CDH1, SHH, and WNT5A had the highest degrees of interaction and were associated with the formation of ACP. CDH1 was verified as a critical gene by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) in ACP and CG samples. We found that CDH1 may play an important role in the pathways in cancer signaling pathway that regulates ACP development. The CDH1 gene may be a target for future research and treatment of ACP.Adamantinomatous craniopharyngioma (ACP) is an epithelial tumor that arises when Rathke’s pouch remains during embryonic development. The pathogenesis of ACP remains unclear, and treatment options are limited. Here, we reveal the critical genes expressed in ACP and provide a basis for further research and treatment. The raw dataset GSE94349 was downloaded from the GEO database. We selected 24 ACP and 27 matched samples from individuals with no documented tumor complications (control group). Then, we screened for differentially expressed genes (DEGs) to identify key signaling pathways and associated DEGs. A total of 470 DEGs were identified (251 upregulated and 219 downregulated). Hierarchical clustering showed that the DEGs could precisely distinguish the ACP group from the control group (CG). Gene Ontology (GO) enrichment analysis indicated that the upregulated DEGs were mainly involved in cell adhesion, inflammatory responses, and extracellular matrix management. The downregulated DEGs were primarily involved in cell junction and nervous system development. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that the critical pathway was pathways in cancer. In the PPI network,  Craniopharyngioma (CP) is a complex and diverse congenital intracranial tumor, and surgical resection is the primary treatment strategy at present. Because this tumor is located near essential brain structures, such as the optic nerve and hypothalamus, it poses significant challenges for surgery. Damage to the hypothalamus and other important brain tissue structures can lead to high fever, electrolyte disorder, obesity, and other severe complications and seriously harm patients’ quality of life after surgery . TreatmeBRAFV600E is observed in 95% of PCPs, and CTNNB1 is observed in 75%–96% of ACPs  in ACP tissues, providing high-throughput gene expression data. Gene expression microarrays are an essential approach for studying the pathogenesis of CP. The National Center of Biotechnology Information GEO database comprises a large amount of genetic data, providing a rich resource for the study and analysis of differential gene expression levels .Zou et al.  analyzedGSE94349, submitted by Donson et al. on 31 January 2017 , containWe selected 24 ACP surgical tumor samples and 27 samples from individuals with no documented tumor complications  from GSE94349. However, as Donson et al.  was a clBy analyzing the gene expression data of GSE94349, DAVID and GSEA were used to conduct GO and KEGG pathway enrichment analyses. Then, the intersection of the two methods was obtained, namely, crucial pathways and genes. Finally, STRING and Cytoscape software were used to construct protein−protein interaction (PPI) networks of critical pathways and genes. The present study identified critical genes in the pathogenesis of CP, which will provide new insight into the treatment of CP.http://www.ncbi.nlm.nih.gov/geo/) database. The quality control and standardization of these data were completed prior to the current study. The data were processed using R software . First, we searched and downloaded GPL570, the corresponding platform of GSE94349, from the official GEO website. GSE94349 was matched with GPL570 in R language to complete gene ID conversion and annotation. Next, the missing values were calculated by KNN in R language to supplement the missing values.The gene expression microarray data of GSE94349_series_matrix.txt were downloaded from the GEO (http://limma.org)  . Only gep < 0.01 was considered statistically significant.The upregulated and downregulated DEGs were input into the online tool DAVID (DAVID 6.8 version) for GO enrichment analysis. The results were analyzed according to biological process (BP), cellular component (CC), and molecular function (MF), and p < 0.01 was considered statistically significant. We imported all the gene data for the GSEA. The Kolmogorov−Smirnov test was used to calculate the value of DEGs in each KEGG pathway by 1,000 repeated permutation tests to conduct the KEGG pathway enrichment analysis of DEGs in GSEA. p < 0.01 was considered statistically significant. Then, the upregulated and downregulated DEG KEGG pathway enrichment analysis results were derived. Finally, the intersection of the results of the two analysis methods was obtained to determine the target DEGs.DEGs were imported into the DAVID online tool for KEGG pathway enrichment analysis, and DAVID was used to conduct KEGG pathway enrichment analysis for all the different genes, yielding more comprehensive results. However, GSEA is a KEGG pathway enrichment analysis of all genes, yielding a complete dataset. The intersection of the two may be more valuable for further research . We usedCDH1, WNT5A, and SHH) were selected.The KEGG pathway-related DEGs from the intersection of the DAVID and GSEA results were imported into the Search Tool for the Retrieval of Interacting Genes (STRING). The gene interaction relationship was derived with a confidence score >0.7 as the cutoff standard. Then, Cytoscape software (Version 3.5.1) was used to construct the PPI network between the KEGG pathway and its related DEGs. CytoHubba was used to predict the top 10 key genes. Three critical genes . ACP samples were collected after endoscopic nasal resection, stored at 4°C for transport and then preserved in liquid nitrogen. Samples from individuals with no documented tumor complications [control group (CG)] were collected from patients undergoing surgery for epilepsy. The mean age of the four epileptic patients was 20 years , including two men and two women. A total of four ACP samples from two men and two women were obtained. The mean age of the ACP patients was 25.5 years . Patients pathologically diagnosed with ACP were included in the present study, while patients with other diagnoses were excluded. All specimens were pathologically and clinically diagnosed as ACP by three pathologists. The present study was approved by the Research Ethics Committee of Nanchang University , and written informed consent was provided by all patients prior to the start of the study.1. Clinical diagnosis of craniopharyngioma and consent to surgery;2. Han nationality, Chinese, right-handed;3. No history of chronic cardiovascular diseases; no history of infectious diseases; previously healthy, no history of kidney disease and liver disease;4. Normal hearing and language functions, no metal implants in the body, no history of surgery for heart, lung, or other vital organs, no history of major diseases such as brain disorders, and no history of mental illness; and5. At least two pathologists diagnosed ACP.6. Only when the above five items meet the requirements can they be included in the sample database of this study.1. Clinically diagnosed epilepsy and consent to surgery;2. Han nationality, Chinese, right-handed;3. No history of chronic cardiovascular diseases or infectious diseases;4. No history of kidney and liver diseases;5. Normal hearing, no metal implants in the body; and6. No history of mental illness.Only when the above six items meet the requirements can they be included in the sample database of this study.1. Oral hormone medication within 2 weeks;2. Speech and hearing impairment;3. Patients who cannot cooperate with researchers in information collection ;4. Lost contact or refused to answer the phone during the follow-up period so that postoperative information could not be collected; and5. Patients with anemia, cachexia, and other blood cells or low hemoglobin.6. Any one of the above five items should be excluded from this study.1. Patients or their family members voluntarily withdraw from the study after surgery;2. Non-craniopharyngioma patients were diagnosed after preoperative collection of craniopharyngioma patient specimens;3. Postoperative complications that require hormone and other related treatments;4. Postoperative complications and inability to cooperate with follow-up investigators; and5. Patients who cannot cooperate with postoperative follow-up.6. If any of the above five criteria are met, the study will automatically stop by default.CDH1, WNT5A, and SHH) were validated in ACP tissues and compared with CG tissues by qRT-PCR using the QuantStudio 7 Flex real-time PCR system . The primer sequences used are shown in −ΔΔCt method was used, and the PCR results were normalized to the ACTIN gene . The total RNA and cDNA of CG samples were obtained by the same method. Finally, 7.5 μl of 2×qPCR Mix, 1.5 μl of 2.5 μm primer (upstream + downstream), 2.0 μl of reverse transcription product (cDNA), and 4.0 μl of ddH2O were added to a 200-µl PCR tube. After the PCR solution was prepared, it was gently mixed by pipetting up and down. A 96-well PCR plate was placed into a special bracket. The researchers avoided touching the bottom of the reaction plate to avoid affecting the data reading. Three wells were prepared for each reaction, and qPCR was conducted using the following reaction conditions: predenaturation at 95°C for 10 min, denaturation at 94°C for 15 s, and annealing at 60°C for 30 s for 40 cycles. When the temperature rose from 65°C to 95°C, the fluorescence signal was collected every 0.5°C to form a melting curve. RT-PCR  was performed on CG samples and ACP samples according to the above steps. Each experiment was repeated three times.Total RNA was extracted from ACP samples according to the instructions of the RNA Extraction Kit . Then, the total RNA of ACP samples was reverse transcribed according to the steps of the Servicebio−K. All experimental data are expressed as the mean ± SD and were statistically analyzed by t-test. p < 0.05 was considered to be statistically significant.The ΔΔCT method was used as follows: A = CT  − CT ; B = CT  − CT ; K = A − B; express multiple = 2The gene expression microarray data of GSE94349_series_matrix.txt were downloaded from GEO, comprising 168 samples. The ACP and control groups were selected from GSE94349_series_matrix.txt, and other samples were removed. Quality control and standardization of these data were completed, and all chip data reached comparable levels.10p > 2 (p < 0.01) and |log2 Fc| > 4. Red dots represent upregulated genes, and green dots represent downregulated genes. Then, based on the cutoff criteria (p < 0.01 and |log2 Fc| > 4), a total of 470 DEGs were identified, including 251 upregulated and 219 downregulated DEGs.Twenty-four ACP samples and 27 control samples in the GSE94349 dataset were analyzed. The volcano plot in All DEGs were analyzed. DEGs were also divided into upregulated and downregulated components for separate analyses. This study aimed to identify the upregulation and downregulation of target DEGs; thus, we divided the upregulated and downregulated genes for analysis. The expression heatmap of the DEGs is shown in p-value in The DEGs were imported, DAVID was used for functional enrichment analysis, and the results were obtained according to the three functions of MF, CC, and BP. The GO analysis results of upregulated and downregulated DEGs are listed in the top 10 items in order of The results showed that the upregulated genes were mainly involved in cell adhesion, inflammatory responses, and extracellular matrix management. The downregulated genes were mainly involved in cell junction and nervous system development.p < 0.01). Twelve KEGG pathways were enriched among the downregulated DEGs (p < 0.01).The upregulated pathway comprised pathways in cancer, the PI3K-Akt signaling pathway, the extracellular matrix (ECM)–receptor interaction, and the Wnt signaling pathway. The downregulated pathways were mainly associated with pathways related to retrograde endocannabinoid signaling, the synaptic vesicle cycle, morphine addiction, and neuroactive ligand–receptor interactions.p < 0.01 as the standard, there were seven upregulated and no downregulated pathways. Detailed results of the 7 KEGG pathways are shown in Upregulated pathways were mainly associated with pathways in cancer and cytokine−cytokine receptor interactions.The KEGG pathway analyses identified 19 significant pathways from DAVID and seven significant pathways from GSEA. The results of the two methods intersected, and only one common upregulated pathway was found, as shown in the Venn diagram in CDH1, WNT5A, and SHH) were selected from the 10 genes for RT-PCR verification.All of the 15 DEGs in the one common pathway were collected using STRING, and a PPI analysis was performed. The 15 DEGs were imported into the STRING database, and their interactions were identified. If the interaction score was >0.7, the PPI network could be formed with 14 nodes and 32 edges. The PPI networks presented in vs. the control group, including CDH1, WNT5A, and SHH, but only CDH1 had statistical significance (p < 0.05) (Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to measure and compare the expression levels of the key genes in the ACP and control groups Figure 8A total of 470 DEGs were identified in the present study, including 251 upregulated and 219 downregulated DEGs. GO enrichment analysis indicated that the upregulated genes were mainly located in the extracellular exosome, extracellular region, and extracellular space and were involved in structural molecule activation and the binding of calcium ions, cell adhesion, inflammatory responses, and extracellular matrix organization bioprocesses. Another study showed that proinflammatory mediators drive the phosphorylation and activation of STAT3 . Persisthttps://www.kegg.jp/pathway/hsa05200) was the major pathway involved in ACP.The KEGG pathway enrichment analysis in this study showed that “pathways in cancer”  of the 15 DEGs were involved in the PI3K-Akt signaling pathway. The FGF family proteins are key regulators of several biological processes, and together with their receptors, they affect the development of many human cancers  have been closely/positively associated with craniopharyngioma. One report showed that SHH is highly expressed in ACPs  plays an important role in canonical Wnt signaling  are a major component of the cancer stroma and promote cancer cell aggressiveness by secreting multiple factors . WNT5A wrognosis . Wnt/β-C lesions . It is tIn conclusion, we hope that these methods can be applied in future studies. DAVID was used to thoroughly analyze the DEGs, while GSEA was performed to comprehensively analyze the genes. The main aim of our study was to analyze the KEGG pathways of all genes, including the DEGs, using GSEA. The analysis of large amounts of ACP gene data requires substantial work, and the analysis results are likely to change as the major databases are updated.CDH1, WNT5A, and SHH genes and “pathways in cancer”, may be associated with ACP. According to the cytoHubba statistical results in Cytoscape software, the first three DEGs were selected for RT-PCR verification. Our results suggest that CDH1 may play an important role in the pathways in cancer signaling pathway that regulate ACP development. However, its specific role in ACP remains to be confirmed in further experiments. Brastianos et al. showed that in PCPs with the BRAFV600E mutation, a BRAF inhibitor combined with MEK inhibitor targeted therapy can reduce tumor volume by 85%–91%  Medical Research Ethics Review (No. 160)]. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.TH and SZ conceived the study and supervised the research. CF performed the bioinformatics analysis and was a major contributor to the writing of the manuscript. LZ prepared the figures and edited the manuscript. HH performed the PCR validation experiments. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (grant no. 82060246), the Natural Science Foundation of Jiangxi Province, and the Science and Technology Research Project of Jiangxi Provincial Education Department (grant nos. 20202BABL206059 and GJJ190128).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-alcoholic fatty liver disease (NAFLD) represents a severe public health problem. Dysbiosis of gut microbiome has been identified as one of the key environmental factors contributing to NAFLD. As an essential nutrition, Vitamin D (VD) plays an important role in regulating gut microbiota based on its receptor  which is highly expressed in the gastrointestinal tract.Rats were fed with HFD (high-fat diet) for 12 weeks. And the rats were treated with VD two times a week by intraperitoneal injection for 12 weeks. H&E staining combined with plasma biochemical index was performed to characterize pathological changes and function of the liver. Fecal microbiota 16S rRNA gene sequencing and metabolomics were taken to reveal the altered gut microbiota and metabolites.The VD alleviates the HFD-induced lipid accumulation in the liver as well as decreases the levels of amlodipine besylate (ALT) and amlodipine aspartate (AST). VD supplement decreased the ratio of phylum Firmicutes/Bacteroidetes (F/B) but increased alpha diversity. In addition, the VD treatment improved the HFD-induced gut microbiota by increasing the Prevotella and Porphyromonadaceae and decreasing Mucispirillum, Acetatifactor, Desulfovibrio, and Oscillospira abundance. Furthermore, the capability of tyrosine metabolism, tryptophan metabolism, arginine biosynthesis, and sphingolipid metabolism was enhanced after VD treatment. Consistently, Prevotella positively correlated with tryptophan metabolism and sphingolipid metabolism. Importantly, the Prevotella abundance was positively associated with serotonin, melatonin, tryptamine, L-arginine, and 3-dehydrosphinganine which synthesize from tryptophan, tyrosine, arginosuccinate, and serine, respectively.VD treatment inhibited HFD-induced NAFLD accompany by dysbiosis gut microbiota and metabolites, suggesting that VD supplement could be a potential intervention used for NAFLD treatment by targeting the specific microbiota. Firmicutes/Bacteroidetes (F/B) and a decreased butyrate-producing bacteria Ruminococcaceae involved in NAFLD progression (Proteobacteria phylum was enriched in patients with NAFLD (Enterococcus metabolize trimethyllysine (TML) to N,N,N-trimethyl-5-aminovaleric acid (TMAVA), which could aggravate fatty liver , the most prevalent chronic liver disease, by regulating host energy and metabolism . Human cgression . The clith NAFLD . Gut micty liver . Also, tesponses . In addiesponses . Furtheresponses . A previof NAFLD . Thus, tVdr knockout mice compared to control mice (Vitamin D (VD) and its active form 1,25-dihydroxyvitamin D  inhibit immune responses and indirectly attenuate immune responses by increasing IL-10 production in macrophages, dendritic cells, and T cells . Our prerol mice . Thus, aPorphyromonadaceae and Prevotella but decreased Mucispirillum, Acetatifactor, Desulfovibrio, and Oscillospira. The tyrosine metabolism, tryptophan metabolism, arginine biosynthesis, and sphingolipid metabolism were enhanced by VD treatments. Of note, sphingolipid metabolism was positively correlated with Prevotella. Taken together, targeting gut microbiota and the sphingolipid metabolism pathway may be a feasible preventive strategy for patients with NAFLD.Here, the high-fat diet (HFD)-induced NAFLD rat model was adopted to treat VD by intraperitoneal injection. VD supplement obviously reversed the serum transaminases, hepatic fat deposits, and hepatocyte membrane destruction induced by HFD. In addition, VD decreased plasma glucose profiles. Furthermore, VD treatments stimulated the growth of 2D3  two times a week by intraperitoneal injection for 12 weeks. Rats in ND and HFD groups were intraperitoneally injected with an equivalent volume of vehicle. During the experiments, body weight and food intake were assayed weekly. All animal procedures used in this study were conducted according to the Guide for Care and Use of Laboratory Animals and approved by the ethics committee of China Medical University (No. 2018162). At the end of the experiment, animals were euthanized to harvest liver, blood, and feces for further analysis.Five-week-old male SD rats were housed in a specific pathogen-free environment at 20–22°C with 12-h light–dark cycles. To establish the NAFLD model, rats were fed with an HFD  after 1 week of adaptation for 12 weeks. Normal diet combined with VD supplement (ND + VD) and HFD with VD supplement (HFD + VD) groups’ rats were treated with 1,25(OH)Plasma glucose, amlodipine besylate (ALT), amlodipine aspartate (AST), triglyceride (TG), and total cholesterol (TC) levels were quantified according to the manufacturer’s instructions .Histological changes in liver tissues were detected using hematoxylin–eosin (H&E) staining. The fixed liver tissues were embedded in paraffin and cut into 5-μm thick slices.The liver sections were analyzed by transmission electron microscopy.Stool DNA was isolated, and the V3 + V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using primers CCTACGGGNGGCWGCAG(F) and GACTACNVGGGTATCTAAT(R). After PCR products were purified, the positive amplicon sequencing was performed on the Illumina MiSeq PE300 platform. After cutting off barcodes, QIIME V.2.0. was used for analyzing raw sequencing reads . Uparse post-hoc Dunn’s test.The OTU numbers of each sample were flattened, and the alpha diversity of fecal bacteria was calculated based on the normalized OTU table by R package Vegan as previous methods . Principp-value (Wilcoxon’s test) < 0.05 was selected for enrichment analysis. The enrichment pathway of differential plasma metabolite profile between the two groups was analyzed by MetaboAnalyst 5.0, respectively.1Blood metabolite signatures were identified by LC-MS between HFD and HFD + VD groups samples. The processed data such as m/z, RT, and normalized peak area percentages were imported into SIMCA to identify metabolites. The HMDB database was adopted to map and identify the metabolites. Partial least squares discriminant analysis (PLS-DA) was used to reveal the metabolite changes in groups by R package ropls, and the abundance of significant metabolites with variable important in projection (VIP) ≥1 and The relationship of KEGG pathways of bacterial predicted function and remarkable changed bacterial, alpha diversity of microbiota and microbiome as well as blood metabolites and gut bacterial were analyzed by Spearman’s correlation and Mantel’s test based on R package dplyr and ggcor. Meanwhile, the co-occurrence network of metabolites of key pathways and changed bacteria was constructed and visualized by R package igraph and Cytoscape, respectively.Post-hoc Dunn’s test, Tukey’s multiple-comparison test, and Wilcoxon’s test were performed to analyze the difference between the two groups. P-values < 0.05 were considered statistically significant. Error bars indicate mean ± standard error of mean (SEM).For statistics in multiple groups, we utilized Kruskal–Wallis and two-way ANOVA tests to evaluate the difference among groups, respectively. via H&E staining  and 1,474 (down) features after VD treatment . As showNext, as illustrated in Clostridium_XlVb, Paraprevotella, and Mucispirillum, which were lower in the HFD + VD group. Prevotella, Prevotellaceae_unclassified, and Alloprevotella were correlated with dopamine, L-arginine, indoleacetaldehyde, and 3-dehydrosphinganine. In addition, Mucispirillum, Desulfovibrio, and Akkermansia maintained a negative association with 4-hydroxyproline and tyramine. The enriched Oscillospira, Robinsoniella, and Acetatifactor in the HFD group were negatively correlated with upregulated 2-aminobenzoic acid, epinephrine, tryptamine, serotonin, and melatonin after VD treatment. It was noted that the integrated analysis of significantly changed pathways revealed that sphingolipid metabolism overlaps with microbiome and metabolite .X-LZ, JiaY, LC, S-SZ, SJ, and NA designed and performed the experiments. LC conducted the analyses and wrote the manuscript. X-LZ measured the fecal bacteria and metabolites. H-XL and JD conceived and supervised the study. All authors read and approved the final version of the manuscript.


Salvia miltiorrhiza Bge., and their targets were standardized using the UniProt platform. The disease targets related to stroke were collected by comparative toxicogenomics database (CTD), GeneCards, and quantitative structure-activity relationships-TargetNet (QSAR-TargetNet). Thereafter, the protein-protein interaction (PPI) network was constructed using the STRING platform and visualized by Cytoscape (3.8.2) software. Then, the Metascape platform was used to analyze the Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Cytoscape (3.7.2) software was also used to construct the network of the “herb-component-target-pathway.” We found that Tanshinol B, Tanshinol A, Przewaquinone C, Tanshinone II, and other main components of Salvia miltiorrhiza Bge. may regulate neurotransmitters and neurological function. Therefore, we speculate Salvia miltiorrhiza Bge. has a neuroprotective effect. For further verification, potential core targets  for ischemic stroke and core active ingredients  for Salvia miltiorrhiza Bge. were further verified by molecular docking.  In this study, SymMap was used to screen the 50 bioactive scored components and 65 putative targets of Salvia miltiorrhiza Bge. may have a neuroprotective effect against ischemic stroke, which provides a new understanding for the development of therapies for the prevention and treatment of ischemic stroke. Our findings revealed that Tanshinol A, Tanshinol B, Tanshinone II A, and Przewaquinone C as the main component of  Salvia miltiorrhiza Bge. has unique advantages in the treatment of stroke, and can also improve symptoms of patients in the recovery period. In addition, it plays an irreplaceable role in improving patients' quality of life [Stroke is one of the three leading causes of death in the world. Ischemic stroke is a common disease with a high incidence rate, mortality rate, and disability rate. It mainly occurs in the elderly, and it has a trend of becoming younger in recent years . A large of life .Salvia miltiorrhiza Bge. compound injection and the control group treated with routine treatment, The total effective rates of the treatment group and the control group were 88.3% and 68.3% respectively. In the treatment group, the consciousness of comatose patients recovered, increased from 33.3% to 61.1%, and the incidence of side effects decreased [An earlier study showed that the experimental group was treated with in vitro [The mechanism of Tanshinone I in treating ischemic stroke have been verified. Infarct volume and neurological deficit were assessed by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and Longa. Tanshinone I was found to reduce the volume of cerebral infarction induced by transient middle cerebral artery occlusion (tMCAO) in a dose-dependent manner. it was observed neuronal death in the hippocampus and cortex by detecting the changes of Neuronal nuclear antigen (NeuN) by Immunohistochemistry. It was found that pretreatment using Tanshinone I could improve the cell viability of HT-22 cells after oxygen-glucose deprivation (OGD) in vitro .The percentage of CD4+CD25+FOXP3+ Treg cells in the peripheral blood of the MCAO group was increased with Cryptotanshinone treatment. The protein level of FOXP3 and the phosphorylation of STAT5 were recovered in the CD4+CD25+ Treg cells of the model group after being treated with Cryptotanshinone [Salvia miltiorrhiza Bge. extract reduced the infarct area of the core, penumbra and subcutaneous, and the neurological deficit score was also decreased (P < 0.05) [Although some experiments have proved that after ischemia-reperfusion, free radicals will be produced, leading to cell death. After middle cerebral artery occlusion, compared with the sham operation group,  < 0.05) .Salvia miltiorrhiza Bge. in alleviating ischemic penumbra. The pharmacological effect and molecular mechanism of Salvia miltiorrhiza Bge. on ischemic penumbra caused by ischemic stroke have not been fully elucidated [Salvia miltiorrhiza Bge. in the treatment of ischemic stroke through evaluation of pharmacological components, prediction of drug binding sensitive binding sites, molecular docking, network, and pathway analysis.However, the above studies have laid a foundation for us to study the molecular mechanism of ucidated . We usedSalvia miltiorrhiza Bge.According to the existing literature reports and network pharmacology knowledge, we constructed the pathway network formed by different pathological characteristics of ischemic stroke candidate biomarkers and selected components of Salvia miltiorrhiza Bge. target proteins were selected and analyzed for GO and KEGG pathway enrichment, PPI interaction network of important proteins was also constructed. We constructed and analyzed the composite targeting pathway (CTP) network between the main components of Salvia miltiorrhiza Bge. and ischemic stroke.Based on their integration scores, 56 possible Salvia miltiorrhiza Bge. and seven ischemic stroke proteins were calculated by molecular docking method, especially potential active compounds  and ischemic stroke related proteins , which has a good binding ability.The binding energies of four components of In this study, we followed the methods of Xu et al.  and explSalvia miltiorrhiza Bge. in stroke are shown in The potential pharmacological with biochemical mechanisms and the details behind each step of using systematic pharmacology to determine the role of Salvia miltiorrhiza Bge. in the treatment of stroke, the drug correlation and potential targets between stroke and Salvia miltiorrhiza Bge. were studied in more detail.In order to further analyze the mechanism of Salvia miltiorrhiza Bge. were found, which can be divided into six categories: terpenes, fatty acids, diphenyl heptane, sterols, flavonoids, and others, as shown in Through the screening of the traditional Chinese medicine databases including the Traditional Chinese Medicine System Pharmacology database (TCMSP), the Traditional Chinese Medicine Integrated Database (TCMID), the Traditional Chinese Medicine Information Database (TCMID), and the Encyclopedia of Traditional Chinese Medicine database (ETCM). More than 50 active components in Similarly, we take the union of the collected data sets and obeyed the rules described in Furthermore, we selected 26 components with a value of oral bioavailability (OB) greater than 36% and a value of drug-likeness (DL) greater than 0.2 .Among these target proteins, 109 key target proteins were screened, and their docking chemical structure, target protein type, binding potential percentage, protein UniProt ID, and common name were summarized, as shown in In addition, the construction and description of PPI networks are based on different classes of compounds and putative protein arrangements .P < 1.0E − 16, indicating that there is a significant difference between this group of interaction targets and random similar target combinations.As shown in Salvia miltiorrhiza Bge. target proteins were selected and analyzed for GO and KEGG pathway enrichment. As shown in Tables P-value cut-off value should be less than 0.01, 824 GO items of terpene and 48 items of KEGG pathway were identified. A total of 824 GO terms are included. The display of GO enrichment used bubble charts, as shown in Figures P-values are arranged in descending order, and more important response to biological regulation (GO:0065007), cellular process (GO:0009987), metabolic process (GO:0008152). In addition, it also contains some genes  in the cell's response to nitrogen compounds. The target pathways of Salvia miltiorrhiza Bge. and stroke are including axon guidance, steroid hormone biosynthesis, nitrogen metabolism, metabolic pathways, Serotonergic synapse, and Alzheimer`s disease  and Genecards database, and collected 6227 protein entries by combining the above data. There are 66 related proteins related to the main components of Salvia miltiorrhiza Bge. complex targeted pathway disease (CTPD) network contains 87 nodes and 180 edges. To get a deeper understanding of the molecular mechanism of the neuroprotective mechanism of Salvia miltiorrhiza Bge., we studied the complex targeting pathway of the main components of Salvia miltiorrhiza Bge. The network of “active ingredient target pathway” is shown in Salvia miltiorrhiza Bge. on the target. The 56 major active components may act on 109 targets and play a role in 15 signaling pathways, which indicates that Salvia miltiorrhiza Bge. may inhibit the occurrence and development of ischemic stroke through multiple components, multiple targets and multiple pathways. The elliptical nodes with red border represent 13 ischemic stroke-related proteins and 4 key proteins. What's more, clustering and topology methods are used to identify individual differences and similarities between different protein targets . Among these target proteins, Fyn and TTR are the overlapping nodes of ischemic stroke-related proteins, which have important value for future research.Good blood-brain barrier permeability, pharmacokinetics, and water solubility play an important role in drug development. In order to better explore the mechanism of action and possible targets of the main components of Salvia miltiorrhiza Bge. has a certain effect on ischemic stroke. In order to further detect and verify the interaction of the selected active components of Salvia miltiorrhiza Bge. with key proteins, molecular docking of the above proteins and drug targets can be carried out. Using PyMOL software, Discovery studio 4.5 client, and Autodock tools 1.5.6 software, the interaction between potential active compounds  and ischemic stroke related proteins  was studied  models, Tanshinone I (TSN I) was found to dose-dependently decrease mice's cerebral infarct volume induced in vivo. Compared with the cerebral ischemia-reperfusion group, the infarct volume significantly decreased in the 25 mg/kg TSN I intervention group, the number of NeuN-positive neurons was markedly increased in the 7 days of the TSN I intervention group, the protein levels of PI3K and t groups . PI3K/AKt groups . TSN I ct groups , 13.In vitro, mainly using Neuro-2A cells cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal bovine serum at 37°C and humidified in a 5% CO2 atmosphere and divided into different groups treated in different ways, pretreatment with Tanshinone I could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD) [P < 0.05) [ < 0.05) . the PI3 < 0.05) . mTOR is < 0.05) .Salvia miltiorrhiza Bge. and its main active ingredients in the treatment of ischemic stroke by integrating the methods of neurobiology, traditional Chinese medicine pharmacology and computational pharmacology, with the classical pharmacokinetic and models conducted by molecular docking.This prompted us to study the possible active components, target proteins, and potential protein-drug mechanisms of Salvia miltiorrhiza Bge. were collected through TCMSP and STITCH database, and the targets of ischemic stroke were downloaded from the website; Based on string platform data, the protein-protein interaction network (PPI) of Salvia miltiorrhiza Bge. on the common target of ischemic stroke was constructed by using Cystoscope software; The key genes of the signaling pathway (hub gene) were screened by using cytohubba in Cystoscope software, and the topological network of the target was constructed; The network diagram of “active ingredient target pathway” was drawn; Schrodinger software was used to docking the active components with the target.The active components and targets of Salvia miltiorrhiza Bge., which were related to 20 signaling pathways.The “active component target pathway” network showed that there were 56 main active components and 38 main targets in Furthermore, KEGG and GO annotation can also be used to infer the specific function and potential role of these proteins. Through the construction of the PPI network, the function of key proteins was predicted.Cell components are mainly enriched in the cell, intracellular, cytoplasm, membrane-bounded organelle, intracellular organelle, intracellular membrane-bounded organelle, membrane, plasma membrane, nucleus, and so on, as shown in Figures Salvia miltiorrhiza Bge. play an important role in regulating the neural rehabilitation, the formation and repair of neurons, and signal transmission of ischemic stroke  or Therapeutic Target Database (TTD) disease enrichment with adjusted P-value smaller than 0.05 are shown.The network graph is drawn based on the known and predicted candidate target proteins with scores not smaller than 20 of each query ingredient of The purple hexagon represents the constituent absorbed into the blood, The blue five-pointed star represents drug target, The yellow circle represents the KEGG pathway, the red square represents OMIM disease, and the green square represents CTD disease.Salvia miltiorrhiza Bge. related with brain injury, vascular disease, neurodegenerative diseases and also depression, heart failure, and visceral pain. Component targeting pathway/disease network reveals the possible association between ischemic stroke and other comorbidities and the theoretical basis of network pathway level.We can find out KEGG enrichment analysis involved 48 signaling pathways, and 15 KEGG pathways were screened by the number of genes more than 4, as shown in As shown in There is still great research value for the four more meaningful stroke-related proteins  in this study, and pointed out the direction for our further research.On the molecular level, STAT3 found in this experiment might have a neuroprotective effect against cerebral ischemic injury through IL-6R-mediated by phosphorylation of signal transducer and activator of transcription 3 (STAT3), and finally induced neuronal cell death via a decrease in manganese-superoxide dismutase (Mn-SOD) .Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that digest the components of the extracellular matrix that form the basal lamina surrounding the neurovascular unit, Several studies have suggested a strong relationship between the induction of MMP-2 and brain edema following cerebral ischemic injury, Matrix metalloproteinases (MMPs) and tissue inhibitors to metalloproteinases (TIMPs) modulate capillary permeability .MMP-mediated proteolysis of neurovascular matrix may also interfere with homeostatic signals between different cell types in the neurovascular unit. Resting matrix signaling via integrins is vital for normal cell function. Disruption of extracellular matrix by MMPs can induce anoikic in neurons and cerebral endothelial cells , 19, indEstrogen receptor alpha (ESR1), not beta, is a critical link in estradiol-mediated protection against brain injury, through knockout animal models experiments . TelomerSalvia miltiorrhiza Bge. and ischemic stroke, it is concluded that Tanshinol B, Tanshinol A, Przewaquinone C, Tanshinone II A of Salvia miltiorrhiza Bge. have good binding effect on the key proteins of ischemic stroke, such as STAT3, MMP2, ESR1, TERT. A large number of main components of Salvia miltiorrhiza Bge. have good molecular docking fraction, and they are highly matched with key proteins. It is of great significance to further test and discuss its specific functions and properties.Based on the network analysis of active components of Salvia miltiorrhiza Bge. from SymMap (https://www.symmap.org/) [https://www.tcmsp-e.com/) [https://www.tcmip.cn/ETCM/index.php/Home/Index/) database [Salvia miltiorrhiza Bge. and Metabolism, excretion, and toxicity for ischemic stroke as the oral bioavailability (OB) standard to evaluate and screen the comprehensive drug similarity classification and pharmacokinetic characteristics of Salvia miltiorrhiza Bge. (OB ≥ 30%), drug-likeness (DL ≥ 0.18) assessment and blood-brain barrier (BBB) ≥ −0.3 respectively. In addition, the compounds of two-dimensional or three-dimensional (2D/3D) structure, also canonical smiles, and PubChem ID calibrated database with PubChem (https://pubchem.ncbi.nlm.nih.gov/).In this study, we collected the chemical composition information of ap.org/) , which i-e.com/) , the Tra-e.com/)  the Trad-e.com/) , and thedatabase . Take thhttps://ctdbase.org/; inference score >1) [https://www.genecards.org/) [Salvia miltiorrhiza Bge.Ischemic stroke-related protein targets were detected by the comparative toxicogenomics database   and the ds.org/) , we selehttps://targetnet.scbdd.com) [Salvia miltiorrhiza Bge. compounds were verified by their unique UniProtKB ID and names in the UniProt database (https://www.uniprot.org/) [Compounds were then scored by the Lipinski Rule of 5, and the putative ischemic stroke-related target protein screening, which was filtered conditionally from the quantitative structure-activity relationships-TargetNet (QSAR-TargetNet: bdd.com) . The iscot.org/) .https://metascape.org/) [P-value less than 0.01 were regarded as significant interest targets.Metascape (pe.org/)  combinespe.org/)  and KEGGpe.org/)  pathway Salvia miltiorrhiza Bge., the compound-target interaction (CT), compound-target-pathway (CTP), and compound-target-pathway-disease (CTPD) network were constructed.To elucidate the pathogenesis of ischemic stroke and the mechanism of https://www.cytoscape.org/) [https://string-db.org/) [The topology parameters are used to analyze, and the subnetworks of bottleneck nodes are filtered and visualized by Cytoscape 3.6.0 software  . Proteindb.org/)  databasehttps://www.pdb.org/) and decorated by removing the ligands and water motifs, revising it and optimizing the mutation sites, and adding hydrogen by the Pymol 2.3 and UCSF Chimera 1.14rc software.The binding ability, site, and interaction between compounds and target proteins were analyzed by classical molecular dynamics using AutoDockTools-1.5.6, Pymol 2.3, and Discovery Studio 4.5 Client , 37. TheIn this study, we used network pharmacology and molecular docking methods to study the mechanism of ischemic stroke.Salvia miltiorrhiza Bge. has multiple targets and multiple pathways, showing the overall performance.We found that as a traditional Chinese medicine, For complex diseases with multiple causes and factors, such as stroke, compound drugs may be a better curative effect. Compound drugs have an effect of multiple targets and biological pathways.Salvia miltiorrhiza Bge. in the treatment of hemorrhage or ischemic stroke.However, our experiment found that a large number of genes have not been verified, which can also provide information for the application of Salvia miltiorrhiza Bge., particularly the main chemical components, not only in the synthesis, release of neurotransmitters during transmission, or the formation and neuronal plasticity, there is a protective effect.Salvia miltiorrhiza Bge. may be well combined with stroke-related proteins, which contributes to the new development in theory and the development of new drugs against ischemic stroke.The main components of


Lithospermum erythrorhizon (LE) is known in Korean traditional medicine for its potent therapeutic effect and antiviral activity. Currently, coronavirus (COVID-19) disease is a developing global pandemic that can cause pneumonia. A precise study of the infection and molecular pathway of COVID-19 is therefore obviously important. The compounds of LE were identified from the Natural Product Activity and Species Source (NPASS) database and screened by SwissADME. The targets interacted with the compounds and were selected using the Similarity Ensemble Approach (SEA) and Swiss Target Prediction (STP) methods. PubChem was used to classify targets linked to COVID-19. The protein–protein interaction (PPI) networks and signaling pathways–targets–bioactive compounds (STB) networks were constructed by RPackage. Lastly, we performed the molecular docking test (MDT) to verify the binding affinity between significant complexes through AutoDock 1.5.6. The Natural Product Activity and Species Source (NPASS) revealed a total of 82 compounds from LE, which interacted with 1262 targets (SEA and STP), and 249 overlapping targets were identified. The 19 final overlapping targets from the 249 targets and 356 COVID-19 targets were ultimately selected. A bubble chart exhibited that inhibition of the MAPK signaling pathway could be a key mechanism of LE on COVID-19. The three key targets  directly related to the MAPK signaling pathway, and methyl 4-prenyloxycinnamate, tormentic acid, and eugenol were related to each target and had the most stable binding affinity. The three bioactive effects on the three key targets might be synergistic effects to alleviate symptoms of COVID-19 infection. Overall, this study shows that LE can play a role in alleviating COVID-19 symptoms, revealing that the three components  are the most significant elements of LE against COVID-19. However, the promising mechanism of LE on COVID-19 is only predicted on the basis of mining data; the efficacy of the chemical compounds and the affinity between compounds and the targets in experiment was ignored, which should be further substantiated through clinical trials.  The outbreak of a new health crisis due to coronavirus disease 2019 (COVID-19) occurred in Wuhan, Hubei Province, China, in December 2019 , and theSalix, Taxol from Taxus brevifolia, and artemisinin from Artemisia annua [The novel coronavirus was designated as severe acute respiratory syndrome coronavirus-2  because of its high genetic similarity around 80%) to SARS-CoV, which caused acute respiratory distress syndrome (ARDS) and high mortality between 2002 and 2003 . The SAR0% to SARia annua ,13. Dongui Bogam, shows that Lithospermum erythrorhizon can be used to treat measles caused by Measles morbillivirus. According to an experiment conducted on Beagle dogs, the data suggested that the no observed adverse effect level (NOAEL) of LE extraction was 100 mg/kg/day. Therefore, LE may have a favorable therapeutic effect and is safe to use on virus-related diseases [The early Korean medical book, diseases . At presdiseases . Moreovediseases .Accordingly, network pharmacology—a multiple analytical mode—can investigate interaction networks such as compounds, genes, protein targets, and diseases . Additiohttp://bidd2.nus.edu.sg/NPASS/, accessed on 21 October 2021), Google Scholar, and SMILES (Simplified Molecular Input Line Entry System). The molecular formula of selective compounds was identified using ChemSpider (https://www.chemspider.com/StructureSearch.aspx) (accessed on 21 October 2021) or PubChem (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 21 October 2021) to confirm compound names or structures. The drug-likeness properties of the identified compounds were confirmed through Lipinski’s rule on SwissADME (http://www.swissadme.ch/) (accessed on 21 October 2021) [https://pubchem.ncbi.nlm.nih.gov/edit3/index.html) (accessed on 21 October 2021) [The compound information of LE was collected by NPASS (http://sea.bkslab.org/) (accessed on 23 October 2021) [http://www.swisstargetprediction.ch/) (accessed on 24 October 2021) [Homo Sapiens” mode. We selected the overlapping targets between SEA and STP databases based on the use of a Venn diagram. Moreover, the COVID-19 targets were adapted from PubChem (https://pubchem.ncbi.nlm.nih.gov/) (accessed on 24 October 2021). The final targets between the selected compounds-related targets and COVID-19 targets were visualized by a Venn diagram plotter.Based on SMILES, targets related to the selected compounds were identified via both SEA (er 2021)  and STP https://string-db.org/) (accessed on 27 October 2021). In the PPI network, the size of circle represents the degree of value. In particular, a target indicated in red color in the most central position was considered the most significant target.The final targets were utilized to construct a protein–protein interaction (PPI) network on STRING (https://string-db.org/) (accessed on 27 October 2021). A bubble chart was constructed according to the rich factor, which is defined as the proportion of the number of genes expressed differentially in a signaling pathway . Thus, wThe STC networks were illustrated the relationships of the three components  and obtained the most critical target in the correlation. In the network, yellow rectangles (nodes) stood for the signaling pathways; blue triangles (nodes) represented targets; and pink circles (nodes) denoted bioactive compounds. The size of the blue triangles marked the number of correlations with the signaling pathways; the size of the pink circles depicted the number of connections with the targets. The merged networks were completed in RPackage. https://www.rcsb.org/) (accessed on 28 October 2021). The targets were converted .pdb format into .pdbqt format via AutoDock (http://autodock.scripps.edu) (accessed on 30 October 2021).The bioactive compounds were associated with the key signaling pathway were extracted .sdf format from PubChem, which were converted into .pdb format using Pymol, and then they were changed into .pdbqt format via AutoDock. The number of three proteins on the MAPK signaling pathway, i.e., TNF (PDB ID: 5YOY), RELA (PDB ID: 2O61), and VEGFA (PDB ID: 3P9W) were obtained using STRING via RCSB PDB , TNF , and VEGFA . The cubic box size of active site was set at x = 40 Å, y = 40 Å, and z = 40 Å. The 2D molecular docking studies were performed in LigPlotWe obtained the 82 bioactive compounds in LE through NPASS database and physicochemical properties of these compounds are listed in As shown in In the PPI networks, the TNF target was considered to be the most significant target with the highest degree of value (15) . MoreoveThe Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that the 19 targets were associated directly with 18 signaling pathways  . The ideHomo sapiens” setting was analyzed. The binding energy of R1-RELA, R2-RELA, R3-RELA, R4-RELA, and R5-RELA demonstrated at −7.1, −6.2, −5.7, −5.5, and −5.4 kcal/mol, respectively  to measure the binding energy. The MDT of the affinity between R1–R5 and RELA target (PDB ID: 2O61) in the “ectively . Methyl ectively A. The biectively . Tormentectively B. The biectively . Eugenolectively C. CollecThe compounds–targets network indicated that LE compounds might be significant ligands that could be used to alleviate COVID-19 symptoms. Of these, RELA has a more significant effect than any other kinds of targets. Furthermore, based on the pathway enrichment, the MAPK signaling pathway is the uppermost mechanism of LE against COVID-19. Thus, three targets  linked to the MAPK signaling pathway might be promising targets for use against COVID-19. Accordingly, the docking score on the three targets suggested that three bioactive compounds  were considered as the most notable compounds of LE against COVID-19. Meanwhile, the results of the KEGG pathway enrichment analysis showed that nine targets might play important roles against COVID-19. The relationships of the 18 signaling pathways with anti-virus were discussed as follows.AGE-RAGE signaling pathway in diabetic complications: Activation of the binding of AGE to its receptor RAGE can stimulate cytokine production, can cause tissue damage, and the suppression of AGE-RAGE can effectively reduce inflammation .Adipocytokine signaling pathway: Most adipocytokines are pro-inflammatory factors and they are closely linked to chronic inflammation ,30.RIG-I-like receptor (RLR) signaling pathway: The RIG-I-like receptors (RLRs), RIG-I, MDA5, and LGP2, play an important role in pathogen recognition of RNA virus infections that instigate and regulate antiviral immunity .IL-17 signaling pathway: IL-17 can play vital roles in responding to pathogenicity in diverse tissues, as well as being important for inflammation balance and tissue cohesion during viral attacks .Toll-like receptor (TLR) signaling pathway: Nucleic acids originating from bacteria and viruses can be recognized by the intracellular Toll-like receptor (TLR) and they are also sensitive to self-nucleic acids in disease conditions such as autoimmunity .HIF-1 signaling pathway: The dysfunction of HIF-1α develops influenza A virus (IAV) replication by triggering autophagy in alveolar epithelial cells .NF-κb (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway: Viruses have evolved to exploit NF-κb-driven cellular functions, and the understanding of molecular mechanisms might be a new strategy against viral diseases .Sphingolipid signaling pathway: Sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate, are signaling messengers that tune a wide range of cellular processes and are essential for immunity, inflammation, and inflammatory disorders .NOD-like receptor (NLR) signaling pathway: NLRs have been linked to human diseases, including infections, inflammatory disorders, and even chronic inflammation .Chemokine signaling pathway: In COVID-19 patients, inhibiting the secretion of cytokines and chemokines dulled the cytokine storm that represented the severity of the disease and was a negative side effect .PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway: The regulation of PPAR-α  with agonists enhanced herpesvirus replication and reactive oxygen species (ROS) production .MAPK (Mitogen-Activated Protein Kinase) signaling pathway: it was reported that the virus’s existence in hosts could activate the MAPK signaling pathway; some viral specific proteins can maintain the persistent activation of the MAPK signaling pathway .T cell receptor (TCR) signaling pathway: The TCR recognizes pathogens on major histocompatibility complex molecules with the cooperation of CD4 (Cluster of Differentiation 4) or CD8 (Cluster of Differentiation 8) co-receptors and produces cytokines .TNF  signaling pathway: TNF-α boosters influenza A virus-induced production of antiviral cytokines by activating RIG-I (Retinoic acid-inducible gene I) gene expression .Relaxin signaling pathway: Relaxin receptor abnormality enhances vascular inflammation and damages external remodeling in arteriovenous fistulas .cAMP (Cyclic Adenosine MonoPhosphate) signaling pathway: cAMP stimulates interleukin-10 production as the anti-inflammatory cytokine .RAS (Renin-Angiotensin System) signaling pathway: The use of RAS antagonists might increase the risk of developing a SARS-CoV-2 infection. However, it is not sufficient evidence for discontinuing RAS blockers in patients with hypertension .Based on the pathway enrichment analysis, RELA was considered as a hub target in LE against COVID-19. The RELA was directly enriched in 17 out of 18 signaling pathways by the MAPK signaling pathway, indicating that the MAPK signaling pathway might be a hub signaling pathway in LE against COVID-19. The other two targets (TNF and VEGFA) that are directly related to the MAPK signaling pathway might be important targets for creating synergistic effects against COVID-19. A report demonstrated that inhibition of RELA of the NF-κB component reduce cytokine production and thus could alleviate inflammation severity . Most reEndothelial cell inflammation is a serious symptom of COVID-19 infection, and its uncontrollable cytokine production in tissues and cells causes a severe immune reaction, which is defined as a “cytokine storm” that results in aggravating pneumonia. Moreover, inhibition of the other two targets (TNF and VEGFA) related to the MAPK signaling pathway contribute to anti-proinflammation and anti-vascular permeability against COVID-19. Therefore, the key mechanism of LE against COVID-19 might be the ability to block inflammation and vascular permeability in tissues and/or cells by inactivating the MAPK signaling pathway . HoweverThus, we need to validate the pharmacological mechanism via in vitro or in vivo tests. Thereby, the usefulness of computational approach might be represented. As a matter of fact, network pharmacology is a holistic perspective to search for multiple factors against specific diseases, which might provide significant clues about the relationships between components such as signaling pathways, targets, and compounds.The bioactive compounds and mechanism(s) of LE against COVID-19 were first explored using network pharmacology. The findings of this study suggest that the final bioactive compounds were methyl 4-prenylcinnamate, tormentic acid, and eugenol and their targets were RELA, TNF, and VEGFA, respectively. The mechanism(s) of LE against COVID-19 might inhibit cell inflammation and permeability against COVID-19 by inactivating the MAPK signaling pathway. This research provides a scientific indication to support LE’s therapeutic effects on COVID-19, and thus, the proper application of these three bioactive compounds against COVID-19 might lead to promising synergistic effects such as anti-inflammation and anti-permeability in order to alleviate COVID-19 symptoms. In parallel, this study needs to be further verified through in vitro or in vivo models.


In addition, glucuronidation, sulfation, methylation, hydroxylation, and reduction were observed as the major metabolic pathways of PF. The constructed compound–target–pathway network revealed that 11 absorbed constituents associated with the 16 relevant targets could be responsible for the protective activity of PF against ALD by regulating nine pathways attributable to glycolysis/gluconeogenesis, amino acid metabolism, and lipid regulation as well as inflammation and immune regulation. In addition, four active ingredients  as well as two target genes (MAO-A and PPAR-α) were screened and validated to play a crucial role with a good molecular docking score. The present results not only increase the understanding on the effective form and molecular mechanisms of PF-mediated protection against ALD but also promote better application of PF as a supplement food and herbal medicine for the treatment of ALD.Puerariae Flos, a representative homology plant of medicine and food for alcoholism, has a long history of clinical experience and remarkable curative effect in the treatment of alcoholic liver disease (ALD). However, its effective forms and hepatoprotective mechanisms remain unknown. In the present study, a strategy based on UPLC-QTOF MS combined with mass defect filtering technique was established for comprehensive mapping of the metabolic profile of PF in rat plasma, urine, bile, and feces after oral administration. Furthermore, the absorbed constituents into plasma and bile with a relatively high level were subjected to the network analysis, functional enrichment analysis, and molecular docking to clarify the potential mechanism. Finally, the therapeutic effect of PF on ALD and predicted mechanisms were further evaluated using a rat model of alcohol-induced liver injury and Western blot analysis. In total, 25 prototype components and 82 metabolites, including 93 flavonoids, 13 saponins, and one phenolic acid, were identified or tentatively characterized   Almeida or P. montana var. lobata (Willd.) Maesen and S.M. Almeida ex Sanjappa & Predeep. As a homology plant of medicine and food, it has been traditionally used to relieve toxic symptoms caused by excessive alcohol consumption, such as hangover, nausea, headache, and red face in China, Japan, and Korea for over 1,500 years , known as “Ge-hua” in Chinese, is botanically from the dried flowers of 00 years . Consequ00 years , hypogly00 years , hypolip00 years , estroge00 years , and ant00 years . With re00 years , among w00 years .in vitro has been reported by some scholars  M.R. Almeida in rat plasma, urine, bile, and feces were first characterized by UPLC-QTOF MS. Furthermore, with subsequent visualization of “ingredient–target–pathway–disease” association network constructed by using a network analysis and the binding interactions between key ingredients with targets performed by molecular docking simulation, the potential active components and underlying pharmacological mechanisms for the effect of PF on ALD were explored. Furthermore, the predicted key targets of PF against ALD were validated in an alcohol-induced liver injury rat model, which would promote better application of PF, which is a medical resource for developing a supplement food or an herbal medicine for the treatment of ALD (Graphic abstract).In this study, the absorbed and excreted prototypes and metabolites of the extract of Pueraria montana var. thomsonii (Benth.) M.R. Almeida by Prof. Dan Yuan . A voucher specimen was deposited at the authors’ laboratory. The reference compounds daidzein  and luteolin  were purchased from Funakoshi Co., Ltd.  and Chengdu Pufei De Biotech Co., Ltd. , respectively.Puerariae Flos (Batch No. 161001) collected from Anhui Province was purchased from the Tong Ren Tang TCM store  in October 2017 and was authenticated as the flower of Pueraria montana var. thomsonii (Benth.) M.R. Almeida or Pueraria lobate (Willd.) Ohwi. in our previous studies , tectoridin (12), genistein (23), tectorigenin (25), and irisolidone (32) were isolated from the extracts of the flowers of  studies . The purPharmaceutical Co., Ltd..HPLC-grade acetonitrile, methanol, and formic acid were supplied by Fisher Scientific Company Inc. . Ultra-pure water (18.2 MΩ) was prepared using a Milli-Q water purification system . All other reagents were of analytical grade and purchased from Shandong Yuwang Rabbit monoclonal antibodies against peroxisome proliferator–activated receptor α , monoamine oxidase type A , and β-actin (Cat. No. AC026) were obtained from Abcam  and ABclonal Inc, , respectively. A protein extraction kit was purchased from KeyGen Biotech Co., Ltd. .Pueraria montana var. thomsonii (Benth.) M.R. Almeida (1 kg) were weighed accurately and reflux-extracted twice with 80% EtOH  for 1 h each time. After filtering with a six-layer absorbent gauze, the two filtered extracts were combined, concentrated under vacuum to 1 L , and finally transformed into a freeze-dried powder.Dried flowers of Now, 50 mg of prepared powder was dissolved again with 10 ml of methanol/water  and extracted for 30 min under ultrasound. After centrifugation at 13,000 rpm for 10 min at 4°C and filtration through a 0.22-μm filter, 1.0 μL of filtrate was injected to UHPLC-QTOF MS for a qualitative analysis.To calculate the administered dose, the contents of the three major ingredients were quantitatively determined by reported HPLC-UV using an external standard method . The resad libitum in metabolic cages for 1 week before the experiments. The animals were divided into three groups at random: a dosed plasma collection group (n = 3), a dosed urine and feces collection group (n = 3), and a dosed bile collection group (n = 3). All animals were fasted for 12 h before the experiments and provided with free access to water and sugar over the period of sample collection. PF extracts were suspended in a 0.5% carboxy-methyl cellulose sodium salt aqueous solution with a concentration of 0.11 g/ml and administered by oral gavage at a dose of 1.1 g/kg body weight  to rats. All experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University (License number: SCXK (Liao) 2015-0001).A total of nine male Sprague–Dawley rats (200 ± 20 g body weight and about 6- to 8-week-old) purchased from the animal center of Shenyang Pharmaceutical University were maintained in ambient houses (22 ± 2°C) with a 12-h light/12-h dark cycle. For acclimatization, rats were allowed soy-free food and water Serial blood samples (approximately 0.5 ml) were collected from the suborbital vein and placed in heparinized polythene tubes at 0, 0.5, 1, 1.5, 2, 3, 4, and 8 h after oral administration and then immediately centrifuged at 3,500 rpm for 10 min at 4°C to obtain plasma. Urine and bile samples were, respectively, collected at 0–2, 2–4, 4–8, 8–12, and 12–24 h after the dosing. Feces samples were collected at 0–12 and 12–24 h and then left in a cool and dry place to dry. The collected plasma, urine, and bile samples were mixed and pretreated using solid phase extraction, while the feces samples were extracted with ultrasound in methanol/water , according to our previous method .The condition of chromatographic separation and mass detection was almost the same as those reported in the literature . Water cin vivo by UPLC-QTOF MS/MS, the chemical structure of absorbed constituents in plasma and bile with relative content higher than 3% was obtained as a SDF format by using ChemBioDraw Ultra 12.0 software, and then it was submitted to the Swiss Target Prediction platform (http://www.swisstargetprediction.ch/) to predict the most probable protein targets. The official gene names of top 100 targets with high matching degrees were selected for subsequent analysis. The targets associated with “alcoholic liver disease” were acquired from OMIM, TTD, CTD, GAD, DisGeNET, and GeneCards databases.After identifying the absorbed and excreted ingredients of PF https://string-db.org/) provides predicted PPI information as well as the data which have been experimentally confirmed. The version 11.0 of STRING was applied to acquire the PPI information, with the species limited to “Homo sapiens” and a confidence score > 0.9. These PPI targets were defined as core targets for further analysis.The Search Tool for the Retrieval of Interacting Genes (STRING) database  to find the signaling pathways related to candidate targets, and then the ALD-related pathways were selected.The KEGG pathway analysis was performed by Database for Annotation, Visualization, and Integrated Discovery . The binding energy could be accomplished by the formation of binding pockets after preparation of ligands and receptors by removal of water molecules and original ligands, addition of hydrogen atoms, and repairmen of amino acids. “Total Score” was used as the indicator and positive correlation with docking preference. A score ≥ 4 was considered meaningful, which mean that there was a binding between the constituents and the targets.The Surflex docking program in Sybyl X2.0 was utilized to evaluate the binding energies and interactions between key active compounds and targets. The crystallographic structures of 16 target proteins were retrieved from the RCSB Protein Data Bank database (−1) group, and tiopronin treatment (60 mg⋅kg−1) group (n = 10 in each group). The medicine-treated rats were pretreated with PF extracts or tiopronin by intragastric administration twice daily for 1 week before the first dose of ethanol and at 1 h before each administration of ethanol doses for 4 weeks, whereas the rats in the control and model groups were given equivalent volume of 0.5% sodium carboxymethylcellulose. Except for the control group, the ALD model group was simultaneously induced by orally feeding 56% Erguotou wine (10 ml/kg/d) by gavage for 4 weeks.SD rats were randomly divided into four groups, including control group, alcoholic liver injury (ALD) model group, PF treatment , aspartate transaminase (AST), and alkaline phosphatase (ALP) in the serum were measured using an automatic biochemistry analyzer . The middle lobe of liver tissues was collected, sectioned, and fixed in 4% paraformaldehyde for at least 24 h. After being dehydrated in ethanol and embedded in paraffin, a series of paraffin sections (5 μm) were stained with hematoxylin-eosin (H&E) for histopathological examination.The liver tissues were lysed with a lysis buffer containing 1% PMSF, phosphatase inhibitors, and protease inhibitors and incubated in an ice bath for 30 min to extract total protein. The concentration of the extracted protein was measured by using the BCA quantitative method. Equal amount of protein was electrophoretically separated by 10% SDS-PAGE. After electrophoresis, the protein was transferred on PVDF membranes and then blocked in 5% milk with TBST for 2 h. The membranes were immersed in primary antibody of PPAR-α , MAO-A , and β-actin , respectively, at 4°C overnight; the next day, they were incubated with horseradish peroxidase–conjugated secondary antibody at room temperature for 2 h. Protein bands were detected with ECL Plus chemiluminescence reagent and quantified using ImageJ software . The values for each target protein were normalized to β-actin.p < 0.05 was considered as statistically significant.All values were expressed as mean ± standard deviation (SD). Differences between different groups were analyzed with one-way analysis of variance (ANOVA) using GraphPad Prism 8.0.1. The value of in vivo metabolites in rats, a total of 43 chemical constituents, including 22 isoflavonoids, 14 saponins, six flavonoids, and one phenolic acid were first identified or tentatively characterized in PF extracts by UPLC-QTOF MS based on our previous literatures  and glycosides (P1∼P8), respectively. In addition, five , 25 (P1∼P25), three (P6∼P8), and six  prototype components were observed separately in rat plasma, urine, bile, and feces samples. As for the metabolites, glucuronidation, sulfation, methylation, hydroxylation, and reduction were their major metabolic pathways. 76 metabolites, including 24 sulfates, 21 glucuronides, 17 aglycones, eight glucuronide–sulfates, four diglucuronides, and two disulfates were identified .2 spectra with high energy showed characteristic 1,3 A− and 1,3 B− ions origin from a retro-Diels-Alder (RDA) cleavage of the C ring as well as losses of CH3 (15 Da), O (16 Da), H2O (18 Da), CO (28 Da), CO2 (44 Da), and/or combination of the fragments mentioned before. Meanwhile, the reduction at 2,3-double bond of the C ring and rearrangement between C-6 and C-8 positions were also common in the in vivo metabolic pathway of PF flavonoids according to the related literature  as the aglycone, and they presented similar fragmentation pathways. P18 and P20 with precursor ions detected at m/z 911 (M−H)− are a pair of isomeric with an identical elemental composition of C47H76O17. Both displayed a series of characteristic fragment ions at m/z 765, 615, 457, and 437 by simultaneously losing sugar units including 146 Da (rhamonose), 132 Da , 176 Da (glucuronosyl), and 18 Da (H2O) at the site of C-3. They were ascribed to astragaloside VIII and soyasaponin II, respectively (m/z 635.4177 (C36H60O9) and m/z 649.4307 (C37H62O9), which were 2Da (2H) and 16 Da (CH2+2H) higher than that of the soyasapogenol B-3-β-D-glucuronide, suggesting that both were reduced and methylated metabolites. The reduction at 12,13-double bond of the C ring is a metabolic pathway for saponins such as α-hederin (m/z 473.3635 (C30H50O4) was 16 Da (O) higher than that of aglycone, which was identified as 1-hydroxyl-soyasapogenol B because C-1 was the active site according to the related research , which was 30 Da (-CH2-O) lower than that of kakkasaponin I (P23). Thus, it was demethylated and dehydroxylated metabolite of P23. Since there is only one hydroxyl substituted at C-22, M77 was deduced as dimethyl-22-dehydroxyl-kakkasaponin I. Analogously, M79 and M80 were identified as methylated and C1, C21-dihydroxyl of aglycone soyasapogenol E based on the similar metabolic pathway of saikosaponin G and glycyrrhetinic acid . Conversely, supplementation of PF could effectively reduce the activities of AST and ALT as well as increase the level of ALP (p < 0.05). Similarly, tiopronin reduced the content of ALT (p < 0.05), while there was no statistical difference in the AST and ALP activities (p > 0.05). H&E staining results indicated that PF and tiopronin markedly alleviated ethanol-triggered microvesicular steatosis with mild swelling and nuclear deviation and red blood cells overflow as well as certain lipid vacuolation in hepatocytes . As hypothesized, treatment with PF significantly recovered the protein expression of MAO-A and PPAR-α compared to the model group (p < 0.05). Notably, the effect of PF was approximately equal to that of tiopronin. The verification results are consistent with the network prediction results.To validate whether the targets MAO-A and PPAR-α were involved in the protective effect of ALD by PF, their protein levels in the liver tissues were examined. As shown in in vivo can narrow the screening scope of effective forms and build a foundation for the follow-up mechanism research of PF in treating various diseases. In the present study, a global metabolic profile of PF including the identification and classification was provided by detecting the metabolites in rat plasma, urine, bile, and feces after oral administration owing to high sensitivity of the UPLC-QTOF MS system. Moreover, the relative content of the prototypes and metabolites in the four biological samples were determined to evaluate the contribution of different metabolic reactions in vivo. As shown in In general, PF is given as a tea drink or compounded preparations with other herbal medicines by oral administration in the daily life or clinic therapy. Thus, exploring the constituents absorbed and excreted in vivo metabolism or biotransformation for flavonoids is a process to transform them into more hydrophilic metabolites, which not only enhanced their oral absorption and bioavailability but also enhanced the excretion from the body via the bile and urine. in vivo and then underwent enterohepatic circulation. Therefore, the metabolites with higher relative content in bile were also selected for a follow-up mechanism analysis.If we evaluate the contribution of different metabolic reactions to the metabolite formation according to their number and relative content, we could find that sulfation and glucuronidation are both major metabolic reactions for PF flavonoids. The route of O-glucuronide; quercetin-3-O-glucuronide; quercetin-4′-O-glucuronide; quercetin-3′-O-sulfate; and luteolin-7-O-glucuronide, have also been proved to possess some pharmacological activities, including anti-inflammatory  and three metabolites genistein-7-glucuronide-4′-sulfate (M11), tectoridin-4′-sulfate (M17), and 6″-O-xylosyltectoridin-4′-sulfate (M16) may be the effective forms in the treatment of PF on ALD. The latter three belong to conjugated metabolites. Over the past two decades, several studies showed that glucuronides, sulfates, or bis-conjugates were the major existent form of flavonoids in systemic circulation and closely associated with their pharmacological actions. Our previous study found that tectorigenin-7S (M55) and tectorigenin-7G (M36) exhibited stronger inhibitory activity against aldose reductase than tectorigenin (P15) . In addiammatory , anti-oxammatory , antitumammatory , and triammatory . TherefoP6), 6″-O-xylosyltectoridin-4′-sulfate (M16), and tectoridin-4′-sulfate (M17) were strongly associated with fatty acid metabolism, adipocytokine signaling pathway, arginine and proline metabolism, NOD-like receptor signaling pathway, and glycolysis/gluconeogenesis via targeting ALDH2 and TNF. 6″-O-xylosyltectoridin (P6) was also involved in the NOD-like receptor signaling pathway and glycolysis/gluconeogenesis via regulating the candidate targets HSP90A1 and TPI1. Remarkably, GOT2 and CHKB, involved in the regulation of lipid and amino acid metabolism, were only found to be related with the metabolites genistein-7G-4′S (M11), 6″-O-xylosyltectoridin-4′-sulfate (M16), and tectoridin-4′S (M17). These results showed that the protective role of PF depended on the interacting and synergetic of both prototypes and metabolites. The prototypes are more likely to act on glycolysis/gluconeogenesis as well as inflammation and immune regulation, while the metabolites are highly involved in regulation of lipid and amino acid metabolism.When compare the difference in connecting targets, 6″-O-xylosyltectoridin , adipocytokine signaling pathway (hsa04920), and PPAR signaling pathway (hsa03320), selected on the basis of the enrichment by five genes ACOX1, CHKB, ALDH2, PPARA, and TNF, participate in the lipid regulation process of PF. Peroxisome proliferator–activated receptor α (PPAR-α), a key nuclear transcription factor abundantly expressed in the liver, plays a major role in lipid metabolism regulation. Several studies reported that PPAR-α agonists were expected to be a treatment for ALD by reducing oxidative stress, regulating fatty acid synthesis and oxidation, inhibiting the production and release of inflammatory and profibrogenic factors, and alleviating liver tissue damage . As the Abnormal amino acid metabolism, including tyrosine metabolism (hsa00350), phenylalanine metabolism (hsa00360), and arginine and proline metabolism (hsa00330), is closely related to intestinal flora metabolism, and bacterial translocation from the gut microbiota into the portal blood stream is an important driver of ALD . MAO-A, An increasing number of studies have shown that inflammation and immune regulation was also critical for the progression of ALD. Heat shock protein 90 (hsp90), an emerging therapeutic target in ALD, was a main effector in the NOD-like receptor signaling pathway (hsa04621). It is involved in initiation of the early phases of ER stress contributing to stimulation and accumulation of hepatic lipids . Hsp90 iin vivo experiments, which preliminarily prove the scientific nature of this method. In addition, some of the compound–target–pathway interactions predicted by the network analysis have also been confirmed in the previous studies. For example, protective effects of genistein against chronic alcohol-induced liver injury in mice were related to regulate expression of inflammatory-related factors TNFα, NF-κB, and PTGS1 (Belamcanda chinensis (L.) DC., could protect against ethanol-induced liver steatosis mainly by modulating the disturbance of the PPAR-α pathway and ameliorating the mitochondrial function (In this study, the bioinformatic method was combined to elucidate the active components and mechanism of PF in the treatment of ALD. Some of the predicted results are confirmed by nd PTGS1 . Tectorifunction . In addifunction . Howeverin vivo metabolites, the phase II metabolites including glucuronides, sulfates, and/or bis-conjugates that account for the most part were difficult to isolate due to their exclusivity distribution in the biological matrix rather than the plant kingdom. Therefore, the absence of metabolite references restricted further affinity evaluation. On the other side, the single dose in the present study was chosen to continue and compare with our previous metabolism research, which determined the pharmacokinetic parameters of tectoridin and tectorigenin after oral administration at dosages of 200 and 130 mg/kg, respectively (There are still two shortcomings in this study. On the one hand, benchwork assessing affinity using several technologies such as the surface plasmon resonance biosensor should be combined with a docking analysis to evaluate and validate the importance of relative compounds and targets. As our research focus on the ectively . Howeverin vivo for the first time. Glucuronidation, sulfation, methylation, hydroxylation, and reduction are the major metabolic reactions. Furthermore, the constructed absorbed constituent–target–pathway–disease network and the docking analysis revealed that phase II metabolites may play more important roles in the PF-mediated protection against ALD. Also, the protective effects and predicted mechanism associated with decreased and elevated expression of MAO-A and PPAR-α in rat ALD models were also validated (The present study has developed a sensitive and rapid method for the separation and identification of the absorbed constituents and metabolites of PF alidated . However


Radix Scutellariae, Fructus Gardeniae, and Pulvis Fellis Suis. Recent study showed that QZZD and its effective components played important roles in anti-inflammation, antioxidative stress and preventing brain injury. It was noted that QZZD had protective effects on the brain, but the mechanism remained unclear. This study aims to investigate the mechanism of QZZD in the treatment of AD combining network pharmacology approach with experimental validation. In the network pharmacology analysis, a total of 15 active compounds of QZZD and 135 putative targets against AD were first obtained. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were then applied to clarify the biological mechanism. The anti-inflammatory mechanism of QZZD was proved, and a synthetic pathway–TNFR1-ERK1/2-NF-κBp65 signaling pathway was obtained. On the basis of the above discoveries, we further validated the protective effects QZZD on neurons with an APP/PS1 double transgenic mouse model. Weight change of the mice was monitored to assess QZZD’s influence on the digestive system; water maze experiment was used for evaluating the effects on spatial learning and memory; Western blotting and immunohistochemistry analysis were used to detect the predicted key proteins in network pharmacology analysis, including Aβ, IL-6, NF-κBp65, TNFR1, p-ERK1/2, and ERK1/2. We proved that QZZD could improve neuroinflammation and attenuate neuronal death without influencing the digestive system in APP/PS1 double transgenic mice with dementia. Combining animal pharmacodynamic experiments with network pharmacology analysis, we confirmed the importance of inflammation in pathogenesis of AD, clarified the pharmacodynamic characteristics of QZZD in treating AD, and proved its neuroprotective effects through the regulation of TNFR1-ERK1/2-NF-κBp65 signaling pathway, which might provide reference for studies on treatment of AD in the future.There is yet no effective drug for Alzheimer’s disease (AD) which is one of the world’s most common neurodegenerative diseases. The Qin-Zhi-Zhu-Dan Formula (QZZD) is derived from a widely used Chinese patent drug–Qing-Kai-Ling Injection. It consists of  The aging population in the world sees increasing incidence of Alzheimer’s disease (AD) . It is eRadix Scutellariae, Fructus Gardeniae, and Pulvis Fellis Suis. It is derived from a traditional Chinese patent drug–Qing-Kai-Ling Injection that is approved by the China Food and Drug Administration   , and has, China) . Clinica, China) . We prel, China) . The speNetwork pharmacology provides a new paradigm in study on traditional medicines . It combWe designed a basic pharmacodynamic experiment and further combined the network pharmacology method for a comprehensive study of the pharmacodynamic mechanisms involved in the treatment of AD .1 3 and PubMed.4 The calculation method of ADME/T  (MW < 500), partition coefficient less than 5 (AlogP < 5), no more than 5 hydrogen bond donors (Hdon < 5), and no more than 10 hydrogen bond acceptors (Hacc < 10) , on the basis of which, related targets were obtained after removing the duplicates.To find active compounds of QZZD and related targets, we utilized Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)1  in combioxicity)  was seleoxicity) . The rulcc < 10) . AlogP volecules . Hdon anrameters . The act5  network  reflects studies . The pur studies . The pot studies  were imp studies  for visup < 0.05, and the biological processes or pathways of the lowest p value were identified after excluding irrelevant pathways. GO enrichment was mainly used to understand the main action process of the target, and the KEGG pathway analysis was used to observe distribution of the targets in the pathway. Then, we chose the top 10 pathways for construction of the “Target-Pathway” network of QZZD in the treatment of AD and used Cytoscape 3.7.1 for visualization. Furthermore, in order to investigate the roles of protein targets in the pathophysiological network of AD and how QZZD acts on AD by regulating certain pathways, an “AD-integrated pathway” was proposed based on our present understanding of AD pathology and target identification (The clusterProfiler package in Bioconductor (version 3.1.2)  and R sofication .Radix Scutellariae and Fructus Gardeniae were purchased from Anxing Chinese Herbal Medicine Co., Ltd. . Pulvis Fellis Suis was purchased from Shengtai Chinese Herbal Medicine Co., Ltd. . Donepezil hydrochloride (DNZ) was obtained from Eisai China Inc. .Radix Scutellariae, Fructus Gardeniae, and Pulvis Fellis Suis) were mixed at a ratio of 10:6:1.5. The extraction and quality control were implemented according to our previous study  was used in a previous study to analyze the composition of the QZZD. It was implemented on an UHPLC system  with a Waters UPLC BEH C18 column (1.7 μm 2.1 *100 mm). The flow rate was set at 0.4 ml/min and the volume of QZZD sample was set at 5 μl. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was as follows: 0–3.5 min, 95–85% A; 3.5–6 min, 85–70% A; 6–6.5 min, 70–70% A; 6.5–12 min, 70–30% A; 12–12.5 min, 30–30% A; 12.5–18 min, 30- 0% A; 18–5 min, 0–0% A; 25–26 min, 0–95% A; 26–30 min, 95–95% A. An Orbitrap Exploris 120 mass spectrometer coupled with an Xcalibur was employed to obtain the MS and MS/MS data based on the information dependent acquisition mode. And initially identified baicalin, geniposide, cholic acid, hyodeoxycholic acid, and chenodeoxycholic acid . The resn = 12), while a total of 36 APP/PS1 double transgenic mice were randomly divided into model group , QZZD group , and DNZ  group . It should be noted that APP/PS1 double transgenic mice present with abnormal expression of Aβ that will lead to extracellular deposition and plaque formation . The animals were euthanized through inhalation of excessive isoflurane after the water maze experiment. Their brains were immediately removed after sacrifice so that follow-up experiments could be carried out.After one week of adaptive feeding, the weight was recorded at the same time per week for subsequent statistical analysis.Water maze experiment is a classic behavioral experiment aiming to detect spatial learning and memory ability and define changes of cognitive functions before and after administration in mice with dementia. The Morris Water Maze (MWM) test includes the place navigation test and the spatial probe test . After 8(1)The place navigation test: the mice were placed into the pool from 4 quadrants successively with their heads toward the wall, and the time they spent reaching the hidden platform was recorded. If they failed to finish in 60 s, we guided them to the platform and allowed them to rest for 10 s. The test lasted for four continuous days.(2)The spatial probe test: the hidden platform was removed on the 5th day, and the mice were put into the pool from the opposite of the target quadrant. The system automatically recorded the time when the mice first reached the platform within 60 s, the number of times they had crossed the original platform, the time they had stayed in the target quadrant, and the total moving distance within the specified time. EthoVision XT software  was then used for analysis.Hematoxylin and eosin (HE) staining was used to observe neuronal morphology in the CA1 region of the mouse hippocampus. Mice were sacrificed and their brain tissues were immediately fixed in 4% paraformaldehyde solution. Afterward, the brain tissues were embedded in paraffin and cut into 5 μm slices. Then the slices were put on a slide and dried at 38°C for 24 h. The dried slices were dewaxed and rehydrated before being stained with hematoxylin for 5.5 min. Then they were rinsed in running tap water for 10 s, and finally stained with eosin for 8 s before dehydration and mounting.After the end of administration, the freshly dissected brain tissue (7 μm) was fixed in formalin and embedded in paraffin for preparation of sections. The sections were then deparaffinized and rehydrated before being repaired in citrate buffer (pH 9.0) at the temperature of 80°C for 50 min. Then 3% hydrogen peroxide was used to quench endogenous peroxidase before being blocked with 10% goat serum in an incubator (37°C) for 30 min. Thereafter, the sections were incubated with anti-NeuN antibody  at the temperature of 4°C for 48 h. The secondary incubation was conducted with goat anti-mouse DyLight 488 antibody  at room temperature (37°C) for 2 h. The next step was DAPI staining, which is followed by quenching of fluorescence and mounting. After that, morphology of sections was observed under a laser scanning confocal fluorescence microscope and photographed at the indicated sites. Quantified analysis was performed to positive cells present in the sections at lens of 20 × and 40 ×, respectively, and the number of NeuN-positive cells was calculated in a random microscope field of each slice by CellF software.After the end of administration, the hippocampal tissues of the mice were collected before lysis in RIPA buffer on ice. The supernatant was acquired after centrifugation. Then protein concentration of the sample was determined using bicinchoninic acid (BCA) assay, which was followed by protein separation by sodium dodecyl sulfate-polyacrylamide denaturing gel electrophoresis (SDS-PAGE). The next step was transferring the separated proteins from the gel onto polyvinylidene fluoride (PVDF) membrane with wet electroblotting. The membrane was blocked with 5% bovine serum albumin (BSA) in a mixture of Tris-buffered saline and Tween-20 (TBST) for 1 h. Then it was incubated with β-actin (1:1000) and primary antibodies against TNFR1 , NF-κB p65 , ERK1/2 , p-ERK1/2 , IL-6 , Aβ  overnight at 4°C. Then the blots were incubated with the secondary antibody at room temperature for 1 h. When the immunodetection was finished, ECL assay kit  was used to visualize the protein bands which were then analyzed by Gel Image software version 4.00 .After a series of routine manipulations including deparaffinization, rehydration, repair and blocking as in 2.2.7, the sections were incubated with antibodies against Aβ , TNFR1 , NF-κB p65 , and IL-6  at 4°C overnight. Then biotin-labeled secondary antibodies were used for the second incubation at room temperature (37°C) for 20 min. The next step was 3,3′-Diaminobenzidine (DAB) staining, which was followed by hematoxylin staining of the nuclei. Then the sections were dehydrated and stabilized with mounting medium. After that, the staining area and the average optical density were calculated, and Image-Pro Plus 6.0 software  was used for analysis.P value less than 0.05 were considered to be statistically significant.SPSS  was used for statistical analysis. Data were expressed as mean ± standard deviation (SD). Experimental data of each group were analyzed by normality test and homogeneity of variance test. One-Way Analysis of Variance (ANOVA)  was usedMW < 500, AlogP < 5, Hdon < 5, and Hacc < 10), a total of 15 active compounds of QZZD were identified through TCMSP database in combination with text mining in Google Scholar, CNKI, and PubMed. See According to the four principles , the top 30 targets genes of the highest degree value were obtained, including tumor protein p53 (TP53), serine/threonine-protein kinase (AKT1), epidermal growth factor receptor (EGFR), beta cytoskeletal actin (ACTB), tumor necrosis factor (TNF), interleukin-6 (IL-6) and so on, which served as core targets in the network .p value  database in terms of molecular function (MF), cell component (CC) and biological process (BP). There were 5 related to MF, 5 to CC and 10 to BP according to p value . To be mp value were selected . After 8 weeks of intervention, the protein levels in QZZD and DNZ groups were significantly decreased compared with those in the model group. For QZZD group, the expressions of IL-6, ERK1/2 and p-ERK1/2 were decreased significantly (p < 0.01), and the expressions of Aβ, TNFR1 and NF-κBp65 were also inhibited (p < 0.05) in QZZD compared with those in the model group. Besides, the levels of proteins in QZZD group were close to those in the control group, which indicated that QZZD tended to restore the levels of these proteins. Moreover, we selected several major protein indicators, including Aβ, TNFR1, NF-κBp65, and IL-6, for immunohistochemical examination  according to the degree value. TP53 is an important tumor suppressor gene which plays a key role in regulating apoptosis, senescence, and DNA damage repair in response to cellular stress . RecentlBased on the 135 common targets, we also conducted GO enrichment analysis and KEGG enrichment analysis. GO enrichment analysis showed that oxidative stress and neuron death were important biological processes in treatment of AD. In terms of oxidative stress, although it is pathologically different from neuroinflammation in neurodegenerative diseases, such as AD, they are also correlated. Oxidative stress can modify the inflammatory response, and it has been known that some ROS and reactive nitrogen species (RNS) could promote intracellular signaling cascades, which leads to increased expression of pro-inflammatory genes . The othin vivo from the following three aspects. Firstly, the effects of QZZD on the digestive system were explored through weight change of the mice during administration; secondly, the spatial learning and memory ability of the mice were clarified through the water maze experiment; thirdly, the pathological and morphological changes in the hippocampus were examined by HE staining and immunofluorescence analysis. It was found that QZZD could not only alleviate impairments in spatial learning and memory but also attenuate neuronal death. Furthermore, we also observed that the development of AD might affect the digestive system of mice as the model group presented with decreased appetite and slow weight growth. The weight growth rate of mice in the QZZD group was higher than that in the DNZ group, which also confirmed that donepezil hydrochloride could affect the digestive system.After the network pharmacology analysis, we evaluated the effects of QZZD by experiment According to the network pharmacology analysis, we mainly focused on the anti-inflammatory mechanism of QZZD in treating AD and assembled it into a synthetic pathway , with thIn this study, the central nervous system (CNS) immune cascade in AD model mice was induced by inflammatory factors and their reactions, as a result of which immunogens were formed, and then activated NF-κB in the brain, and further promoted secretion and release of TNF-α, IL-6 and other inflammatory factors. In the process ROS was also released and mediated the inflammatory response. Thus a cycle was formed. The chronic inflammatory response produced direct or indirect toxic effects on neuronal cells, eventually leading to the development of AD. Network pharmacology analysis and experiments confirmed that QZZD regulates the TNFR1-ERK1/2-NF-κBp65 inflammation pathway in the treatment of AD. It is of great significance to study regulation of inflammatory mechanisms, which can help to understand how to reduce pathological apoptosis of neurons through modulating neuroinflammation in the brain.In this paper, we comprehensively analyzed the mechanism of QZZD against AD combining network pharmacology analysis and experimental validation. In the first part, we identified 15 important compounds of QZZD, and 135 putative targets of QZZD against AD. GO analysis and KEGG enrichment analysis revealed the anti-inflammatory mechanism of QZZD, and a synthetic pathway–TNFR1-ERK1/2-NF-κBp65 signaling pathway was obtained. When it comes to the experiment, it was found that QZZD could not only alleviate impairments in spatial learning and memory, but also improve neuroinflammation and attenuate neuronal death without influencing the digestive system in APP/PS1 double transgenic mice with dementia. These findings further confirmed that QZZD had therapeutic effects on AD through inhibiting inflammatory response in regulating the synthetic signaling pathway. The formula is characterized by multi-target and multi-pathway action and comprehensive regulation, which might provide reference for regulatory mechanism of treating dementia.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by the Ethics Review Committee for Animal Experimentation of Beijing University of Chinese Medicine (BUCM-4-2018122101-1018).QW, FC, and XW conceived the research theme. WX and BR participated in the research design and wrote the manuscript. ZZ and CM performed target prediction and analysis as well as related enrichment processes. SL and CC contributed to animal experiments. TX conducted the data analysis. XW revised the language and supervised the implementation. All authors have read and approved the final manuscript.