after PAL and 4CL were involved in the phenylpropanoid metabolism pathway, and many secondary metabolites in this pathway were changed, including decreases in the accumulation of phenylalanine and cinnamic acid, and an increase in p-coumarate accumulation. We found that many enzymes and metabolites were involved in the process of infecting wheat embryos by Agrobacterium in related synthetic pathways, such as phenylpropionin and lignin  is a tripeptide composed of glutamic acid, cysteine, and glycine, which is involved in the tricarboxylic acid cycle and glucose metabolism to obtain energy, and is able to participate in the metabolism of products arising from oxidative processes [Agrobacterium infection of immature embryos through the TCA cycle.Many genes in the TCA cycle were downregulated, including rocesses . GSH andPKM, gapN, and gpmI led to the accumulation of the important intermediate 2PGA, while the upregulation of pyk reduced the accumulation of pyruvate—an end-product of the glycolytic pathway. Acetyl-CoA is the starting substrate of the tricarboxylic acid cycle and is an intermediate of sugar metabolism, as well as a metabolite of fats and certain amino acids. The genes encoding the enzyme of acetyl-CoA were downregulated, including PDHA, PDHB, DLAT, and DLD. The shikimate pathway starts from the pep of the glycolytic pathway, which leads to the biosynthesis of aromatic amino acids that are essential for protein biosynthesis and the production of a wide array of secondary plant metabolites. Among them, quinate is an astringent feeding deterrent that can be formed in a single-step reaction from 3-dehydroquinate, catalyzed by quinate dehydrogenase (QDH) [Agrobacterium-treated immature embryos. It has been hypothesized that the accumulation of quinate may be involved in the metabolic pathway of shikimate, affecting the synthesis of alanine (Phe), tyrosine (Tyr), and tryptophan (Trp). In addition, many amino acids were detected to be involved in the metabolic activities of the infected immature embryos through the TCA cycle and the glycolytic pathway, including D-glucuronide, D-serine, L-histidine, isoleucine, D-aspartate, and β-alanine. Numerous studies have shown that actively growing tissues are involved in pathways related to energy metabolism, including the TCA cycle, glycolysis, amino acid metabolism, etc. Immature embryos stimulated with Agrobacterium were also involved in these processes, as in previous descriptions.Meantime, many genes and metabolites were also altered in the glycolytic pathway connected by the TCA cycle. The upregulation of se (QDH) . 5-O-, which affected several metabolic pathways, including lignin biosynthesis and benzoxazinoid biosynthesis.Agrobacterium tumefaciens did not choose to enter the TCA cycle after the glycolytic pathway was decomposed but entered the phenylpropanin pathway through the shikimic acid pathway, resulting in changes in the metabolites.The upregulation of several genes increases the accumulation of 2PGA in the glycolytic pathway, which prompts the accumulation of 5-o-(1-carboxyvinyl)-3-phosphoshikimate, a key metabolite in the shikimic acid pathway. Moreover, most genes in the TCA cycle linked by acetyl-CoA were downregulated in the experiment. We speculated that the immature embryos treated with F5H gene led to increased accumulation of 5-hydroxyferulic acid and sinapic acid, while the upregulated expression of the CCR gene led to increased accumulation of coniferaldehyde, and aldehyde accumulated under 24 h treatment. These metabolites are important precursors of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). Scanning electron microscopy showed that the surface of the immature embryos was damaged after centrifugation, and Agrobacterium could enter the intercellular space through these damaged holes and directly contact the cell wall. We hypothesized that the cell wall of immature embryos would be attacked by Agrobacterium, which would affect the response mechanism of lignin in the cell wall. Unfortunately, no changes in lignin were detected in this study, which still needs to be confirmed by further experiments.The lignin pathway is one of the branches of phenylpropanoid metabolism downstream of the general phenylpropyl pathway. In this study, the downregulated expression of the ASMT in Arabidopsis leads to an increase in melatonin content [ASMT was significantly increased. The results suggest that melatonin is involved in the response of immature embryos to Agrobacterium tumefaciens. Moreover, transcriptome analysis revealed that a variety of plant hormones were involved in this process, and changes in POD, SOD, and other oxidases were detected, further indicating that Agrobacterium infection caused biological stress on immature embryos, inducing the occurrence of the melatonin synthesis pathway. In addition, we also detected a large accumulation of benzoic acid in immature embryos, and the functional groups of benzoic acid may induce tolerance to multiple stresses in plants. Combined with lignin biosynthesis, melatonin biosynthesis, benzoxazinoid biosynthesis, and benzoic acid biosynthesis, we used the phenylpropanin synthesis pathway as the pivot pathway to analyze the wheat embryos infected by Agrobacterium. A large amount of evidence suggests that wheat embryos will respond to oxidative stress, biological stress, and other related pathways after infection by Agrobacterium.Melatonin is ubiquitous in plants and promotes tolerance to adverse environmental conditions . In addi content . We founAgrobacterium EHA105 strain were preserved in our laboratory. Seeds were cultivated in pots  containing field-collected soil in a greenhouse with a relative humidity of 70 ± 5%, at 20 ± 2 °C and 16 ± 2 °C during the day and night, respectively. Moreover, air conditioning and supplementary lights were needed to ensure stable growing conditions. Good immature embryos of 2.0 and 2.5 mm in length along the axis were collected at the developmental stage from panicles about 14 days after anthesis, on an extremely clean bench. The infection with Agrobacterium tumefaciens was carried out in strict accordance with the protocol provided by Ishida et al.  after gold-spraying treatment [To observe the adsorption status of reatment .Physiological indicators were measured with reference to previous studies, and the data were collected and analyzed ,58,59,60E. coli DNA polymerase I , RNase H , and dUTP solution . An A base was then added, the sequencing connector was connected, and the fragments were purified using the AMPure XP beads to select cDNA fragments of 300 bp in length. Finally, we performed the 2 × 150 bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 , following the vendor’s recommended protocol. Reads obtained from the sequencing machines including raw reads containing adapters or low-quality bases were removed before downstream analyses.Total RNA was extracted using TRIzol reagent  following the manufacturer’s procedure. The total RNA quantity and purity were analyzed using a Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit , and high-quality RNA samples with RIN number > 7.0 were used to construct the sequencing library. After total RNA was extracted, mRNA was purified from total RNA (5 μg) using Dynabeads Oligo (dT) , with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature. Then, the cleaved RNA fragments were reverse-transcribed to create the cDNA using SuperScript™ II Reverse Transcriptase , which was then used to synthesize U-labeled second-stranded DNAs with https://daehwankimlab.github.io/hisat2/, version:hisat2-2.2.1, accessed on 10 August 2022) package [http://www.bioconductor.org/packages/release/bioc/html/ballgown.html, accessed on 10 August 2022) were used to estimate the expression levels of all transcripts and determine the expression abundance for mRNAs by calculating FPKM (fragments per kilobase of transcript per million mapped reads) values [The filtered reads were mapped to the wheat reference genome using the HISAT2 ( package . FPKM (f) values ,62. Gene) values ,64. The ) values ,66.Agrobacterium infection. The specific primers were designed using Primer 6.0 , according to the manufacturer’ s instructions.To verify the accuracy of transcriptome data, quantitative reverse-transcription PCR was performed on 9 DEGs altered after imer 6.0 . First-sTM RT reagent kit with gDNA Eraser  according to the manufacturer’ s instructions. Quantitative reverse-transcription PCR was conducted using the QuantStudio 3 Real-Time PCR system  with TB Green™ Premix Ex Taq™ II . The programmer performed the following steps: 10 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 31 s at 55 °C. The actin gene was employed as the reference gene for the experiments  was used for the reversed-phase separation. The column oven was maintained at 35 °C. The flow rate was 0.4 mL/min, and the mobile phase consisted of solvent A  and solvent B . The gradient elution conditions were set as follows: 0~0.5 min, 5% B; 0.5~7 min, 5% to 100% B; 7~8 min, 100% B; 8~8.1 min, 100% to 5% B; 8.1~10 min, 5%B. The injection volume for each sample was 4 μL.m/z) of samples with those from the database. The metaX software (http://metax.genomics.cn) was used to quantify and screen the differential metabolites. Principal component analysis (PCA) was used to normalize the metabolite profiling data. The differential metabolites were detected based on the following parameters: metabolites with VIP ≥ 1 and a fold change ≥ 2 or fold change ≤ 0.5. The different metabolites were subjected to data normalization.The acquired MS data pretreatments—including peak picking, peak grouping, retention time correction, second peak grouping, and annotation of isotopes and adducts—were performed using XCMS software (v3.22.0). Raw LC−MS data files were converted into mzXML format and then processed by the XCMS, CAMERA, and metaX toolbox implemented with R software (v4.2). The intensities of each peak were recorded, and a three-dimensional matrix containing arbitrarily assigned peak indices, sample names, and ion intensity information was generated. The online KEGG HMDB database was used to annotate the metabolites by matching the exact molecular mass data (A joint analysis of DEGs and DAMs was used to determine the degree of enrichment of the pathways. Pearson’s correlation tests with a PCC > 0.8 were used to examine the associations between gene expression and metabolite content, and the annotated metabolites and genes were then mapped to the KEGG pathways.SPSS Statistics 22.0 software was used for the data analysis. The difference between the two treatments was determined by one-way analysis of variance and Duncan’s multiple range test at the 0.05 and 0.01 significance levels. The data were displayed using Microsoft Excel 2016.Agrobacterium infection. Our results showed that immature embryos infected by Agrobacterium mainly activated energy- and stress-related signaling pathways, and in this process, redox substances such as SOD, POD, CAT, GSH, proline, and MDA were changed. We also identified several genes that play key roles in this process, including F5H and CCR. Overall, integrated transcriptomic and metabolomic analyses revealed that immature embryos respond to Agrobacterium invasion through changes in energy-metabolism-related pathways, shikimic acid pathways, phenazine synthesis pathways, and phenylpropanoid metabolic pathways; this is a complex signaling regulation process, and further studies should focus on how these candidate genes or metabolites are involved in the process.This investigation explains the response of immature wheat embryos to


In this review, we investigated the potential mechanism of Total Salvianolic Acid Injection (TSI) in protecting against myocardial ischemia reperfusion injury (MI/RI). To achieve this, we predicted the component targets of TSI using Pharmmapper and identified the disease targets of MI/RI through GeneCards, DisGenNET, and OMIM databases. We constructed protein-protein interaction networks by analyzing the overlapping targets and performed functional enrichment analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Our analysis yielded 90 targets, which were implicated in the potential therapeutic effects of TSI on MI/RI. Seven critical signaling pathways significantly contributed to TSI’s protective effects, namely, PI3K signaling, JAK-STAT signaling, Calcium signaling, HIF-1 signaling, Nuclear receptor signaling, Cell Cycle, and Apoptosis. Subsequently, we conducted a comprehensive literature review of these seven key signaling pathways to gain further insights into their role in the TSI-mediated treatment of MI/RI. By establishing these connections, our study lays a solid foundation for future research endeavours to elucidate the molecular mechanisms through which TSI exerts its beneficial effects on MI/RI. Ischemic heart disease persists as a prominent causative factor of mortality in cardiovascular disorders, encompassing both coronary heart disease and ischemic heart failure . The cliHowever, the very act of restoring blood flow (reperfusion) post-ischemia unwittingly contributes to an exacerbation of damage at the hallowed site of myocardial ischemia, leading to a discernible escalation in the infarct area and a subsequent, consequential impact on myocardial function. This intricate phenomenon, widely recognized as myocardial ischemia reperfusion injury (MI/RI), bequeaths its tribulations upon the afflicted cardiac tissues .Salvia miltiorrhiza, a herb renowned for its traditional use in treating cardiovascular diseases like coronary heart disease, atherosclerosis, myocardial infarction, and angina pectoris, belongs to the class of herbs known to stimulate blood circulation and alleviate blood stasis  is a water-soluble in MI/RI .Due to the rapid progress in bioinformatics and pharmacology, network pharmacology has emerged as a cost-effective approach for conducting pharmacological research for drug development. This methodology involves investigating the intricate relationships among compounds, proteins/genes, and diseases from a holistic network standpoint. It has garnered significant attention in drug discovery as it facilitates the exploration of potential “compound-protein/gene-disease” pathways .In this study, we aimed to investigate the underlying mechanism of TSI against MI/RI and provide valuable insights for treating MI/RI. We employed a network pharmacology approach to comprehensively analyze the interactions between TSI and its potential targets to systematically understand the molecular pathways underlying its anti-MI/RI effects to achieve this goal. Insights gained from this study will pave the way for further experimental studies and ultimately aid in developing new therapeutic strategies for treating myocardial infarction and reperfusion injury.The particular steps consist of the following : using lThe literature was consulted to determine the compound of TSI; three articles described the compound of TSI. 14 competc. Intercellular contacts, cell cycle processes, signal transduction, and metabolic pathways are biological processes in which PPI plays a significant role. It is essential to investigate the PPI of TSI with MI/RI for pharmaceutical efficacy. The intersection of disease targets and drug targets. We imported the targets obtained in step one into String (Version 11.5) (Protein-protein interaction (PPI) is a physical contact formed when two or more protein molecules interact via electrostatic forces, hydrogen bonds, on 11.5)  to generon 11.5) , deletedon 11.5) .2+) binding, transcription factor binding, and signaling receptor regulator activity.The PPI targets are imported into Metascape for Gene Ontology (GO) enrichment analysis. The GOs of the most abundantly enriched genes are rated, and the top 10 are selected see . The tophttps://www.genome.jp/kegg), as shown in Kyoto Encyclopedia of Genes and Genomes (KEGG) is an online database on genomic, enzymatic pathways, and biochemistry developed in Japan. To better understand the probable mechanism of TSI on MI/RI, we imported the PPI-acquired targets into Metascape for KEGG enrichment analysis to determine the pathways with which the targets are connected. Finally, 119 pathways were enriched, ranked according to logP, with the top 10 KEGG pathways displayed in in vitro and in vivo experiments, Peng Yu also revealed that Elabela could prevent cardiac fibrosis, increase mitochondrial function, and reduce apoptosis and oxidative stress via the PI3K/AKT signaling pathway in MI/RI (Phosphatidylinositol-3-kinase (PI3K) and the downstream target serine/threonine kinase (Akt) constitute a signaling pathway with critical roles in various physiological processes that promote cell survival in response to noxious external stimuli . In addiin MI/RI . See Fig2+, and the above effect will be eliminated by AG490 (JAK2/STAT3 pathway inhibitor) , signal transducer and activator of transcription proteins (STATs), and receptors  . JAK conhibitor) . Similarhibitor) . Muntasihibitor) . In addihibitor) . See Fig2+ to communicate and drive intracellular processes, often as a step in signal transduction. Ca2+ is a critical second messenger in regulating mitochondrial tasks and a key link in the coupling of excitation metabolism and excitation contraction in the heart. The inner mitochondrial membrane has a complex system of Ca2+ uptake and efflux channels and transporters that decode cytoplasmic Ca2+ signals and store Ca2+ in the mitochondrial matrix , located in the inner mitochondrial membrane, is the essential unidirectional channel responsible for Ca2+ influx into mitochondria, and the MCU regulates mitochondrial Ca2+ homeostasis  and m-calpain  are the major isoforms of calpain expressed in cardiac myocytes. During myocardial reperfusion, mitochondrial Ca2+ overload caused by MCU activates calpain, which causes MI/RI through different mechanisms, including increased membrane fragility and mitochondrial dysfunction  is an oxygen-sensitive transcription factor. It consists of a beta subunit  and an alpha subunit. Under normoxic conditions, HIF-1α is degraded via the ubiquitin-protease pathway . During The cell cycle is a series of processes that lead to dividing a single cell into two daughter cells. In cardiac myocytes, dysregulation of cell cycle control has been linked to MI/RI . The prointrinsic pathway and extrinsic pathway. The extrinsic pathway is activated by pro-inflammatory ligands  via plasma membrane receptors, and with the assistance of FADD (Fas-associated death domain protein), the TNF receptor family continuously collects procaspase-8 collected in the cytoplasm and a high density of procaspase-8 autocatalyzes the formation of caspase-8 which will trigger a caspase cascade reaction. In the intrinsic pathway, caspase-8 can mediate the hydrolysis of the BH3-only protein BID to form a truncated BID (tBID), promoting the release of cyt c and the assembly of apoptotic vesicles Apaf-1 and caspase-9. The intrinsic pathway usually requires the activation of BH3-only proteins of the Bcl family. When BH3-only protein activation reaches a certain amount, it can overcome the inhibition of Bcl-2 family members and promote the assembly of granule outer membrane Bax-Bak oligomers, where mitochondrial outer membrane permeabilization occurs, allowing the release of pro-apoptotic factors such as cyt c, which enters as pre-apoptotic substances in the cytoplasm, which together with Apaf-1 and caspase-9 form the apoptosome and cause the caspase cascade reaction (MI/R aggravates myocardial injury and leads to apoptosis of cardiomyocytes, which can lead to a decrease in cardiac function. The apoptosis process is divided into two phases: the initiation phase and the effector phase. The initiation phase is divided into two pathways: the reaction . The proreaction . p53-uprreaction . FOXO3a reaction . It was reaction . Xiangwereaction . See FigIn this study, we employed a network science approach to explore potential TSI and MI/RI targets. We performed functional enrichment analysis to elucidate the underlying mechanism of TSI’s effect on MI/RI. In addition, we extensively reviewed and discussed vital signaling pathways, including PI3K signaling, JAK-STAT signaling, calcium signaling, HIF-1 signaling, nuclear receptor signaling, cell cycle, and apoptosis, to lay a literature foundation for future studies on the mechanism of TSI in the treatment of MI/RI. However, it is essential to acknowledge that our study lacks experimental validation, and further investigation is required to confirm our findings. Specifically, measuring protein or mRNA expression levels of potential targets identified in our network analysis would provide crucial experimental evidence supporting our predictions. Ultimately develop more effective treatment strategies for MI/RI management.


Gandoufumu decoction (GDFMD) is a traditional Chinese medicine that has been widely used to treat Wilson's disease (WD) liver damage patients. However, its specific molecular mechanism currently remains unclear. Autophagy as a key contributor to WD liver damage has been intensely researched in the recent years. Therefore, the aim of this present study is to explore the effect of GDFMD on autophagy in WD liver damage, and the final purpose is to provide scientific evidence for GDFMD treatment in WD liver damage.  The molecular mechanisms and autophagy-related pathways of GDFMD in the treatment of WD liver damage were predicted using network pharmacology. Copper assay kit was used to determine copper content in serum. Enzyme-linked immunosorbent assay (ELISA) was utilized to quantify serum levels of liver enzymes and oxidative stress-related indicators. Hematoxylin-eosin (HE), Masson, and Sirius red staining were used for the characterization of liver pathological changes. Transmission electron microscopy, immunofluorescence, and Western blot analyses were used to evaluate autophagy activity. The impact of the GDFMD on typical autophagy-related pathway (PI3K/Akt/mTOR pathway) molecules was also assessed via Western blot analysis.  GDFMD effectively attenuated serum liver enzymes, oxidative stress, autophagy, and degree of hepatic histopathological impairment and reduced serum copper content. Through network pharmacological approaches, PI3K/Akt/mTOR pathway was identified as the typical autophagy-related pathway of GDFMD in the treatment of WD liver damage. Treatment with GDFMD activated the PI3K/Akt/mTOR pathway, an effect that was able to be counteracted by LY294002, a PI3K antagonist or Rapa (rapamycin), an autophagy inducer.  GDFMD imparted therapeutic effects on WD through autophagy suppression by acting through the PI3K/Akt/mTOR pathway. Wilson's disease (WD) is an inherited metabolic disease culminating in pathological progressive copper accumulation in the liver and other tissues and is known to be caused by a genetic defect in the ATPase copper transporting beta gene (ATP7B) . WD is a−/− rats as well as in those of WD patients. Moreover, in vitro experiments reveal that high cellular copper loads trigger autophagy [Autophagy is an omnipresent, highly conserved catabolic cellular recycling pathway and has been documented in organisms ranging from yeast to humans , 5. Seveutophagy . RegulatReynoutria multiflora (Thunb.) Moldenke, Lycium barbarum L., Panax notoginseng (Burkill) F.H.Chen, Curcuma aromatica Salisb., Smilax glabra Roxb., Bupleurum chinense DC, and Paeonia lactiflora Pall. Our group has demonstrated that the GDFMD could improve serological markers of liver function as well as the spleen pachydiameter value and liver ultrasound integral in patients with WD-mediated liver injury [Traditional Chinese medicine (TCM) has been trialed as a novel WD therapy for several years. Investigations on TCM highlighted its abilities to reduce hepatic fibrosis, enhance urinary copper excretion, and protect the kidneys and brain . A TCM pr injury . GDFMD hIn the past decade, with the development of bioinformatic methods, network pharmacology has become a dominant paradigm in investigating the multitarget pharmacological effects and complicated action mechanisms of TCM –16. Thishttps://tcmspw.com/tcmsp.php) database and related literature [https://www.uniprot.org/) database and Perl language were used to query gene targets corresponding to the active compounds of GDFMD [https://www.genecards.org/) database. The intersection between the targets of active compounds in GDFMD and WD liver damage-related genes was visualized using VENNY 2.1 (https://bioinfogp.cnb.csic.es/tools/venny/) online platform.The active compounds of each herb in the GDFMD formula were obtained by searching the Traditional Chinese Medicine Systems Pharmacology  and Cytoscape software (version 3.8.2) [The intersection targets were used to establish a protein-protein interaction (PPI) network by the STRING database (n 3.8.2) , 24. TheP values for the biological processes (BP), cellular components (CC), and molecular functions (MF) were selected to plot circle graphs, and the top thirty items with the smallest P values for the KEGG signaling pathways were selected to plot bubble charts. As P represents the significance of enrichment, the screening condition was limited to P < 0.05.To clarify the possible biological functions and key pathways of GDFMD in the treatment of WD liver damage, the R software (version 4.2.1) was used to perform ID conversion on the names of the intersection targets, following which Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out. Moreover, the top ten items with the smallest Reynoutria multiflora (Thunb.) Moldenke, 20 g of Lycium barbarum L., 3 g of Panax notoginseng (Burkill) F.H.Chen, 12 g of Curcuma aromatica Salisb., 12 g of Smilax glabra Roxb., 12 g of Bupleurum chinense DC, and 15 g of Paeonia lactiflora Pall. All herbs were supplied by the pharmacy of the First Hospital Affiliated to Anhui University of Chinese Medicine . The medicinal herb mixture was immersed in water (10 times the weight of medicine) for 1 h, boiled twice for 1 h, and then concentrated to 0.2/mL to obtain GDFMD suspension [The GDFMD comprises of 4 g of spension .Atp7btx−J/J and control C3HeB/FeJ mice were procured from the Experimental Animal Center of the Jackson Laboratory . All mice weighed between 20 and 40 g and were reared in specific-pathogen free (SPF) conditions in temperature-controlled standardized cages under a 12 h : 12 h light : dark cycle at the Academy of Life Sciences of Anhui Agricultural University . Animals were grouped into 5 cohorts (n = 10 per group) as follows: model, Gandoufumu , and penicillamine (PCA) cohorts .C3HeB/FeJIntragastric administrations of 0.2 mL/10 g normal saline were given to the control and model groups. Mice in the PCA and Gandoufumu groups were treated with daily doses of 0.1 g/kg PCA and GDFMD , respectively, for a total of 28 successive days. At the end of the treatment period, all mice were anesthetized with sodium pentobarbital , and venous blood was collected from the retroorbital sinus, and liver tissue was excised and rinsed with 0.9% NaCl solution. The Animal Ethics Committee of Anhui University of Chinese Medicine approved all protocols regarding animal care and experiments that were used in this study (2018AH-08).μm sections for staining with hematoxylin-eosin (HE) and Masson's trichrome. Structural liver changes were then visualized under an optical microscope . The liver damage score was assessed by three separate pathologists based on the following: 0: no damage; 1-3: mild damage; 4-6: intermediate damage; 7-9: severe damage; and 10: damage of liver architecture [Liver samples were treated with 4% formaldehyde prior to production of 5 itecture . Sirius Serum samples were prepared, and serum copper content was determined according to the copper assay kit instructions (complexation colorimetry). The kit was purchased from the Jiancheng Biotechnology Research Institute .2 in a humidified tank at 37°C.HepG2 cells were purchased from the Wuhan Pu-nuo-sai Life Technology Co. Ltd. , maintained in with foetal bovine serum  (20%), and supplemented minimal essential medium , which also contained penicillin/streptomycin (1%). Cells were cultured at 5% COμm microporous membrane . The resultant GDFMD-containing serum was maintained at −80°C until further use. Serum of 10% GDFMD concentration was used for further experiments [Treated mouse serum samples were prepared by extracting blood from the abdominal aorta, which was then centrifuged for 20 minutes at 3500g. The supernatant was incubated for 30 min in a 56°C water bath before being filtered with a 0.22 eriments .2) in 100 μM was used as the model group [For studying the modulatory effect of GDFMD in HepG2 cells, copper chloride  and sectioned  for observation under a transmission electron microscope .Cells were stained for immunofluorescence on coverslips. Cells were first fixed and permeablized before they were exposed to primary antibodies against LC3A/LC3B (1 : 100) overnight at 4°C and secondary antibodies at 37°C for 50 minutes. Slides were rinsed with PBS thrice and counterstained with DAPI. A positive fluorescence microscope  was used to visualize DAPI fluorescent signals.μg total protein was first electrophoresed with a 5% SDS-PAGE prior to Western blotting that was performed based on standard protocols. β-Actin protein was used as a reference. Primary antibodies were used as follows : Anti-PI3K, anti-phospho-PI3K, anti-Akt, anti-phospho-Akt (1 : 2000 dilution), anti-mTOR, anti-phospho-mTOR, anti-LC3A/LC3B, anti-p62, anti-beclin-1, β-actin, goat anti-rabbit, and goat anti-mouse-IgG HRP. All antibodies were purchased from Cell Signaling Technology Inc. . The ImageJ software was used to analyze band densities .RIPA lysis buffer was used for total protein extraction from liver tissues and HepG2 cells. Protein concentrations were measured with a BCA protein assay kit. 10 The levels of ALT, AST, ALB, TP, ROS, SOD, MDA, and GSH-Px were quantified by ELISA based on protocols stipulated by the manufacturer.P values less than 0.05 were interpreted to be statistically significant.All quantitative data is depicted in terms of mean ± standard deviation (SD) and processed using SPSS version 22.0 . Significant differences were analyzed using the one-way analysis of variance (ANOVA), and A total of 67 active compounds of GDFMD were screened from the TCMSP database and related literature corresponding to the 254 potential targets. After converting the drug targets to standard gene names using UniProt database and Perl language, a total of 215 gene targets of GDFMD were obtained. In addition, a total of 687 WD liver damage-related targets were retrieved from the GeneCards database.Using VENNY 2.1 online platform, a total of 55 intersection targets between GDFMD and WD liver damage were presented . PPI netA total of 1935 GO biological function items were obtained. The BP mainly involved response to extracellular stimulus, response to xenobiotic stimulus, and response to metal ion; CC mainly involved membrane raft, vesicle lumen, and membrane microdomain; MF mainly involved nuclear receptor activity, ligand-activated transcription factor activity, and signaling receptor activator activity . A serieTo explore the impact of GDFMD on the liver function in mouse models of WD, we examined the serum levels of ALT, AST, TP, and ALB. The ALT and AST levels were lowered, while the levels of TP and ALB were raised in the GDFMD and PCA groups . Next, lNext, we evaluated the impact of GDFMD on autophagy in mouse models of WD. Based on immunofluorescence staining, there appeared to be a notable increase in immunostaining intensity of LC3 and Beclin-1 protein in liver tissues in the model group in contrast to controls; however, GDFMD and PCA treatments appeared to attenuate these changes . Similarin vitro experiments were conducted to determine the effect of GDFMD on oxidative stress. With reference to 2 stimulation increased levels of MDA while decreasing GSH-Px and SOD activities in HepG2 cells. GDFMD exposure appeared to mitigate this effect.Further Autophagosomes were visualized in the HepG2 cells from each group using a transmission electron microscope. The number of autophagosomes was obviously increased in the model groups, while decreased after treatment with GDFMD or 3-MA, whereas increased in the LY294002 or Rapa group .Next, we observed LC3 protein expression in HepG2 cells using immunofluorescence analysis. LC3 proteins appear as an intracytoplasmic collection of red spots and was greatly raised in the model group in contrast to the control group. LC3 protein production appeared to be suppressed upon exposure to GDFMD and 3-MA, and further pretreatment with LY294002 or Rapa attenuated the inhibitory effect of GDFMD .2 exposure triggered suppression of PI3K, Akt, and mTOR phosphorylated protein levels. These effects were reversed with GDFMD which was in turn reversed again through the addition of LY294002 or Rapa , Lycium barbarum L., Panax notoginseng (Burkill) F.H.Chen, Curcuma aromatica Salisb., Smilax glabra Roxb., Bupleurum chinense DC, and Paeonia lactiflora Pall., which has been reported to be effective in treating WD-related liver disease [WD is a genetic disease characterized by aberrant copper metabolism that presents with psychiatric, neurological, and hepatic derangements. Although clinical manifestations of WD vary widely, the most common ones are those of liver disease. Reports demonstrate that patients with WD often present with hepatic copper accumulation coupled with cirrhosis . The rec pathway . Similar3A cells . The GDF disease , 31, 32.2-induced oxidative stress in HepG2 cells appeared to be alleviated by GDFMD. This compound also reduced autophagy through modulation of the PI3K/Akt/mTOR pathway activity.In the present study, network pharmacology-based approaches were used to identify the active compounds in GDFMD and the interactions between the GDFMD and WD liver damage-related genes. The further analysis  demonstrated that GDFMD plays an active role in the treatment of WD liver damage. The results of subsequent experiments show that the hepatoprotective effect of GDFMD in WD liver damage is brought about by inhibiting autophagy, which manifests as a lesser degree of histopathological liver damage, improved liver enzymes, reduced oxidative stress, and decreased serum copper content. In addition, our results uncovered that CuClAutophagy is a lysosomal self-digestion phenomenon that is vital in development, differentiation, and tissue remodeling across various organisms. A plethora of liver diseases has been shown to demonstrate deranged autophagy processes . Autophavitro experiments [in vitro. CuCl2 promoted oxidative stress and autophagy of HepG2 cells by blocking the PI3K/AKT/mTOR pathway, while treatment of GDFMD reversed this effect. The antiautophagy effect of GDFMD was antagonized by LY294002 or Rapa.The PI3K/Akt/mTOR pathway is notable for regulating autophagy in mammalian cells . It appeeriments , 41. SimIn conclusion, the current investigation demonstrated that GDFMD significantly protects against WD liver damage by inhibiting overwhelming cellular autophagy through activation of the PI3K/Akt/mTOR signaling pathway.


Huangqi Guizhi Wuwu Decoction (HGWD) has a definite effect on neuropathic pain (NP), whereas the specific mechanism has not been elucidated. The components and targets in HGWD were collected and identified through System Pharmacology Database . Genecards and Online Mendelian Inheritance in Man databases were used to search for NP‐related genes. The Venn diagram was drawn to get the intersection target. Cytoscape 3.8.0 software was used to construct the compound‐disease‐target‐pathway networks. STRING database was applied to analyze protein–protein interaction of potential targets. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were used to identify the function of genes related to NP. Finally, molecular docking was performed to visualize the binding mode and affinity between proteins and active ingredients. According to the intersection target of the Venn diagram, the network graph is constructed by Cytoscape and the results show the five compounds, β‐sitosterol, (+)‐catechin, quercetin, Stigmasterol, kaempferol, and 15 genes  were highly correlated with NP. KEGG and GO of 15 genes results that TNF, IL‐17 and MAPK signaling pathway were Significantly related to the pathological mechanism of NP. Molecular docking showed that core genes in this network were IL‐6 , ICAM1 , and CASP3 . This study found that the five active compounds, three core genes, and three signaling pathways may be the key to the treatment of NP by HGWD. The intersection genes of Huangqi Guizhi Wuwu Decoction (HGWD) and neuropathic pain (NP) were analyzed by the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology to obtain the relevant pathway network map, and then the key genes in the signaling pathway were molecularly docked to explain the potential effect of HGWD in the treatment of NP. OB is the most momentous parameter in pharmacokinetics, which is used to reflect the speed, degree, and dosage ratio of drugs entering the blood circulation.2.3The identified target genes of the active ingredients were collected from the UniProt2.4The treatment target gene information of NP comes from the GeneCards database2.5A Venn diagram was created using the R software to show the overlap between the effective molecule and disease‐related genes. Thus, the bioactive compounds in the HGWD formula in NP treatment were obtained, and their potential targets were identified.2.6A pharmacological network is composed of nodes and edges. The entities constituting the network nodes are the networks of five TCMs of HGWD and their bioactive compounds, related targets, and related diseases. Cytoscape, an open‐source Java software,2.7In addition, the KEGG pathway2.8The STRING database2.9To further elaborate on the interactions between molecules  and to predict their binding mode and affinity, we selected five active compounds and three targets for molecular docking. From the Protein Data Bank database, we collected the three‐dimensional (3D) structure of the protein; the two‐dimensional (2D) structure of the ligand molecule was downloaded from the PubChem database; and ChemBio 3D software was used to calculate, minimize the molecular energy, and store as a 3D structure. The receptor protein was performed dehydration and removal ligand in Chimera 1.15 software, and executed hydrogenation and charge calculation with Autodock software. Then, setting the active pocket site of small molecules ligand binding, SeeSAR 11.2.0 was applied to, respectively, dock three proteins and five active compounds.33.1HGWD is composed of five kinds of TCMs , each Chinese medicine contains dozens or even hundreds of compounds. Through OB and DS, 790 compounds were screened by the ADME model Table ; after r3.2Using the UniProt database, 63 compounds contained 112 genes, of which 41 were potential target genes, that is, the intersection between 63 compounds and disease‐related genes ‐catechin, quercetin, stigmasterol, and kaempferol and the centrical pathways that MAPK, IL‐17, and TNF signaling pathways, which related to central genes such as IL‐6, ICAM1, and CASP3. According to the analysis results, it is speculated that HGWD may play a role in treating NP and provide a reference for subsequent experimental studies to further verify its mechanism of action.Bo‐Yan Luo wrote the manuscript, Hong‐Su Zhou and Qiu‐Xia Xiao helped in revising the manuscript, and Yu‐Qi He conceived and reviewed the manuscript. All authors had read and approved the final manuscript.The authors declare no conflicts of interest. Qiu‐Xia Xiao is a reviewer of Ibrain but is not involved in the peer review process of this article.Not applicable.


Shanyaotianua decoction (STT), a typical traditional Chinese medicine prescription, positively affects psoriasis, although its molecular targets remain unknown. To elucidate its molecular mechanisms, a combination of network pharmacology, bioinformatics analysis, and drug similarity comparisons were employed. Participants were separated into 3 groups: non-lesional (NL), lesions after medication (LM), and psoriasis lesion groups (LS). Based on the Gene Ontology/kyoto encyclopedia of genes and genomes enrichment analyses, the key targets were mainly enriched for biological processes  with the relevant pathways , thus identifying the possible action mechanism of STT against psoriasis. Target prediction for 18 STT compounds that matched the screening criteria was performed. Then, the STT compounds were intersected with the differentially expressed genes of the psoriatic process, and 5 proteins were potential targets for STT. Based on the open-source toolkit RDKit and DrugBank database, and through molecular docking and drug similarity comparisons, spinasterol, diosgenin, and 24-Methylcholest-5-enyl-3belta-O-glucopyranoside_qt may be potential drugs for psoriasis.Psoriasis is an immune-mediated inflammatory skin disease with a complex etiology involving environmental and genetic factors. Psoriasis patients often require long-term treatment.  Epidemiological surveys indicate a global incidence of approximately 2% and a Chinese population incidence of 0.5%.,3 Previous studies have implicated abnormal expression of psoriasis-susceptible genes, autoimmune disorders, obesity, and dysregulation of multiple inflammatory signaling pathways in the development of psoriasis. However, the underlying pathogenesis remains unclear. For patients with mild psoriasis, topical agents, including corticosteroids, vitamin D analogs, calcineurin inhibitors, and keratolytics, remain the mainstay of treatment. Although Western medicine has a specific curative effect, the safety of these immunotherapies poses significant concern. Traditional Chinese medicine (TCM) provides advantages including prolonged remission, stable conditions, reduced recurrence, and lower toxicity and side effects.–8 Nonetheless, the precise mechanisms by which TCM combats psoriasis have yet to be fully elucidated, limiting its widespread adoption.Psoriasis is an easily diagnosed common skin disorder; however, it is refractory to treatment and prone to recurrence.,9–12 Heat-clearing and blood-cooling methods are commonly employed in TCM clinical treatment for psoriasis.Shanyaotianhua decoction (STT) is a fundamental formula in Chinese medicine frequently used for psoriasis treatment. It consists of 2 representative traditional Chinese herbal medicines, namely the dry root of Trichosanthes kirilowii Maxim  and Dioscorea opposita . These herbs exert heat-clearing and toxicity-removing effects, cool the blood, and disperse blood stasis.,9–12 Skov et al have highlighted that staphylococcal subclinical infections, as well as upper respiratory tract streptococcal infections, are among the causes of psoriasis. Phellodendrine, a component of THF, exhibits significant broad-spectrum antibacterial effects, effectively inhibiting staphylococci and enhancing the clinical treatment outcomes for patients. Psoriasis is associated with abnormalities in essential fatty acid metabolism, lymphokine release, free radical generation, and lipid peroxidation.,16 Trillin, a secondary metabolite identified in Dioscorea spp., may alleviate psoriasis symptoms in patients by increasing superoxide dismutase (SOD) activity and reducing lipid peroxidation and oxidative stress.,18 However, the scientific basis and potential pharmacological mechanisms of STT in psoriasis treatment remain unclear and require further investigation.,20According to TCM, psoriasis is characterized by 3 predominant syndromes: blood heat, blood stasis, and blood dryness. In this study, multiple algorithms and network-based computational methods were employed to predict the active components, identify various drug targets, and establish core networks of targets and elements for the treatment of psoriasis using STT. Network pharmacology and molecular docking analyses were conducted to elucidate potential mechanisms of STT and lay the groundwork for further investigation.Network pharmacology, aligning with the “holistic” concept of TCM, utilizes a “multi-component, multi-target, multi-pathway” network construction approach. It has gained widespread application in exploring the action mechanisms of Chinese medicine pairs and compound drugs, particularly in target prediction.https://www.ncbi.nlm.nih.gov/geo). Non-lesion (NL) was used as a reference for differential expression analysis between the NL-psoriasis lesion (LS) groups. In total, 780 differentially expressed genes (DEGs) were identified, of which 532 were up-regulated and 248 were down-regulated. The possible occurrence of batch effects was ruled out . STT compounds and target proteins were obtained from the TCM systems pharmacology database and analysis platform (TCMSP) (https://tcmspw.com/) and Pharmmaper server, respectively.We obtained the corresponding clinical data and RNA-seq data of skin diseases (including 269 skin tissue samples) from the Gene Expression Omnibus dataset. The 2 cohorts used as validation datasets (GSE30999 and GSE53552) were downloaded from the Gene Expression Omnibus database  in all 3 possible comparisons -LS, and NL-LM) were considered psoriasis-specific genes.Differential expression analysis was conducted using the R package “limma.” The screening conditions for the DEGs were |log2FoldChange| > 2.0 (NL-LS), |log2FoldChange| > 1.0 (LM-LS), and P- and q-values of <.05 were considered significantly enriched. For psoriasis, GO and KEGG enrichment analyses were based on up-and down-regulated genes. For STT, “org.Hs.e.g.db” was used to convert the target protein into a gene, followed by GO and KEGG enrichment analyses. The required GMT file was downloaded from the Molecular Signatures Database (MSigDB), and gene set enrichment analysis was performed using the R package “clusterProfiler” .Gene Ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed using the R packages “clusterProfiler,” “enrichplot,” and “ggplot2.” Only terms with  The STT compounds that met the screening criteria are shown in Supplementary Table 1, http://links.lww.com/MD/J579.All the chemical ingredients of STT were obtained from TCMSP. STT is composed of 2 TCM herbs, THF and SY. The active STT compounds were mainly filtered with oral bioavailability, drug-likeness, and Caco-2 permeability, whereas STT compounds with oral bioavailability ≥20%, drug-likeness ≥0.18, and Caco-2 permeability ≥−0.04 were preserved.http://lilab.ecust.edu.cn/pharmmapper/) utilized the compounds’ pharmacophore as the probe molecule recognition group to predict target proteins. A stringent threshold (z-score = 1.0) was selected, and all protein targets above this threshold were extracted for further analysis. Existing drug therapeutic targets were retrieved from the DrugBank database (https://go.drugbank.com/).Cytoscope 3.7.0 software was employed to construct the active compound-target network, and compounds with a larger number of nodes were selected for key analysis. The STT compound 3D structure was acquired from TCMSP. The PharmMapper service  was used to build compound descriptors and PubChem fingerprints (https://pubchem.ncbi.nlm.nih.gov) and to calculate the structural similarities between compounds.The “full database” document was downloaded from the DrugBank database,https://www.rcsb.org/). RMSD and RMSF values were calculated using the Python smartools VMD. The structure of the core gene protein was dehydrated and hydrogenated, and pymol 2.3 was used to insert hydrogen atoms. The targets were set to rigid and saved in a PDBQT file format using AutoDock Tools 1.5.6. The Vina software was applied to do molecular docking. Based on the top minimal binding energy of each target, STT compounds were chosen for further analysis of their binding mode, binding affinity, and critical interactions using pymol2.3.According to the results from Network analysis of active STT compounds, the 3D structure of proteins: serum S100 calcium-binding protein A9(S1000A9) , GM2 activator protein (GM2A) , remote electrical neuromodulation (REN) , macrophage elastase (MMP12) , and Retinol binding protein 4 (RBP4)  were selected and downloaded from the Protein Data Bank (PDB) database (http://links.lww.com/MD/J580). Figure http://links.lww.com/MD/J581 shows DEG expression in NL-LM-LS.NL was used as a reference for differential expression analysis between the NL-LS groups. In total, 780 DEGs were identified, of which 532 were up-regulated and 248 were down-regulated . In total, 137 GO entries of up-regulated genes, including 134 biological processes (BPs) and 3 cellular components, and 560 GO entries of down-regulated genes, including 462 BPs, 79 molecular functions (MFs), and 19 cellular components, were obtained.The degrees of NL-LS progression were categorized according to the up-and down-regulated expression, and GO enrichment analysis was performed ( The enriched GO-BP terms of up-regulated genes (p.adj < 0.01) are primarily associated with fatty acid metabolic processes, lipid catabolic processes, and cornification  were mainly mapped to receptor ligand, signaling receptor activator, cytokine activities, cytokine receptor binding, and other immune stress-related pathways, similar to the BP results  of down-regulated genes in NL-LS mainly included type I interferon (IFN) signaling pathway, response to virus, skin development, and cellular response to type I IFN Fig. A, suggesion Fig. B. This rlts Fig. E. Down-regulated genes were enriched in IL-17 signaling pathway, cytokine-cytokine receptor interaction, influenza A, and nucleotide oligomerization domain like receptor signaling pathways  cells and an important role of IL17 appears to be the regulation of local inflammation through the upregulation of other pro-inflammatory cytokines and chemokines. Recently, the FDA approved multiple highly effective psoriasis therapies that disrupt interleukin-17  and interleukin-23 (guselkumab and tildrakizumab) signaling in the skin, all with promising therapeutic results. Cytokine-cytokine receptor interaction and nucleotide oligomerization domain like receptor signaling pathways regulate proliferation and differentiation.This is consistent with the clinical features described in other published studies. KEGG enrichment analysis results were consistent with that of GO enrichment, enhancing the reliability of GO enrichment results and providing more evidence that inflammation, hyperproliferation and lipid metabolism disorders as the cause of LS.Up-regulated gene expression enrichment (p.adj < 0.05) was observed in the peroxisome proliferator-activated receptor (PPAR) signaling pathway in the NL-LS group Fig. A. PPARs ays Fig. B. An exthttp://links.lww.com/MD/J579) and the statistical analysis of the predicted targets are shown in Figure The interaction between the predicted targets and drug-like compounds  revealed significant enrichment in inflammation-related pathways such as the Janus kinase/signal transducers and activators of transcription (JAK-STAT) and adipocytokine signaling pathways, Th17 cell and osteoclast differentiation, leishmaniasis, parathyroid hormone synthesis, secretion and action, staphylococcus aureus infection, and necroptosis, among others  has been identified as an elastolytic metalloproteinase secreted by inflammatory macrophages; the levels of MMP12 messenger RNA (mRNA) were 17-fold higher in psoriatic lesions than in NL skin. RBP4, a protein in the lipocalin family, has been associated with various metabolic complications, such as metabolic syndrome, obesity, and insulin resistance, and is closely associated with psoriasis. Previous clinical studies have identified increased serum levels of RBP4 in patients with psoriasis compared to controls, suggesting that RBP4 may have a protective effect against chronic inflammation and psoriatic complications. Therefore, these may be the potential targets by which STT combats psoriasis.Five target proteins were identified by intersecting the predicted STT target with the intensity of all psoriatic stages Fig. A and B. Small-molecule compounds Fig. A, such aThe RDkit tool ranked the small molecular compounds in the DrugBank database and recommended the top 10 drug compounds with similarities. The drug similarities included spinasterol and lanosterol 0.83139), cholesterol (0.79056), beta-sitosterol (0.79056), 25-Hydroxycholesterol (0.77456), and androstenediol (0.73333)  to systematically elucidate the molecular and pharmacological mechanisms underlying the amelioration of psoriasis by STT. The network pharmacology results revealed that the primary targets of the active ingredients in STT were S100A9, GM2A, REN, MMP12, and RBP4, all of which were primarily associated with psoriasis. The potential therapeutically active compounds in STT include spinasterol, diosgenin, and 24-M5. These findings may provide the basis for the therapeutic effectiveness of STT. However, further experiments and additional clinical trials are necessary to validate the above findings.In conclusion, this study is the first to perform gene enrichment analysis, target prediction, network analysis, integrate active components, and utilize a network pharmacology and molecular docking techniques approach (flowcharts are shown in Supplementary Fig. 3, Formal analysis: Chen Yue.Project administration: Chen Yue.Software: Jiahao Feng.Supervision: Aili Gao.Writing – original draft: Chen Yue.Writing – review & editing: Aili Gao.