Nelumbo nucifera Gaertn) is a common medicinal-food dual-use vegetable. In this study, the effects of lotus root extract on acute alcoholism were investigated.Lotus root  was used to construct protein-protein interaction network graphs. Gene ontology (GO) functional, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes common to lotus root and alcoholism by Metascap database. Molecular docking simulations were performed using AutoDock 1.5.6 software. Animal experiments verified the relieving effect of lotus root extract on acute alcoholism after intervention.The Walle-Hoch method was used to determine the ADH activity of lotus root extracts in vitro (18.87%). The 433 compounds of lotus root methanol extract were identified by UPLC-QTOF-MS/MS based metabolomics analysis. Bioinformatics analysis indicate that there were 224 intersectioning targets between lotus root extract and acute alcoholism. KEGG enrichment analysised shows that lotus root extract may play a role in treating acute alcoholism by intervening with the neuroactive ligand-receptor interaction pathway. The protein-protein interaction network (PPI) analysis found that HSP90AA1, MAPK1 and STAT3 played a key role in lotus root extract-modulated PPI networks. Molecular docking showed that -dihydrodihydrodipine cypressol had the best binding ability with MAPK1. Experiments in mice indicate that lotus root extract improved the activity of liver alcohol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), increase glutathione (GSH) and reduce malondialdehyde (MDA) levels, decrease glutamate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (AKP) in the serum of mice with acute alcoholism, and accelerate the metabolic rate of alcohol after drinking. This study reveals the mechanism of lotus root to alleviate acute alcoholism, which provides a basis for further research on functional foods using lotus root and offers new possibilities for the treatment of acute alcoholism.Results indicated the methanol extract of lotus root showed the highest activation rate of ethanol dehydrogenase The results of the current study showed that the methanolic extract of lotus root had the highest activation rate of ethanol dehydrogenase. Network pharmacology results suggest that lotus root extract may play a role in the treatment of alcoholism by regulating signaling pathways, such as neuroactive ligand-receptor interactions, as well as biological processes, such as regulation of secretion, regulation of ion transport, response to lipopolysaccharides, and response to alcohol. Animal experiments confirmed the therapeutic effect of lotus root on acute alcoholism mechanistically through activation of alcohol catabolic enzyme, reduction of oxidative stress and protection of liver function. With the increase in alcohol consumption worldwide, alcohol has been recognized as a cause of many liver diseases. The International Institute for Humanitarian Environment has listed alcohol consumption as one of the most essential factors endangering human health . Acute aNelumbo nucifera Gaertn) is China's largest and most productive aquatic vegetable. Lotus root mainly contains polyphenols, triterpenes, flavonoids, steroid compounds, and other active substances  and Tandem mass spectrometry (MS/MS) to analyze the main chemical components of the lotus root extract to provide more comprehensive information for its specific active components. In this paper, based on the analysis of the active ingredients of lotus root, we used the bioinformatics research method to establish the active ingredient-target-disease-pathway from the holistic and systematic interactions among chemical components, targets, and diseases by using the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), PubChem database, GeneCard database, and other bioinformatics platforms. The network diagram of active ingredient-target-disease-pathway was established, and the main active ingredients and core targets were docked and validated by molecular docking technology. Finally, we elaborated on the lotus root for the mechanism of action of rescuing acute alcohol poisoning.Lotus roots were purchased from a local farmers' association organization in Hangzhou, Zhejiang province, China. Fresh lotus root samples were placed in a mortar and pestle and ground into powder form. Several lotus root samples were weighed to 20 g each, and distilled water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, anhydrous ethanol, 20% methanol, 40% methanol, 60% methanol, 80% methanol, and methanol were added at a material-to-liquid ratio of 1:10 (g:mL), respectively. The extraction method was assisted by ultrasonic extraction with power of 800 W and extraction temperature of 50°C for 1 h. The solvent extracts of lotus roots with different volume fractions were obtained by filtration. The extract was concentrated with a rotary evaporator and dried into a lyophilized powder using a vacuum freeze dryer  and stored at −20°C for backup.in vitro. In a test tube, 0.375 mL of sodium pyrophosphate buffer (pH = 8.8), 0.25 mL of 0.027 mol/L NAD+, 0.125 mL of 12% ethanol, and 0.025 mL of different concentrations of lotus root extract was added in turn, mixed well and then incubated for 5 min at 25°C in a sealed incubator. After that, 0.025 mL of 3 U /mL ADH solution was added to the test tube. After shaking well, the solution was measured using the absorbance A at 340 nm by a microplate reader , and the absorbance value was read every 10 s for 10 min, and the data were recorded, while the blank group was replaced by 0.025 mL of the sample solution with the solvent of the extract, and the rest of the steps were unchanged.The Walle-Hoch method  was used1 was the sample group enzyme activity, E0 was the blank group enzyme activity, A was the value added of absorbance at 340 nm per 1 min for the initial linear part of the reaction, Ew was the enzyme content in the enzyme solution, 0.8 was the total volume of the reaction solution, 6.2 was the .Where E was the ADH enzyme activity, Ein vitro activation rate of ethanol dehydrogenase was measured by different solvents of lotus root extracts, and the best active extract was screened for subsequent experimental analysis.The After thawing the samples from the refrigerator at −80°C, mixed them with vortex for 10 s. Took 50 mg of the sample after mixing, and placed 2 mL of the centrifuge tube. 1,200 μL 80% methanol internal standard extract was added. Scroll for 3 min Centrifuge  for 10 min. The supernatant was filtered with a microporous filter membrane (0.22 μL) and stored in a sample flask for UPLC-Q-TOF-MS/MS. The model number of UPLC and MS/MS is Nexera X2 (SHIMADZU), and Biosystems 4500 QTRAP (AppliedBiosystems) .* 100 mm). The mobile phase A was ultrapure water (with 0.1% formic acid) and phase B was acetonitrile (with 0.1% formic acid). The elution gradient: 5% of phase B at 0.00 min, linearly increasing to 95% of phase B at 9.00 min and maintaining at 95% for 1 min, decreasing to 5% of phase B at 10.00–11.10 min and equilibrating at 5% until 14 min. The flow rate was maintained at 0.35 mL/min, the reaction column temperature was 40°C, and the injection volume was 4 μL.Chromatographic conditions: The chromatographic column was an Agilent SB-C18 model  5,500 V (positive ion mode)/-4,500 V (negative ion mode). Ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) are set at 50, 60, and 25.0 psi, respectively. Monitoring of a specific set of MRM ion pairs at each stage based on metabolites eluted during each period.https://www.tcmsp-e.com/) for screening, with settings of oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18 were used to screen the eligible compounds as the effective compounds in lotus root  was used to obtain the Canonical SMILES name of the active compound of lotus root. The compound Canonical SMILES name was entered into SwissTargetPrediction database (http://old.swisstargetprediction.ch/), and the species origin was restricted to humans to predict the possible targets of the compound. Cytoscape 3.8.2 software (PubChem database  (https:/database  (http://software  was usedhttps://omim.org/); GeneCards database (https://www.genecards.org/); Drugbank database (https://www.drugbank.com/); PharmGkb database (https://www.pharmgkb.org/); DisGeNET database (https://www.disgenet.org/) to obtain disease targets. The disease targets obtained from each database were merged, and duplicate targets were removed to obtain the targets related to alcoholism. The target names were converted to protein names by uniprot database (https://www.uniprot.org/), and the species' origin was limited to humans. The disease targets obtained above were intersected with the active ingredient targets of lotus root obtained by screening in 2.3.2 to obtain the active targets of lotus root for the treatment of acute alcoholism.We used “alcoholism” and “acute alcoholism” as search terms and entered them into the Online Human Mendelian Genetic Database   was used to construct protein-protein interaction network graphs. The targets of lotus root for the treatment of alcoholism were imported into the database. The scoring value was set at a high confidence level of 0.900 to obtain the interaction relationships among the targets. The results were saved in TSV format and imported into Cytoscape 3.8.0 software, and the “Network Analysis” function was used to analyze and map. The interaction network was obtained by removing duplicate and isolated edges, using node size and color settings to reflect the size of the degree of network connectivity and edge thickness settings to reflect the size of the binding fraction.STRING database  (https:/The CytoNCA plugin in Cytoscape 3.8.0 software was used to filter the core targets in the PPI network. The node degree (DC), betweenness (BC), closeness (CC), network (NC), eigenvector (EC), and local average connectivity-based centrality was calculated. Eigenvector centrality (EC) and Local Average Connectivity-based method (LAC) is used to filter the important targets in the interacting topological networks.https://metascape.org), GO enrichment analysis including biological process (BP), molecular function (MF), and cellular composition (CC). P < 0.05 was considered statistically significant, and a minimum number of enriched genes of 5 and enrichment factor >1.5 were used as screening criteria. The top20 GO functional categories and significantly enriched KEGG signaling pathways were selected. The network diagram of the lotus root component-target-pathway was constructed with Cytoscape 3.8.0 software.Gene ontology (GO) functional, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes common to lotus root and alcoholism by Metascap database  (https:/https://www1.rcsb.org/) and saved as PDB files. The core components of lotus root were downloaded as SDF files of the 3D structures of small molecules in the PubChem database. And the downloaded small molecules were energy minimized using Chem3D software and saved as mol2 files. Before docking, polar hydrogen atoms were added to the target protein using PyMOL software . The animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.After all the mice adapted to the controlled conditions for 1 week, mice were randomly divided into five groups of eight each. Groups were named and treated as follows: (1) control group; (2) alcohol model (Alcohol) group, 0.85% normal saline oral administration once daily; (3) high concentration lotus root extract group (HD), 200 mg lotus root extract per kg body weight oral administration once daily; (4) middle concentration lotus root extract group (MD), 100 mg lotus root extract per kg body weight oral administration once daily; (5) low concentration lotus root extract group (LD) group, 50 mg lotus root extract per kg body weight oral administration once daily. Before ethanol treatment, all groups were fed once a day for seven consecutive days. On the 8th day, mice in each group were gavaged for 30 min. The mice in all groups except the control group were orally administered 52% edible alcohol at a dose of 12 mL per kg body weight to establish an acute alcoholism model. At the same time, the control group was orally administered distilled water. After 60 min of alcohol gavage, all the mice were sacrificed under anesthetization with diethyl ether asphyxiation. The liver tissues and serum were harvested and used for visceral index testing.The blood ethanol content of each group of mice were determined using the detection kit provided by Nanjing Jiancheng Bioengineering Institute  following the manufacturer's instructions.Detection kits were used to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AKP) activity in mouse serum. The liver tissues were used for the determination of ethanol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) by UV colorimetric method, superoxide dismutase (SOD) activity by WST-1 method, glutathione peroxidase (GSH-Px) activity by colorimetric method, catalase (CAT) activity by ammonium molybdate method and malondialdehyde (MDA) activity by TBA method. These detection kits were provided by Nanjing Jiancheng Bioengineering Institute  following the manufacturer's instructions.P < 0.05).The data were expressed as mean ± standard deviation. Data processing and mapping using Excel and Origin-2021 software. ANOVA statistical analysis was performed using SPSS 25.0 with a 95% confidence interval  of the non-volatile components of the methanolic extract of lotus root are shown in We analyzed lotus root methanol extract based on the above conditions of liquid chromatography and mass spectrometry. Four hundred and forty-three compounds were identified: 44 terpenoids, 72 alkaloids, 4 tannin, 43 lignan, 138 flavonoids, and 98 phenolic acids.Screening of each compound is required to comply with the basic principles of network pharmacology. Using the TCMSP database, compounds that met the conditions of oral bioavailability (OB) ≥30% and drug-like properties (DL) ≥0.18 were used as candidates, yielding a total of 109 compounds, We obtained 51 relevant targets from the DisGeNET database, 778 alcohol-related targets were obtained from the GeneCards database, 6 relevant targets were obtained from the OMIM database, 49 relevant targets were obtained from the PharmGkb database and 16 alcoholism-related targets were obtained from the Drugbank database . By inteThe PPI network reveals the potential connection between targets. To explore the possible mechanism of lotus root as a treatment against alcoholism, we imported the gene names of the 224 anti-alcoholism targets of lotus root to the STRING database to construct a PPI network. After hiding the unlinked protein nodes, we obtained 185 nodes and 592 edges . CytoscaThe CytoNCA plugin of Cytoscape 3.8.0 software was used to analyze the PPI network by calculating the Degree Centrality, Betweenness Centrality, Closeness Centrality, Network Centricity, and Local Average Connectivity-based method values of the network, respectively. Eigenvector Centrality and Local Average Connectivity-based method values were used to filter out the targets with each condition greater than the median value, and all targets were screened three times by this method . A totalp < 0.05 -Dihydrodehydrodiconiferyl alcohol has the lowest binding energy to MAPK1, which means that the formed protein-small molecule complexes are more stable . Among tEthanol dehydrogenase (ADH), acetaldehyde dehydrogenase (ALDH), and catalase (CAT) are essential enzymes in the metabolism of ethanol in the body, and the measurement of the activity of these enzymes in the liver can reflect the rate of alcohol metabolism to some extent. As p < 0.05). This result indicates that lotus root extract can significantly reduce the blood alcohol content in mice with acute alcohol intoxication.p < 0.05), indicating that the acute alcoholic liver injury in mice was successfully established; the high, medium, and low dose of methanolic extract of lotus root significantly decreased the activities of AST, ALT, and AKP in the serum compared to those on the control diet. The results showed that lotus root extract could reduce liver injury caused by alcohol in mice.From p < 0.05) and the MDA level was increased (p < 0.05). Lotus root extracts increased the SOD activity and GSH-Px level and decreased the lipid peroxidation products caused by oxidative stress in the liver tissue homogenate of mice (p < 0.05). The levels of MDA decreased (p < 0.05), indicating that lotus root extract could enhance the antioxidant capacity of the liver of mice with acute alcoholic liver injury and it had a certain protective effect on acute alcoholic-caused liver injury.From In this paper, we screened ethanol dehydrogenase activation activity of the different lotus root extracts and concluded that the methanol extract of lotus root had the highest activation capacity for ethanol dehydrogenase. UPLC-QTOF-MS/MS can be used to obtain high-resolution and high-precision mass spectrometry data, and the relevant chemical components can be characterized more accurately according to the structures of the components and their bond-breaking patterns, which have been widely used for the identification of natural herbal ingredients. In this study, 109 potentially active compounds in the methanolic extract of lotus root were obtained by relevant screening conditions.The targets of these components had a total of 224 intersecting targets with the targets of acute alcoholism. It is suggested that lotus root extract may help to alleviate the symptoms of acute alcoholism. The mechanism of action of lotus root in treating acute alcoholism was further investigated by enrichment analysis. The results indicated that the neuroactive ligand-receptor interaction pathway is likely to be the key pathway for treating acute alcoholism. The network diagram of the component-target-metabolism pathway was constructed to obtain the relationship between the three interactions, and the top seven components of lotus root with Degree value obtained were Diosmetin, 6-methoxy-2-(2-phenylethyl)chromone, -Dihydrodehydrodiconiferyl alcohol, Armepavine, N-Methylcorydaldine, Boldine, Methylcoclaurine. These were mainly alkaloids and flavonoids, which suggested that alkaloids and flavonoids might be the main alcoholic and hepatoprotective components in lotus root. The intersecting targets were subjected to protein-protein interaction analysis, and these targets were screened in combination with the cytoNCA plugin of Cytoscape software. The three core targets were obtained were heat shock protein 90 (HSP90AA1), signal transducer and activator of transcription (STAT3), and mitogen-activated protein kinase 1 (MAPK1).MAPK1 is an essential component of the MAP kinase/signal transduction pathway. It is a major member of the MAPK family, which plays an important role in cell growth, differentiation, inflammatory response, and other pathological processes. Inhibition of MAPK protein expression could effectively inhibit alcoholic liver injury . Our molFinally, the protective effect of lotus root extract on acute alcohol intoxicated mice was investigated. Alcohol is mainly metabolized in the liver. After heavy alcohol consumption, the alcohol metabolite acetaldehyde and the reactive oxygen radicals generated during its metabolism can damage liver cells, resulting in a large depletion of endogenous SOD and GSH , 30. ReaThe results of the current study showed that the methanolic extract of lotus root had the highest activation rate of ethanol dehydrogenase. Network pharmacology results suggest that lotus root extract may play a role in the treatment of alcoholism by regulating signaling pathways, such as neuroactive ligand-receptor interactions, as well as biological processes, such as regulation of secretion, regulation of ion transport, response to lipopolysaccharides, and response to alcohol. Animal experiments confirmed the therapeutic effect of lotus root on acute alcoholism mechanistically through activation of alcohol catabolic enzyme, reduction of oxidative stress and protection of liver function.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Ethics Committee of Zhejiang Academy of Agricultural Sciences .ZY: methodology, validation, data curation, and writing—original draft. YG: conceptualization and writing—review and editing. WW, HM, RL, and XF: formal analysis and resources. HG: conceptualization and methodology. HC: conceptualization, project administration, data curation, and supervision. All authors contributed to the article and approved the submitted version.


Using transcriptomic and metabolomic approaches to understand the repressive mechanism of ClO2 in potato sprouting is yet to be reported. Sequencing the transcriptome and metabolome of potatoes treated with ClO2 in this study revealed a total of 3,119 differentially expressed genes, with 1,247 and 1,872 genes showing down- and upregulated expression, respectively. The majority of the downregulated genes were associated with plant hormone signal transduction, whereas upregulated differential genes were associated primarily with biological processes, such as phenylpropanoid biosynthesis and the mitogen-activated protein kinase (MAPK) signaling pathway. Metabonomic assays identified a total of 932 metabolites, with 33 and 52 metabolites being down- and upregulated, respectively. Downregulated metabolites were mostly alkaloids, amino acids, and their derivatives, whereas upregulated metabolites were composed mainly of flavonoids and coumarins. Integrated transcriptomic and metabolomic analyses showed that many different metabolites were regulated by several different genes, forming a complex regulatory network. These results provide new insights for understanding the mechanism of ClO2-mediated repression of potato sprouting.Sprouting is an irreversible deterioration of potato quality, which not only causes loss in their commercial value but also produces harmful toxins. As a popular disinfectant, ClO Potato is one of the most important food crops in the world, with a high nutritional value. However, its postharvest storage remains a challenging step to maintaining quality and minimizing market losses  has broad and high-biocidal activity because of its strong oxidizing and sanitizing properties ., 2 reduced air-dried Salmonella in tomato and Escherichia coli O157:H7 strain in apples .The tubers of the “Atlantic” cultivar were obtained from Kaikai Potato Seed Co., Ltd. . After discarding the tubers with injuries or infections, potatoes were transferred to the laboratory and stored at 20 ± 3°C until use. A formulation with 12% ClO2H3 for 15 min, and a control group (CKH3) was soaked in sterile water. After drying, all treated tubers were stored in the dark at 20 ± 2°C with a relative humidity of 75–85%. Experiments were carried out in three replicates per treatment, with each containing 30 tubers.After dormancy, tubers were cleaned and disinfected by immersion in 1% sodium hypochlorite for 3 min, rinsed with sterile water, and air-dried. Tubers were then soaked in 180 mg/L ClO2H3-treated tubers with no sprouts was performed after detecting sprouting in the CKH3 treatments. About 5 g of tuber tissue was cut 1 cm deep under the skin at the bud growth site (bud eye), with a 1 cm stainless steel punch. Samples were wrapped in tin foil paper, frozen in liquid nitrogen, and stored at −80°C until use.Sampling of the ClOSolanum tuberosum reference genome (DM_1-3_516_R44_potato.v6.1), downloaded from the Potato Genomics Resource (http://spuddb.uga.edu/dm_v6_1_download.shtml).Total RNA extraction (three duplicate samples) was performed following the method described by Woolfson et al. . cDNA li−5 to obtain the functional annotations. The KEGG pathway analyses and GO functional classifications were visualized using the Web Gene Ontology Annotation Plot (WEGO) and KEGG automatic annotation servers, respectively.The unigenes were queried against the Kyoto Encyclopedia of Genes and Genomes (KEGG), Non-redundant (Nr), Eukaryotic Orthologous Groups of protein (KOG), Uniprot, and Gene Ontology (GO) public databases, with a threshold e-value of <10g at 4°C for 15 min. The supernatant was filtered through a membrane, the filtrate was diluted twice with methanol–water (3:1), and then transferred to glass vials; 20 μl of this solution was pooled as QC sample for ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses . The samples were homogenized and sonicated for 15 min at 0°C, extracted with a shaker for 24 h at 4°C, followed by centrifugation with 13,800 × Ultra-high-performance liquid chromatography-mass spectrometry separation was performed using an EXIONLC System, with mobile phases A and B as formic acid (0.1%) and acetonitrile, respectively. The column and the auto-sampler temperatures were set at 40°C. A Sciex QTrap 6500+ (Sciex Technologies) was applied. Ion source parameters were set as follows: temperature, 400°C; curtain gas, 35 psi; ion source gas 2, 60 psi; DP, ±100 V; ion source gas, 1:60 psi; and ion spray voltage, +5,500/−4,500 V , then the raw (.wiff) MS data files were converted to.txt format using the MSconverter. MS peak detection and annotation were performed in the R program  content of each sample was above 42% after filtration . All cleour data .2 treatment  were screened by pairwise comparison with Fold Change (FC) ≥2 and False Discovery Rate (FDR) <0.05 and Fragments per Kilobase of transcript per Million fragments mapped (FPKM) values were calculated for each sample to normalize the gene expression. Hierarchical clustering analysis was performed on the filtered differentially expressed genes, and the genes with the same or similar expression behaviors were clustered, and the results of the clustering of differentially expressed genes are shown in reatment .2. As a result, 43 functional DEG categories were identified, including 12 “cellular components”, 10 “molecular functions”, and 21 “biological processes”  were selected for further analyses to screen DAMs . As a rereatment .2-treated tubers relative to that of control tubers. The most decreased metabolites were alkaloids, followed by amino acids and their derivatives, nucleotides and their derivatives, carboxylic acids and their derivatives, and phytohormones (The most abundant DAMs identified were flavonoids, followed by nucleotides and their derivatives, amino acids and their derivatives, alkaloids, coumarins, phenols, miscellaneous, and carboxylic acids and their derivatives . A totalhormones . Most inhormones .The regulatory networks were analyzed by correlating DEGs and DAMs from each treatment. Results showed that multiple genes were positively or negatively regulated by multiple metabolites. For example, 17 DAMs were negatively or positively regulated by 20 or 30 DEGs, respectively, whereas 33 DAMs were negatively or positively regulated by 30 or 20 DEGs, respectively . About t2 -mediated repression of potato tuber sprouting. RNA-seq results identified 3,119 DEGs after ClO2 treatment, with the majority showing enrichment in phenylpropanoid biosynthesis, the MAPK signaling pathway plant, and plant hormone signal transmission pathways. Phenylpropanoid biosynthesis and MAPK signaling pathways were significantly activated, whereas the plant hormone signal transduction pathway was significantly inhibited. Consistently, ClO2 treatment could activate phenylpropane metabolism to improve apple tolerance (Zhao et al., 2 treatment hindered signal transduction during potato tuber sprouting. Phytohormones are crucial endogenous regulators of tuber sprouting, and dynamic changes in endogenous hormones related to stages of sprouting were observed in hormone-treated tubers (Aksenova et al., 2 repressed potato sprouting by limiting plant hormone signal transduction. In addition, ClO2 could improve the stress tolerance of tubers by activating phenylpropanoid biosynthesis and MAPK signaling pathways. This could be because of the ability of ClO2 to induce the production of reactive oxygen species, which damage cell membranes, eventually leading to the obstruction of signal transduction during tuber sprouting.Potato tuber sprouting caused commercial losses and results in increased content of solanine in the tuber, which poses a potential threat to human health (Liu et al., 2 could improve the antioxidant capacity and the safety of potato tubers by promoting the accumulation of flavonoids and inhibiting the synthesis of alkaloids, respectively (Deng et al., Metabolites are important determinants of potato tuber sprouting (Aksenova et al., 2-repressed sprouting is achieved by a coordinated complex process involving several metabolic pathways, which inhibit signal transduction, plant hormones, and alkaloid synthesis in potato tubers, leading to repressed sprouting. The activity of ClO2 thus differs from other sprout inhibitors, such as chlorpropham, which is a mitotic inhibitor used to control tuber sprouting during storage (Vaughn and Lehnen, 2 is a safer and inexpensive sprout inhibitor compared to chlorpropham and essential oils, which are environmentally harmful and expensive (Fu et al., 2-repressed sprouting; however, its specific regulatory roles still need further clarification.Integrated metabolomic and RNA-seq were performed to determine the correlation between identified DEGs and DAMs. The results showed that different metabolites were regulated by different genes. For example, 4-methylumbelliferyl acetate and methoxyindoleacetic acid were regulated by nine genes, with seven and two genes as positive and negative regulators, respectively. The results suggest a complex regulatory mechanism between the changes in metabolite accumulation and gene expression abundance in potato tubers. Thus, we speculated that the mechanism of ClO2-treated and control tubers to analyze the dynamic changes in metabolite accumulation and gene expression levels using UHPLC-MS and RNA-seq methods. The results revealed a total of 3,119 DEGs between the treated and control tubers. Most DEGs were enriched in phenylpropane metabolism, the MAPK signal pathway, and plant signal transduction pathways. ClO2 could inhibit potato tuber sprouting by altering plant signal transduction. Metabolomic analyses identified 85 differential metabolites, with flavonoids and coumarins as the most upregulated differential metabolites, and alkaloids and amino acids and their derivatives as the downregulated differential metabolites. The ClO2-mediated suppression of potato tuber sprouting is controlled by a complex regulatory network involving several different genes and metabolites.This study used ClOThe authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository, prior to publication. Frontiers cannot accept an article that does not adhere to our open data policies.XZ contributed to writing the original draft and writing, reviewing, and editing the manuscript. ML contributed to project administration and funding acquisition. ST contributed to supervision. SL contributed to formal analysis. JC contributed to project administration. XZ investigated the study. XW contributed to methodology. XG contributed to methodology software. JT contributed to data curation. YM contributed to resources. JS conceptualized the study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflictof interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Shenqi pill (SQP), a traditional Chinese prescription, has proven to be effective in treating nonalcoholic fatty liver disease (NAFLD). However, its bioactive ingredients and underlying mechanisms remain elusive.  We aimed to predict the active compounds, potential targets, and molecular mechanisms of SQP anti-NAFLD by applying network pharmacology and molecular docking methods.  Active ingredients and related targets of SQP were obtained from the TCMSP database. Potential targets of NAFLD were acquired from OMIM and GeneCards databases. The STRING database and Cytoscape software analyzed the protein-protein interaction (PPI) network and core targets of overlapping genes between SQP and NAFLD. GO enrichment analysis and KEGG enrichment analysis were performed in the DAVID database. Finally, molecular docking was employed to find possible binding conformations of macromolecular targets.  15 anti-NAFLD bioactive ingredients and 99 anti-NAFLD potential targets of SQP were determined using Network pharmacology. Quercetin, kaempferol, stigmasterol, diosgenin, and tetrahydroalstonine were the major active ingredients and AKT1, TNF, MAPK8, IL-6, and VEGFA were the key target proteins against NAFLD. The KEGG analysis suggested that the main pathways included PI3K/Akt signaling pathway, HIF-1 signaling pathway, MAPK signaling pathway, and TNF signaling pathway. Molecular docking predicted that quercetin, kaempferol, stigmasterol, diosgenin, and tetrahydroalstonine could bind with AKT1, TNF, and MAPK8.  This study successfully predicts the active compounds, potential targets, and signaling pathways of SQP against NAFLD. Moreover, this study contributed to the application and development of SQP. Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive fat accumulation in liver cells and has been recognized as a leading cause of chronic liver disease worldwide , 2. NAFLShenqi pill (SQP) is a famous traditional Chinese medicine (TCM) prescription for treating various liver and renal diseases in China. It was first described in the book named JinGuiYaoLue written by Zhongjing Zhang. The formula consists of eight herbs: Rehmannia Glutinosa (Shu Di Huang: SDH), Chinese Yam (Shan Yao: SY), Cornus Officinalis (Shan Zhu Yu: SZY), Alisma Orientalis (Fu Ling: FL), Poria (Ze Xie: ZX), Moutan Bark (Mu Dan Pi: MDP), Cassia Twig (Gui Zhi: GZ), and Aconite (Fu Zi: FZ). Previous studies found that SQP has a series of effects, such as ameliorating renal fibrosis , regulatNetwork pharmacology is a promising tool to identify the scientific basis and mechanism of TCM at the systemic level –19. We uhttp://tcmspw.com/tcmsp.php) [https://www.uniprot.org/).All bioactive compounds of eight herbs in SQP were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  . ADME crmsp.php) . Drug-limsp.php) . The highttps://www.omim.org/) [https://www.genecard.org/) [The related targets of nonalcoholic fatty liver disease (NAFLD) were acquired from two major databases: OMIM (im.org/)  and Generd.org/) .In the beginning, we used venny2.1 to obtain overlapping genes of SQP and NAFLD as hub genes. Subsequently, an herb-active compound-disease-target interaction network (C-D-T) of treatment with SQP against NAFLD was built by Cytoscape 3.7.2.https://www.string-db.org/) to construct the protein-protein interaction (PPI) network. The species was set to “Homo sapiens” and an interaction score >0.7. Finally, the TSV format file was input into Cytoscape 3.7.2 for graphical visualization.We imported the hub targets into the STRING database (https://david.ncifcrf.gov/), the Gene Ontology (GO) enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of overlapping target proteins were obtained. GO enrichment analysis was applied to show the functions of gene targets, including three parts: biological process (BP), molecular function (MF), and cellular component (CC). The KEGG enrichment analysis described the distribution of hub targets in relevant pathways. In this study, we set p < 0.05 as statistically significant. We select the top 10 most enriched BP, MF, CC, and pathways to draw bar charts and bubble plots by using the WeChat online software.Based on the DAVID database (http://www.rcsb.org/). The PyMOL software was used to dehydrate and remove ligand residues of receptor proteins. The receptor protein was hydrogenated using the AutoDockTool 1.5.6 software and saved in the pbdqt format. The ligand was also saved in the pdbqt format. The active pocket site was built to cover the entire protein. Finally, we used AutoDock Vina for docking and looked for the optimal conformation. A total of 20 conformations were generated for each ligand-protein docking study. The lower the docking score, the more stable the binding between the protein and the molecule. The best scoring conformer with a minimum energy of a drug molecule and target was visualized in PyMOL.We applied AutoDockTools-vina to perform molecular docking to reveal the interaction between active ingredients (ligands) and target proteins (receptors). We downloaded the 2D structures of the compounds from the PubChem Database and used Chem3D software to transform them into 3D structures with minimizing energy. The 3D structure of proteins was downloaded from the Protein Data Bank  networks. As shown in The PPI networks were constructed by importing 99 overlapping targets into the STRING database and then visualized in the Cytoscape software. As shown in p < 0.05, including 508 biological processes (BP), 55 cellular components (CC), and 92 molecular functions (MF). We selected the top 10 terms from BP, CC, and MF, respectively, based on the −log10 , as shown in GO enrichment analysis was constructed by the DAVID database. In total, 655 GO terms meet the demand p value), as shown in KEGG enrichment analysis indicated that 111 terms were related to liver disease, including cancer pathway, PI3K/Akt signaling pathway, non-alcoholic fatty liver disease (NAFLD), MAPK signaling pathway, HIF-1 signaling pathway, and TNF signaling pathway. We selected the top 20 entries as core pathways based on −log10  and three key target genes  were selected to perform molecular docking. The docking energy score is listed in β-sitosterol can regulate the expression of lipid metabolism genes, thus improving lipid metabolism and reducing the level of bile acid in the intestine [In this study, network pharmacology and molecular docking method were employed to clarify the active compounds and molecular mechanism of SQP for NAFLD treatment. A total of 99 overlapping target genes and 15 active compounds were selected for SQP against NAFLD. The top 5 highest degree ingredients include quercetin, kaempferol, stigmasterol, diosgenin, and tetrahydroalstonine, as shown in ntestine , 28. Theα protein, finally resulting in lipid metabolism disorders and insulin resistance, promoting the process of NAFLD [PPI network shows AKT1, TNF, MAPK8, IL6, TP53, JUN, CASP3, CXCL8, VEGFA, PTGS2, especially AKT1, play a major effect on SQP anti-NAFLD. By GO and KEGG enrichment analysis, we found that the major signaling pathways related to NAFLD were pathways in cancer, PI3K-Akt signaling pathway, and HIF-1 signaling pathway. A recent study has found that the activation of the PI3K-Akt signaling pathway could inhibit the expression of SREBP1c and PPARof NAFLD . Hypoxiaof NAFLD . A previof NAFLD .α expression [Furthermore, molecular docking was applied to simulate the binding ability of different compounds and proteins. Compared with other compounds, the binding energy of quercetin to AKT1 was the lowest, which was about −9.4 kcal/mol. The lower the energy score, the stronger the binding capacity. Similarly, the combined energy of kaempferol-TNF was −6.4 kcal/mol and that of diosgenin-MAPK8 was −8.8 kcal/mol. These results strongly indicate that the active ingredients of SQP can effectively treat NAFLD through major binding genes. Huang et al.  also foupression .This study reveals the theoretical molecular mechanism of the ingredients of SQP for the treatment of NAFLD through network pharmacology and verified by molecular docking. However, further pharmacological and clinical studies are needed to validate the therapeutic mechanism of SQP.The present study demonstrated the active compounds, potential genes, and signal pathways of SQP in treating NAFLD based on network pharmacology. We found that quercetin, kaempferol, stigmasterol, diosgenin, and tetrahydroalstonine are the main active ingredients of SQP. The core target genes of SQP for the treatment are AKT1, TNF, and MAPK8. SQP plays a major effect in NAFLD treatment by regulating the PI3K-Akt signaling pathway, TNF signaling pathway, and MAPK signaling pathway. Overall, this study provides a promising and scientific basis for further investigation of SQP for NAFLD treatment.


Andrographis paniculata (Burm.f.) Nees is a medicinal plant that has been traditionally used as an anti-inflammatory and antibacterial remedy for several conditions. Andrographolide (AG), the active constituent of A. paniculata (Burm.f.) Nees, has anti-lipidic and anti-inflammatory properties as well as cardiovascular protective effects. The present study aimed to explore the effects of AG on the progression of atherosclerosis and to investigate related mechanisms via network pharmacology.Objective: Materials and methods: Compound-related information was obtained from the PubChem database. Potential target genes were identified using STITCH, SwissTargetPrediction, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and Comparative Toxicogenomics Database. Genes involved in atherosclerosis were obtained from DisGeNet and compared with AG target genes to obtain an overlapping set. Protein–protein interactions were determined by STRING. Gene ontology (GO) analysis was performed at WebGestalt, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was analyzed using Metascape. The final network showing the relationship between compounds, targets, and pathways was constructed using Cytoscape. After that, oxLDL-induced RAW264.7 cells were used to further validate a part of the network pharmacology results.Result: Eighty-one potential AG target genes were identified. PPI, GO, and KEGG enrichment revealed genes closely related to tumor progression, lipid transport, inflammation, and related pathways. AG improves the reverse cholesterol transport (RCT) through NF-κB/CEBPB/PPARG signaling in oxLDL-induced RAW264.7 cells.Conclusion: We successfully predict AG’s potential targets and pathways in atherosclerosis and illustrate the mechanism of action. AG may regulate NF-κB/CEBPB/PPARG signaling to alleviate atherosclerosis. Reverse cholesterol transport (RCT) is one of the important mechanisms associated with atherosclerosis (AS) and various cardiovascular diseases. Lipid infiltration is a key risk factor for the increased incidence of atherosclerosis, which leads to stroke, coronary artery disease (CAD), and carotid intima-media thickness . IschemiAndrographis paniculata is a medicinal herb from Acanthaceae used in traditional Chinese medicine (TCM) with thousands of years of history. Andrographolide (G), an active ingredient with diterpene lactone structure in A. paniculata  Nees as a high purity crystalline powder  by the Sichuan Wenlong Pharmaceutical Co., Ltd , with inspection Report No. C-01-19013.AG was extracted from https://pubchem.ncbi.nlm.nih.gov). PubChem is an open chemistry database mainly for small molecules, but larger molecules also are included. The records of PubChem are accumulated from hundreds of data sources, such as chemical vendors, journal publishers, and government agencies [The chemical and physical properties related to AG structure and toxicological properties were obtained from the PubChem database  were administered to two different groups of mice. Animals were provided with free access to food and water. Throughout the 14-day experiment, the mice were monitored for any mortality or behavioral changes. The behavioral changes observed included hyperactivity, tremors, ataxia, convulsions, salivation, diarrhoea, lethargy, sleep, and coma. The study was performed in compliance with the Animal Experimental Ethics Committee of Guang'anmen Hospital, China Academy of Chinese Medical Sciences.Acute toxicology tests in mice were carried out following the protocol of Liao et al. We divided male http://stitch.embl.de/cgi/input.pl?UserId=BzUtaFPWtqdq&sessionId=l7vk7fHggCm6) enables users to view the binding affinities of a chemical in an interaction network and the potential effects of the chemical on its interaction targets [http://old.swisstargetprediction.ch/) measures the similarity between new molecules and known ligands to predict their targets accurately [http://bionet.ncpsb.org/batman-tcm/) is an online Bioinformatics Analysis Tool for TCM ingredients’ target prediction and functional analysis of targets, including biological pathways and enrichment analyses and visualization [http://ctdbase.org/) [Different types of targets matched to AG were collected from the following databases, which are often used in the literature for monomer research. (1) STITCH  Swicurately ; (3) Biolization ; (4) Comse.org/)  is a prehttps://www.uniprot.org/) for gene names and Uniprot IDs. Uniprot ID is an important identification when performing protein alignment between different databases. Genes related to atherosclerosis were obtained from the following databases: (1) DisGeNet (http://www.disgenet.org/search), a versatile platform for various research purposes like investigating the molecular underpinnings of specific human diseases and their comorbidities and analysing the properties of disease genes, etc. [https://www.drugbank.ca/), a robust and comprehensive bioinformatics database containing data on drugs and disease [https://www.omim.org/), an authoritative, timely, and comprehensive database about human genes and genetic diseases [http://db.idrblab.net/ttd/), which provides data on therapeutic protein and nucleic acid targets for the targeted disease [Homo sapiens was selected as the species. After targets from databases were compared with targets matched to AG, the final81 targets were successfully identified.After collection, we input the gene symbols to Uniprot  Dru disease ; (3) Onldiseases ; (4) The disease . Homo sahttps://string-db.org/) is a protein-related database that collects, scores, and integrates many publicly available sources of protein–protein interaction information and complements information with computational predictions. Finally, a global network of PPI, including direct  and indirect  interactions, is generated [STRING  [pe.org/) .http://www.webgestalt.org/option.php) supports 324 gene identifiers from various platforms and computationally analyses 150937 functional categories from public databases [The gene ontology (GO) project is a result of efforts to align the functional description of gene products in various databases. GO has developed a standard language (ontologies) with a tertiary structure, which is defined according to the gene product's relevant molecular functions, biological pathways, and cytological components. WEB-based GEne SeT AnaLysis Toolkit  is a web-based platform designed to provide a comprehensive gene list annotation and pathway analysis resource for researchers [Metascape . RAW264.7 cells were maintained in a 25-cm2 cell culture flask with a 2 μm vent cap at 37°C in a CO2 incubator . Cells were cultured in 8 ml DMEM  supplemented with 10% Defined Fetal Bovine Serum , 1.25% L-glutamine, and 2% penicillin/streptomycin. RAW 264.7 macrophages were seeded in 6-well plates (2 × 105/well) in 10% FBS DMEM as described [RAW264.7 cells were acquired from the Cell Resource Center, Peking Union Medical College  on March 15, 2018. The cell line was routinely tested for the absence of mycoplasma contamination by PCR and culture. Its species origin was authenticated using PCR. The identity of the cell line was confirmed with STR DNA profiling . All the results are available online at the website  was used according to the BioVision manual. After the indicated treatments, cells were fixed with 4% paraformaldehyde  in the indicated groups for 30 min and stained with Oil Red O solution for 15 min at 25 ± 2°C. Finally, The cells were observed with a microscope (Olympus IX70) equipped with a camera at 200× magnification. The percentage of Oil Red O positive area was measured using the ImagePro Plus software.Cell supernatant was collected following each treatment. Meanwhile, the Bio-Plex System was warmed up for at least 30 min. A single vial of standards was reconstituted in 500 μl of a diluent similar to the final sample type or matrix. Standards were incubated on ice for 30 min with vortexing for 5 s. A 4-fold standard dilution of the series and blank were prepared. Samples were vortexed for 5 s between liquid transfers. The 10x single beads were vortexed for 30 s and diluted to 1× in the Bio-Plex Assay Buffer. They were protected from light. The assays were run following the manufacturer’s instructions . The assay measurements were conducted on the Bio-Plex MAGPIX System plate-reader . Finally, the C-C motif chemokine ligand 2 (CCL2), interleukin-6 (IL-6), and tumor necrosis factor α (TNFα) concentration were calculated by the standard curve.www.bio-rad.com, U.S.A.) and immunoblotting with antibodies  using ImageLab™ software. Quantification was performed with Image Pro Plus software.The methods for protein extraction from RAW264.7 cells, electrophoresis, and subsequent Western blotting are as described. The cells were lysed in RIPA buffer (high)  containing halt protease and phosphatase inhibitor single-use cocktail for 30 min on ice. The lysates were centrifuged at 12,000 rpm for 30 min at 4°C. Each sample protein was separated on TGX Stain-Free FastCast Acrylamide Kit, 10% , total RNA samples were extracted from the frozen mice liver tissues using TRIzol Reagent . Total RNA was measured by NANODROP 2000 Spectrophotometer . cDNA synthesis was carried out with the High Capacity cDNA Reverse Transcription Kit . Real-time qPCR was performed with the ABI 7900 system  using an Power SYBR Green PCR Master Mix . The primers were synthesized by Thermo Fisher and the sequences were listed in −△△Ct method and normalized to the β-actin mRNA in each sample.NF-κB-p65, CEBPB, and PPARG mRNA levels were quantified by Real Time Quantitative PCR (RT-qPCR). According to the manufacturer’s instructions from Direct-zol™ RNA MiniPrep . For multiple comparisons, one-way analysis of variance (one-way ANOVA) was performed. All results were expressed as the mean ± SD. 50 in mice has been reported .Oil Red O staining was performed to identify the foam cell formation under different drug concentrations, as shown in P<0.01); however, CEBPB (P<0.05) and NF-κB-p65 (P<0.01) were up-regulated. AG reversed the trend, as shown in P<0.05). In the RT-qPCR experiment, the mRNA of PPARG in the RAW264.7+oxLDL group is significantly down-regulated (P<0.01) compared with that in the RAW264.7 group. In contrast, CEBPB (P<0.001) and NF-κB-p65 (P<0.01) are up-regulated. AG reversed the trend, as shown in P<0.05, CEBPB: P<0.001, NF-κB-p65: P<0.05). Besides, the RAW264.7+oxLDL+AG group also showed significantly lower PPARG mRNA than the RAW264.7 group (P<0.05).To assess the mechanism of AG for promoting the RCT, the protein expression and transcript levels of related genes were measured. In the Western blotting experiment, a clear separation of electrophoretic bands was observed E. ComparP<0.01, IL6 and TNF-α: P<0.001). Additionally, AG inhibited the increase in CCL2, IL-6 and TNF-α in RAW264.7+oxLDL group. Besides, the TNF-α levels in the RAW264.7+oxLDL+AG group were also significantly higher than those in the RAW264.7 group (P<0.05).The levels of cytokines are shown in A. paniculata, could ameliorate lipid metabolism disorder and atherosclerosis. It is known to exert multifaceted antiarteriosclerotic effects, such as anti-inflammatory, inhibiting the formation of foam cells, antiplatelet and protecting the endothelium [Atherosclerosis is a crucial underlying pathology of diseases including coronary heart disease, myocardial infarction, stroke, and peripheral artery disease . The aetothelium –46. The othelium , inhibitothelium  are repoothelium ,48. In tFirst, Lipinski’s rule of five is generally referred to as a guideline for drug optimization. It provides relevant scope to help assess the properties of drugs ,50. As sSecond, pathogenic mechanisms and interactions of potential targets were also elucidated in our study. As shown in Third, the GO project and KEGG pathway enrichment analysis revealed the correlation of 81 target genes with their response to stimulus, biological regulation, protein binding, metabolic process, and cell communication. They regulate lipid transport , ameliorThe compound-target-pathway network shown in Fourth, AG-treated foam cell formation seemed to be significantly decreased, as demonstrated by Oil Red O staining. Thus, AG may be involved in macrophage cholesterol efflux, which is the first step of RCT. AG inhibits inflammation by interacting with NF-κB, the factor which regulates the transcription of pro-inflammatory cytokine genes, such as those coding for TNF-α, IL-6, and CCL2 . SimilarIn conclusion, we successfully predict the potential targets of AG for application in atherosclerosis and help illustrate the mechanism of action on a systematic level. This study not only provides new insights into the chemical basis and pharmacology of AG but also demonstrates a feasible method for evaluating potential molecular drugs from herbal medicine. Our results show that AG may regulate NF-κB/CEBPB/PPARG signalling to play a therapeutic role in atherosclerosis.


Shu Gan Jie Yu (SGJY) capsule has a good effect on relieving depressive symptoms in China. However, the mechanism of action is still unclear. Therefore, systemic pharmacology and molecular docking approaches were used to clarify its corresponding antidepressant mechanisms. SGJY capsule and their antidepressive targets. Mild to moderate depression (MMD) related genes were obtained from GeneCards and DisGeNET databases. A network of bioactive components-therapeutic targets of the SGJY capsule was established by STRING 11.5 and Cytoscape 3.9.0 software. Gene function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed by utilizing Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Active components were taken to dock with the hypothetical proteins by iGEMDOCK and SwissDock, and the docking details were visually displayed by UCSF Chimera software. Then, the related research literature of the SGJY capsule was reviewed, summarized, sorted, and analyzed, including experimental evidence and clinical experience.  Traditional Chinese Medicine Database and Analysis Platform (TCMSP), the Encyclopedia of Traditional Chinese Medicine (ETCM), and Swiss Target Prediction servers were used to screen and predict the bioactive components of the AKT1, HSP90AA1, ESR1, EGFR, and PTGS2. KEGG pathway analysis showed that the mechanism of the SGJY capsule on MMD was mainly involved in the PI3K-Akt signaling pathway.  Seven active components and 45 intersection targets were included in the study. PPI network had genuinely uncovered the potential therapeutic targets, such as SGJY capsule for treating MMD and provided a basis for subsequent experiments. In this study, we have successfully predicted the biochemically active constituents, potential therapeutic targets, and comprehensively predicted the related drug-gene interaction of the  Depression is one of the most disabling disorders worldwide with poor quality of life. It affects human social function, and imposes a heavy economic burden on individuals, families, communities, and countries. Due to the increasing social pressure and the role of various other factors, mild to moderate depression (MMD), as an early stage of depression, shows a trend of younger age . CurrentSGJY) capsule showed an effective intervention for essential hypertension patients with insomnia, anxiety or depression in recent years. And it is widely used in the treatment of MMD. The SGJY capsule mainly concludes two Chinese herbs, Acanthopanax senticosus (Rupr. & Maxim.) Harms (ASH) and Hypericum perforatum L. (HPL). The antidepressant mechanism of ASH may be mediated via the central monoaminergic neurotransmitter system and cAMP response element-binding (CREB) protein expression. Therefore, administration of ASH may be beneficial for patients with depressive disorders [St. John's wort, is commonly used in clinical practice for its antidepressant properties, as well as anxiolytic its properties[SGJY capsule has achieved good clinical efficacy in the treatment of MMD, its mechanism of action is still unclear.Systematic review and meta-analysis have provided effective evidence that Shu Gan Jie Yu , and has been widely used in screening bioactive components in TCM. In general, systemic pharmacology technology could evaluate the pharmacological effects and reveal the underlying relationship among active components, potential targets, and multiple diseases .SGJY capsule on depression treatment. The flowchart of research approach is shown in In this study, a systemic pharmacology-based strategy combined with molecular docking approach had been employed to predict bioactive components, potential gene targets, and related signal pathways of the https://www.tcmsp-e.com/) and the Encyclopedia of Traditional Chinese Medicine  were used to screen components of ASH and HPL [https://pubchem.ncbi.nlm.nih.gov/) and saved in .mol2 and SMILES format for further study.Traditional Chinese Medicine Database and Analysis Platform  based on SMILES format with parameter Probability ≥0.6 in prediction results in order to obtain high-quality targets [Homo sapiens” was used as selected species. After removing duplicate genes, potential targets related with active components of the SGJY capsule were obtained. MMD-related targets were collected individually from the DisGeNET (https://www.disgenet.org/home/) and GeneCards (https://www.genecards.org/) databases with the keywords “psychotic depression, mental depression, depressive disorder, mild depression, and moderate depression.” All the targets were standardized in the UniProt database (https://www.uniprot.org/).Active components were submitted to Swiss Target Prediction platform . The intersection targets were selected as the potential targets for further analysis. A protein–protein interaction (PPI) network was constructed by using the STRING 11.5 platform (https://string-db.org/), “Organism” was set to “Homo sapiens.” An interaction with medium confidence (0.4) was collected. The network was visually displayed by the Cytoscape 3.9.0 software. Then GO function and KEGG enrichment analyses were performed with the DAVID platform (https://david.ncifcrf.gov/tools.jsp). The identifier and species were selected as “official_gene_symbol” and “Homo Sapiens,” respectively. The enrichment results, including molecular functional (MF), cell component (CC), biological process (BP), and KEGG pathway enrichment, were obtained and visualized by using imageGP platform (https://www.ehbio.com/ImageGP/index.php/Home/Index/index.html) as the bubble graph with p value <0.05 [https://www.rcsb.org/) with high resolution and score. Water molecules were removed from the structure. Potential candidate components of the SGJY capsule in .mol2 format were taken as ligands. Molecular docking was mainly completed by iGEMDOCK 2.1 with default parameters. Afterward, the most potential protein with associated active ingredients at the low energy was used to dock on the SwissDock platform (https://www.swissdock.ch/docking/), and the results were visually displayed by the UCSF Chimera 1.15 software.Crystal structures of core proteins were obtained from the RCSB Protein Data Bank  with the term “Shu gan jie yu.” All relevant literature were collected, organized, categorized, and divided into experimental evidence and clinical practice.Literature search was performed SGJY capsule were screened out via the TCMSP and ETCM database with the thresholds of OB ≥ 30% and DL ≥ 0.18 properties , as shown in Forty-five intersection genes correlated with MMD were analyzed by the STRING database, and PPI network was established . A totalp value <0.05. The main biological processes contained signal transduction, negative regulation of apoptotic process, and protein autophosphorylation -epicatechin, kaempferol, luteolin, quercetin, and sesamin). Fluoxetine, which was a SSRI and widely used in clinical practice, was used as a positive control [Etotal = EVDW + EH-bond + Eelectrostatic. It was interesting to note that most compounds had a better bonding ability to potential targets than fluoxetine, as shown in MMP9, and sesamin had the better bonding mode with the MMP9 protein than fluoxetine based on binding energy  Harms (ASH) and Hypericum perforatum L. (HPL). Many evidences show that ASH and HPL play an important role in the treatment of MMD.Shu Gan Jie Yu capsule mainly contains two Chinese herbs, via the central monoaminergic neurotransmitter system and CREB protein expression [In vitro studies had shown that ASH extract significantly increased the cell viability, suppressed the apoptosis of PC12 cells, and up-regulated CREB protein expression. Neuroprotective effect might be one of the acting mechanisms that accounts for the in vivo antidepressant activity of ASH [Jin et al. found that ASH extract significantly elevated the levels of 5-hydroxytrylamine, norepinephrine, and dopamine in the whole brain of mice and up-regulated the level of CREB protein. It might exert antidepressant effects pression . In vitry of ASH . The indy of ASH . Moreovey of ASH .St. John's wort, is used as a medicinal plant for MMD, and is more effective than placebo or some antidepressant drugs. Di Pierro, et al. found that multifractionated hypericum extract has better clinical outcomes in subjects with depression without determining an increased risk of toxicity or reduced tolerability [St John's wort has comparable efficacy and safety when compared to SSRIs [HPL, popularly called rability . HPL courability . For patto SSRIs . Most ofto SSRIs –21.SGJY capsule is widely used in clinical practice and has achieved very good clinical results in MMD. Clinical efficacy and safety of the SGJY capsule in patients with acute myocardial infarction and depression. Significantly lower adverse event rate was observed in the Shu Gan Jie Yu group. The SGJY capsule has a reliable effect and high safety in patients with depression [The pression . In addipression .SGJY capsule is also an effective intervention for essential hypertension patients with insomnia, anxiety, and depression [SGJY capsule significantly reduced the depressive symptoms and improved cognitive functions in poststroke depressive patients through alteration of brain dynamics [The pression . Yao et dynamics .SGJY capsule, including betulinic, cianidanol, (+)-epicatechin, kaempferol, luteolin, quercetin, and sesamin, had been successfully obtained by systemic pharmacology strategy. Recent studies also confirmed the antidepressant effects of these compounds. Kaempferol and quercetin had been reported to relieve symptoms of depression and exhibited antidepression effects through acting on interleukin-6 (IL6), mitogen-activated protein kinase 1 (MAPK1), signal transducer, and activator of transcription 3 (STAT3) and transcription factor AP-1 (JUN) [iNOS, COX-2, TNF-α, and IL-1β in stressed mice hippocampus and cortex [SGJY capsule may exert therapeutic effects on MMD.Seven bioactive components of the -1 (JUN) . Betulin-1 (JUN) . Cianida-1 (JUN) . Luteoli-1 (JUN) . Sesamind cortex . TherefoAKT1, HSP90AA1, ESR1, EGFR, PTGS2, GSK3B, MMP9, MMP2, IGF1R, KDR, APP, MCL1, PIK3R1, and MAPT. Some studies had reported that the AKT1 gene was strongly associated with antidepressant treatment [PTGS2 [EGFR [ESR1 [APP [IGF1R [KDR [GSK3B [MAPT [PIK3R1 [HSP90AA1 was up-regulated in patients with depression, which correlated with elevated levels of VEGF, VEGFR2, PI3K, and AKT1 [MMP-2 and MMP-9 genes had relative lower expression on both mRNA and protein levels in depression [MMP9, a key protein for extracellular matrix degradation, was significantly correlated with depressive symptoms [SGJY capsule had good binding capacity to MMP9, which indicated that MMP9 played a very important role on SGJY-treated MMD.PPI network analysis showed that 14 core targets correlated bioactive components had been determined, such as reatment , 31, whit [PTGS2 , 33, EGFS2 [EGFR , ESR1 [3FR [ESR1 , 36, APPSR1 [APP , IGF1R [P [IGF1R , 39, KDRF1R [KDR , GSK3B [R [GSK3B , 42, MAP3B [MAPT , and PIK [PIK3R1  played vand AKT1 . Some stpression , 47. MMPsymptoms , 39. OurSGJY capsule in the treatment of MMD was the PI3K-Akt signaling pathway, KDR, CDK6, IGF1R, EGFR, INSR, GSK3B, HSP90AA1, PIK3CG, MCL1, PIK3R1, and AKT1 genes were enriched in it. Quercetin, luteolin, and kaempferol had been confirmed to be effective in the treatment of MMD by in vivo experiments. The potential PI3K-Akt signaling pathway, a classic signaling pathway in cells, closely relates to the biological process of depression [SGJY capsule might exert therapeutic effects on MMD via Ras, estrogen, and Rap1 signaling pathways. Homologous Ras-family small GTPases, including Ras, Rap2, and Rap1, played a different role and presented signal diversity and specificity. Ras signals long-term potentiation via endoplasmic reticulum PI3K and lipid raft ERK, whereas Rap2 and Rap1 signal depotentiation and long-term depression via bulk membrane JNK and lysosome p38MAPK, respectively [Ras-Raf-MAPK [Ras-ERK-MAPK [Rap1-MKK3/6-p38 MAPK [SGJY capsule and different related signaling pathways.Signal transduction has been reported to be closely involved in antidepressant treatment. Gene function and KEGG results indicated that the main molecular mechanism of the pression , 48. It ectively . Thus, RRaf-MAPK , Ras-ERKERK-MAPK , and Rapp38 MAPK , may be p38 MAPK . Increasp38 MAPK . Hence, in vivo and in vitro experiments are still needed for verification. In short, our study portrayed the ground view of the SGJY capsule in the treatment of mild to moderate depression.Due to the limitations of compounds screening and accuracy of target prediction, the results obtained in this study are general . AlthougSGJY capsule have been identified by a systemic pharmacology-based strategy and the intersection targets corresponding to these components and their therapeutic mechanism of MMD have been revealed in detail by the PPI network and pathway enrichment analyses. The result of molecular docking showed that sesamin had a better bonding mode with the MMP9 protein than fluoxetine. In general, bioactive components and the main therapeutic mechanism of the SGJY capsule in the treatment of MMD were successfully predicted, which might provide valuable guidance for further pharmacological research of the SGJY capsule on MMD.In this study, seven bioactive components of the


Background: Qifenggubiao granules (QFGBG) is a new Chinese medicine independently developed by Heilongjiang Academy of Traditional Chinese Medicine, which combines the essence of Yupingfeng powder and Shengmai yin , and has been included in the 2020 edition of Chinese Pharmacopoeia. It has remarkable pharmacodynamic results and conclusive clinical effects in the treatment of allergic rhinitis, chronic cough and other diseases. Previous pharmacological studies have shown that it has immunomodulatory effect, but its immunomodulatory mechanism is still unclear.Methods: In this study, cyclophosphamide (CTX) was used to establish the immune hypofunction model in mice, and the weight change, index of immune organs in spleen and thymus, pathological sections of immune organs and inflammatory factors were used to evaluate the model. Based on the metabolic biomarkers obtained by metabonomics technology, the potential targets of Qifeng Gubiao Granule immunomodulation were obtained by integrating the targets of blood components, metabolites and diseases through network pharmacology. Meanwhile, GO enrichment analysis and KEGG pathway analysis were carried out on the potential targets.Results: QFGBG can increase body weight and organ index, and recover immune organ damage caused by CP. Metabonomics identified 13 metabolites with significant changes, among which the level of phospholipid (PC) metabolites decreased significantly in the model group. Sphingosine -1- phosphate, 1- palmitoyl phosphatidylcholine [LysoPC (16:0/0:0)] and other metabolites were significantly increased in the model group, and 98 targets of Qifeng’s external immune regulation were obtained by intersecting 629 component targets, 202 metabolite targets and 1916 disease targets. KEGG pathway analysis obtained 233 related metabolic pathways, and the top 20 metabolic pathways mainly involved IL-17 signaling pathway, TNF signaling pathway, Sphingolipid signaling pathway, and so on.Conclusion: QFGBG may act on AKT1, IL6, MAPK3, PTGS2, CASP3, MAPK1, ESR1, PPARG, HSP90AA1, PPARA and other targets, acting through Sphingolipid signaling pathway and signaling pathway. Combined with pharmacodynamic evaluation, the immunomodulatory effect of QFGBG was confirmed, and the immunomodulatory mechanism of QFGBG with multiple targets and multiple pathways was preliminarily clarified. Astragalus mongholicus Bunge (Fabaceae), Eleutherococcus senticosus (Rupr. & Maxim.) Maxim , Atractylodes macrocephala Koidz (Asteraceae), Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk (Apiaceae), Ophiopogon japonicus (Thunb.) Ker Gawl (Asparagaceae), and Schisandra chinensis (Turcz.) Baill (Schisandraceae), with a ration of 6:3:2:2:2:1. It combines the essence of Yupingfeng powder and Shengmai Yin, and replaces Panax ginseng C.A.Mey  with Heilongjiang genuine medicinal material Eleutherococcus senticosus (Rupr. & Maxim.) Maxim , which has the effects of tonifying lung and kidney and invigorating spleen, and is included in the 2020 edition of Chinese Pharmacopoeia (3b receptor garland (Qifenggubiao granules (QFGGB) is a new Chinese medicine independently developed by Heilongjiang Academy of Traditional Chinese Medicine , which is included in Chinese Pharmacopoeia in 2020 edition, and is composed of six medicinal herbs: acopoeia . It mainacopoeia , Modern  garland , and res garland , but itsIn order to study the immunomodulatory mechanism of QFGBG, cyclophosphamide (CTX) was used to establish a mouse model of hypoimmunity. CP is an alkylating agent, which can destroy immune cells, interfere with the proliferation and differentiation of T and B cells, reduce the number of normal T and B cells, and inhibit cellular and humoral immune responses . BecauseMetabonomics studies the human body as a complete system, which has the characteristics of wholeness, dynamics, non-trauma, close to physiology, etc. It is very similar to the principle of wholeness and dynamics of traditional Chinese medicine, and provides a new and powerful technical means for traditional Chinese medicine research . NetworkThis study will combine metabonomics and network pharmacology methods, metabonomics identifies potential biomarkers, and at the same time, network pharmacology is used to find biomarker targets and correlate component targets and disease targets, Narrow the target range of QFGBG immunomodulation, and provide technical support for the follow-up research.QFGBG , Cyclophosphamide injection , Levamisole hydrochloride tablets , Watsons water, Formic acid (Fisher Company), Acetonitrile and methanol , UltraSYBR One Step RT-qPCR Kit , themouse serum IL-4 (EK0405), IFN-γ (EK0375) ELISA kit , Mouse ACTB Endogenous Reference Genes Primers, 10 μM .Nitrogen generator , Cryogenic centrifuge , Upright white photo microscope , Scanning software , Panoramic slice scanner , Ultra-high performance liquid chromatograph , Mass spectrometer , Infifinite M200 PRO , Bioer Line gene 9600 flfluorescence Quantitative PCR instrument .There are 90 male ICR mice (about 26 g) with animal certificate number SCXK (Liao) 2020-0001, which are provided by Liaoning Changsheng Biotechnology Co., Ltd. The study was conducted strictly according to the ethical guidelines for using experimental animals in Heilongjiang province, guided and approved by the animal ethics committee of the academy of traditional Chinese medicine of Heilongjiang province [(2011)93]. Adaptive feeding for 7 days, ICR mice were randomly divided into 6 groups, with 15 mice in each group, they are normal control (NC), model control (MC), QFGBG low-dose(L), QFGBG medium-dose(M), QFGBG high-dose(H) and postive control levamisole hydrochloride (PC).NC group administered normal saline as well as MC, low-dose group administered QFGBG (13 g/kg), medium-dose group administered QFGBG (26 g/kg), and high-dose group administered QFGBG (52 g/kg). positive control administered levamisole hydrochloride (25 mg/kg). On the 8th day, mice in each group except the blank group were injected with CP 80 mg/kg intraperitoneally for three consecutive days, From the 11 days, each group was given corresponding doses of drugs, 15 consecutive days.Mice in each group fasted for 12 h after the last administration time, The mice were weighed before modeling and after the experiment, and the weight changes of mice in each group were measured. and whole blood was collected, Approximately 1 mL of whole blood was collected, The serum was obtained through centrifuged at 3000 rpm and 4°C for 15 min, the spleen and thymus of each group of mice were collected and weighed, And then fixed with 4% neutral formaldehyde. The liver tissue was washed and frozen.The fixed spleen and thymus organs were washed with distilled water and dehydrated with ethanol gradient. Xylene was transparently treated twice, soaked in wax, and embedded in a paraffifin embedding machine. The embedded tissue was fixed on a microtome for sectioning and placed on a clean carrier. On the glass slides, the slides are dried in a 60°C constant temperature oven, and then dewaxed, HE stained, dehydrated, and finally sealed with neutral gum.After the mouse serum was thawed, it was centrifuged at 3000 rpm for 10 min. The supernatant was taken for ELISA detection, and a microplate reader was used for detection.Serum samples were thawed at 4°C before preparation. 100 μL serum samples were diluted with 300 μL methanol (precooled at 4°C), swirl at 2500 rpm for 1.5 min, Centrifuge at 4°C and 13000 rpm for 10 min blow dry the supernatant with nitrogen, and freeze at −80°C. Before running on the machine, 150 μL of 80% methanol (precooled at 4°C) was re-dissolved, and centrifuged at 4°C 13000 rpm at 4°C for 10 min. The supernatant was taken and put into the inner liner tube, and the liquid was sampled. 10 μl of serum was drawn from all samples, which were mixed and used as QC samples. QC samples were tested every 5 samples to evaluate the stability of the system.The Waters Acquity UPLC BEH C18 column , aquityupl-cbeh C18 vanguard pre-column , the column temperature is 35°C, and the mobile phase is 0.1% formic acid solution (A) −0.1% formic acid acetonitrile (B). 2–5 min, 60–40% B, 5–11 min, 30–70% B, 11–13 min, 10–90% B, 13–14 min, 100% B, 14.1–17 min, 95–5% B.ESI ion source is used, ionization mode is positive and negative ion mode, ion source voltage is 5500 V, ion source temperature is 550°C, cracking voltage is 80 V, collision energy is 40 V, CES is 20 eV, atomizing gas is N2, auxiliary gas and atomizing auxiliary gas are both 55 PSI, air curtain gas is 35 PSI, and scanning range of sub-ions is 80–1600 Da. Eight peaks with IDA response value exceeding 100 cps were set for secondary mass spectrometry scanning, and the scanning range of Produc-tIon was 80–1600 Da, and dynamic background subtraction was started. The data acquisition software is Analyst TF 1.6 software, and the image processing system is Peakview 2.0.ESI ion source is used, ionization mode is positive and negative ion mode, ion source voltage is 5500 V, ion source temperature is 550°C, cracking voltage is 80 V, collision energy is 40 V, CES is 20 eV, atomizing gas is N2, auxiliary gas and atomizing auxiliary gas are both 55 PSI, air curtain gas is 35 PSI, and scanning range of sub-ions is 80–1600 Da. Eight peaks with IDA response value exceeding 100 cps were set for secondary mass spectrometry scanning, and the scanning range of Produc-tIon was 80–1600 Da, and dynamic background subtraction was started. The data acquisition software is Analyst TF 1.6 software, and the image processing system is Peakview 2.0.http://www.swisstargetprediction.ch/). The target of the final composition is obtained after removing the duplicate target from the Database. The Online Mendelian Inheritance in Man , The TTD (http://db.idrblab.net/ttd/), the database , Drug and Target Database (https://go.drugbank.com/), DisGeNET Database (https://www.disgenet.org/) were used to search for the immune dysregulation-related targets. Enter the target points of components into Cytoscape 3.8.2 to construct the network visualization diagram of “medicinal materials-components-targets”.Enter the SMILE format of blood component  into SwiBased on metabolomics, the obtained differential metabolites were input into HMDB database to obtain the SMILE format of substances and input into Swiss Target Prediction database to obtain metabolite targets. Enter the target into Cytoscape 3.8.2 to construct the “metabolite-target” network diagram.https://string-db.org/) website. Furthermore, the nodes in the network are ranked according to the Degree by using the CytoHubba plug-in of Cytoscape 3.8.2, and the top 10 targets are selected as potential targets.The component target, disease target and metabolite target were input into Venny 2.1 software to obtain the intersection target, and the PPI network diagram was obtained by importing the intersection target into String (http//metascape.org/) website for GO and KEGG analysis, with p < 0.05. GO function analysis is mainly used to describe the functions of gene products, including biological process (BP), cell composition (CC) and molecular function (MF). KEGG is used to analyze the possible action pathway of the target and to explore the mechanism of immune enhancement of QFGBG.The intersection targets were input into Metpascape  value of OPLS/PLS-DA model, VIP >1, t test, and the p value < 0.05 showed statistical significance.GraphPad Prism version 8.0.2 software was used for all statistical analysis. All data are expressed as average ± standard deviation (‾X±SD) and the difference between the two groups was compared by p < 0.001), and all of them recovered after administration, and PC had the most obvious recovery effect (p < 0.001). The index changes of thymus and spleen are shown in p < 0.001), and both showed significant recovery after administration (p < 0.001). HE staining of spleen and thymus is shown in The change in body weight and organ index is shown in in vivo  online database, and finally 13 differential markers were determine  is used to draw the top 10 analysis results of three parts, as shown in , cellular component (CC), and molecular function (MF). GO analysis showed that BP included 1139 enrichment results, including regulation of defense response, regulation of inflammatory response, regulation of secretion. CC includes 76 enrichment results, mainly involving neuronal cell body, external encapsulating structure, cell body, and secretory granule lumen. The MF includes 108 enrichment results, including lipid binding, nuclear receptor activity, protein kinase activity etc. The enrichment results are sorted according to shown in . 233 patshown in .Immunity is a physiological function of the human body. The human body relies on this function to identify “self” and “non-self” components, thus destroying and rejecting antigens entering the human body or damaged cells and tumor cells produced by itself, so as to maintain human health. The immune system mainly has three functions: defense, stabilization, and surveillance. Once these functions are out of balance, pathological reactions will occur. Inflammation is a manifestation of immune response to infection or injury, and is the result of activation of innate and/or acquired immune response , that isp < 0.001), which indicated that Sphinganine might be phosphorylated to S1P by SPHKs catalysis, and then play an immunosuppressive role. After administration, the content of each group increased. It shows that QFGBG can play an immunomodulatory role by reducing the contents of Sphinganine and S1P through sphingolipid signaling pathway.Studies have shown that cyclophosphamide can cause liver injury . Liver pMitogen-activated protein kinases (MAPKs) are a kind of serine/threonine protein kinases, which widely exist in mammalian cells. MAPK pathway is an important way to transfer extracellular signals into cells and cause cell biological reactions  , which mInflammation plays an important role in many kinds of diseases, which mainly refers to various inflammatory diseases caused by complex reactions caused by tissue injury or pathogen infection . TRP chaNetwork pharmacology provides key technical support for revealing the scientific connotation of traditional Chinese medicine compound, discovering drug targets and guiding the research and development of new traditional Chinese medicine drugs . Traditi


We aimed to analyze the possible molecular mechanism of Xihuang pill (XHP) in the treatment of pancreatic cancer based on methods of network pharmacology, molecular docking, and bioinformatics.  The main active components and targets were obtained through the TCMSP database, the BATMAN-TCM database, and the Chemistry database. The active ingredients were screened according to the “Absorption, Distribution, Metabolism, Excretion” (ADME) principle and supplemented with literature. We searched GeneCards, OMIM, TTD, and DrugBank databases for pancreatic cancer targets. The targets of disease and ingredients were intersected to obtain candidate key targets. Then, we constructed a protein-protein interaction (PPI) network for protein interaction analysis and a composition-key target map to obtain essential effective ingredients. Metascape was used to perform functional enrichment analysis to screen critical targets and pathways. The expression and prognosis of key targets were examined and analyzed, and molecular docking was carried out. β-estradiol, ursolic acid, and daidzein. The core targets were EGFR, ESR1, MAPK1, MAPK8, MAPK14, TP53, and JUN, which were highly expressed genes of pancreatic cancer. Among them, EGFR and MAPK1 were significantly correlated with the survival of pancreatic cancer patients. The key pathway was the EGFR/MAPK pathway. The molecular docking results indicated that four active compositions had good binding ability to key targets.  A total of 52 active ingredients of XHP, 121 candidate targets, and 52 intersecting targets were obtained. The core active ingredients of XHP for the treatment of pancreatic cancer were quercetin, 17- The molecular mechanism of XHP for the treatment of pancreatic cancer involved multiple components, multiple targets, and multiple pathways. This research theoretically elucidated the ameliorative effect of XHP against pancreatic cancer and might provide new ideas for further research on the treatment of pancreatic cancer. Pancreatic cancer is one of the most lethal malignant neoplasms, which causes over 331000 deaths per year worldwide and ranks as the seventh most common cancer death . TherefoBovis Calculus), She Xiang (Moschus), Ru Xiang (Olibanum), and Mo Yao (Commiphora myrrha). Modern pharmacological studies demonstrated that XHP could enhance chemosensitivity, relieve side effects of conventional therapies, and ameliorate prognosis when used as a complementary therapy [Xihuang pill (XHP), as a famous TCM formula, has been used to clear heat, relieve toxicity, and expel stasis of blood since the Qing dynasty. It is composed of Niu Huang  was used to search the active ingredients of XHP. The filter criteria were oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 according to the “Absorption, Distribution, Metabolism, and Excretion” (ADME) principle. Only when the ingredients in XHP met both of these two prerequisites were they considered as possible active ingredients of XHP. For the herbs in XHP whose information of active ingredients cannot be acquired from TCMSP database, they were screened in other authoritative websites such as BATMAN-TCM database [http://bionet.ncpsb.org/batman-tcm/) and Chemistry database (http://www.organchem.csdb.cn). A literature search was also conducted to get supplements for the active ingredients of XHP which have been reported to have definite pharmacologic action for pancreatic cancer. The target information of the active ingredients was mainly acquired from the TCMSP database and was recorded by the DrugBank database [https://www.uniprot.org/).The Traditional Chinese Medicine Systems Pharmacology Platform  (TCMSP) database  , Online Mendelian Inheritance in Man [https://www.omim.org/), Therapeutic Target Database [http://db.idrblab.net/ttd/), and DrugBank database (https://go.drugbank.com/) to comprehensively find relevant targets of pancreatic cancer. After getting the target information of pancreatic cancer, duplicate removement was conducted. Among them, the screening of the targets from the GeneCards database was conducted according to the median of their relevance to pancreatic cancer.We searched the keyword “pancreatic cancer” in GeneCards   website to construct the PPI network analysis by choosing “multiple sequences” in function units and setting “Homo sapiens” as the organism specie with the interaction confidence retaining the acquiescent value of 0.4. The result of the PPI network analysis was saved as a TSV document and was imported into Cytoscape 3.8.1 software. Then, the MCODE plug-in [We employed the STRING  (https:/ plug-in  was usedhttps://metascape.org/) was used for the gene enrichment analysis to get the function of the targets in the key gene module and to explore the biological process and corresponding pathways. Results were expressed as logP values, reflecting the significance of protein biological functions, and all results were considered significant at P < 0.01.Metascape  (https:/http://gepia.cancer-pku.cn/) [p value cutoff value was set to be 0.01. The KM plotter  [p < 0.05.GEPIA  (pku.cn/)  is a datalysis/)  was emplhttp://cao.labshare.cn/cb-dock/) [Molecular docking is a reliable approach for drug design based on the characteristics of receptors and the interaction between receptors and ligands, and ligands are typically drug molecules. We searched the 3D structures of the proteins corresponding to the key targets and used them as receptors for further docking. The ligands were the main active ingredients of XHP which we obtained after screening by the above methods, which were potentially effective for the treatment of pancreatic cancer. After that, CB-Dock (b-dock/)  was usedBezoar and musk have different main components due to different varieties, but it is not documented in detail in the TCMSP database, and musk is not recorded in the database. Therefore, we referred to relevant literature and databases to confirm the components of cultured bezoar in vitro and artificial musk in XHP sold commercially. Moreover, in the published literature, no pharmacological effect directly related to pancreatic cancer was collected in bezoar, musk, frankincense, and myrrh. In addition, the ingredients of XHP that did not meet the filter criteria in the TCMSP database were also included in this study because the related targets information of which in the database was linked to pancreatic cancer.After normalization of the target names in the UniProt database and duplication omitting, there were 6 active ingredients and 24 related targets in bezoar, 6 active ingredients, and 55 related targets in musk. In frankincense, six active ingredients were identified, corresponding to 35 targets. We found 35 active ingredients and 93 corresponding targets in myrrh, respectively. After removing duplicates, we collected 52 active ingredients in the XHP and a total of 121 related target genes. Among them, bezoar contained CLRs (Mol ID : MOL000953) as same as musk. The results are presented in Through four consecutive screenings, 731 pancreatic cancer targets were identified in the GeneCards database. Other targets information obtained from the OMIM and TTD disease databases were combined, resulting in 1224 disease targets after removing duplicates. After Venn diagram analysis, 52 intersection targets between XHP and pancreatic cancer were finally confirmed . The resWe downloaded the interaction network in TSV files and imported it into Cytoscape software. An interactive network consisting of 52 points and 533 edges was obtained. The Mcode plug-in was utilized to acquire the key module. The Mcode is an algorithm that calculates the information of individual nodes in the network. The final module, obtained after processing according to the parameters of the nodes, is the key functional module in the interaction network. The results yielded a total of 27 key targets, which were ranked according to the degree value, as shown in β-estradiol, ursolic acid, and daidzein.An ingredient-key target network diagram was constructed in Cytoscape software, as shown in The Metascape platform is a gene functional annotation analysis tool that enables cognition of gene and protein function by applying bioinformatic analysis methods to analyze bulk genes and proteins. After importing the 27 potential key targets of XHP for pancreatic cancer into the Metascape platform, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and gene ontology (GO) analysis were performed, respectively. The results of GO analysis included three sections: cellular components (CC), molecular functions (MF), and biological processes (BP). The cellular component result section reflects the location in the cell of the gene, the molecular function result section reflects the effect of a single gene product, and the biological process result section comprises various biological processes in which genes are involved, such as apoptosis and so on.Further identification of the module yielded key functional modules . The modκB signaling pathway, the JAK-STAT signaling pathway, and the IL-17 signaling pathway. Taking the MAPK signaling pathway as an example, the mechanism and potential targets of this pathway are shown in Finally, the cellular components of the genes involved in the vesicle lumen, membrane rafts, extracellular matrix, adherent junctions, the outer side of the plasma membrane, and so on. The molecular functions included cytokine receptor binding, heme binding, MAP kinase activity, serine-type endopeptidase activity, phosphatase binding, and so on. Biological processes involved positive regulation of cell migration, positive regulation of the MAPK cascade, positive regulation of protein serine/threonine kinase activity, response to hypoxia, and apoptosis. The biological pathways mainly included pathways in cancer, the MAPK signaling pathway, the NF-P > 0.05).As shown in β-estradiol, ursolic acid, and daidzein, were used as ligands for docking to verify the potential active components and targets of XHP on pancreatic cancer . The CB-Dock website can predict the binding site of a given protein, calculate the center and size using a new curvature-based cavity detection method, and perform AutoDock Vina molecular docking. The lower the Vina score value, the higher the affinity between the receptor and the ligand. The docking results indicated that 17-beta-estradiol was verified the strongest binding activity with both EGFR and MAPK1 among the four potential key active ingredients.Combining the enrichment and module analysis, the inhibition of the EGFR/MAPK pathway by XHP should be noted. EGFR is a transmembrane protein closely related to multiple pathological processes such as the proliferation, invasion, and metastasis of pancreatic cancer cells. Clinical studies have shown that the overexpression of EGFR could shorten the average overall survival rate of patients after PDAC resection and can be used as an independent predictor . AccumulHigh-throughput omics techniques, including proteomic and transcriptomic analysis, have enabled further comprehension of the TCM . Though β-estradiol, ursolic acid, and daidzein are potential bioactive ingredients of XHP, which act on key genes such as EGFR and MAPK1 and regulate many pathways . It also should be noted that the XHP formula possibly exhibits an anti-tumor effect by regulating multiple biological pathways. The prediction results theoretically elucidated the ameliorative effect of XHP against pancreatic cancer and might facilitate further research on the mechanism of XHP or its bioactive compounds as an alternative therapy for pancreatic cancer.In conclusion, this comprehensive network pharmacological analysis proposes numerous testable surmises about the potential molecular mechanisms of XHP formula for the treatment of pancreatic cancer and predicts that quercetin, 17-