AH Plus penetrated significantly deeper into the dentinal tubules compared to TFHF at each root level (p < 0.05). Dentinal sealer penetration was deeper in the bucco-oral direction compared to the mesio-distal direction. AH Plus penetrated deeper into dentinal tubules than TFHF. Warm vertical compaction exerting high pressure on the root canal filling material is not able to press sealers deep into dentinal tubules as penetration depth values did not exceed a mean of 110 µm in SEM.Sealer staining using rhodamine B dye to investigate the penetration depth of endodontic sealers was proven unsuitable for this purpose. This study aimed to investigate the sealer penetration depth into dentinal tubules by scanning electron microscopy (SEM). Root canals of 52 human upper central incisors were instrumented using the ProTaper Gold NiTi system  up to size F3. After irrigation with sodium hypochlorite and citric acid combined with ultrasonic activation, the root canals were either filled using the epoxy resin sealer AH Plus (Dentsply Sirona) or the calcium silicate-based sealer Total Fill BC Sealer HiFlow  by warm vertical compaction. Root slices of 1 mm thickness were obtained at 2 to 3, 5 to 6 and 8 to 9 mm from the apex. The root slices were investigated for sealer penetration into the dentinal tubules using SEM according to four root quadrants . Statistical analysis was performed by the Kruskal-Wallis test ( Warm vertical obturation of the root canal space was introduced to increase the amount of gutta-percha core material in a root canal filling and to use the pressure applied on the thermo-plasticized material to press endodontic sealer into isthmuses and dentinal tubules . No otheRegarding sealer penetration depth into dentinal tubules, results inconsistent with the above-mentioned theorem have been published in the past. While some studies could not find a relationship between the filling technique and the penetration depth ,3,4,5, aNo reliable data exist about the penetration depth of endodontic sealers others than from studies where sealers were labelled with rhodamine B ,8. StudiThe present study was hence designed to investigate penetration depth of epoxy resin and calcium silicate-based sealers recommended for warm vertical root canal obturation into dentinal tubules. The investigation was performed using scanning electron microscopy as sealer staining using fluorescent dyes and was shown to be unsuitable to precisely indicate sealer penetration depth. The null hypothesis tested was that penetration depth would not differ between epoxy resin and calcium silicate-based sealers.Sample size calculation was performed using G*Power  and indicated a group size of at least 26 samples per group . Thus, 52 freshly extracted central upper incisors were selected for the study and randomly divided into two experimental groups. All subjects gave their informed consent for inclusion of their extracted teeth. The study was conducted in accordance with the Declaration of Helsinki.p = 0.05) as data were not normally distributed according to the Shapiro–Wilk test. Prior to root canal treatment, all teeth were investigated under a stereomicroscope  to exclude cracks or root resorption. Buccal and proximal radiographs were taken to ensure a single root canal with an intact apical region and a single apical foramen. All teeth were cut coronally to a total length of 20 mm and access cavities were prepared when necessary. Apical patency of the root canal was checked using K-Files #10 and 15, and a working length of 19 mm was established. All root canals were instrumented using the ProTaper Gold NiTi system  up to size F3. The root canal was irrigated with 2.5 ml NaOCl 3% after each file and patency was checked after each instrument. After instrumentation, all root canals were irrigated twice with 2.5 mL NaOCl 3% combined with 30 s of ultrasonic activation , 5 ml EDTA 17% and a final irrigation with 5 mL NaCl 0.9%. The roots were placed into plastic vials  and embedded into alginate  as described in a previous study ,16,17. Tp < 0.05)  .p < 0.05), while no statistical difference occurred in the other quadrants (p < 0.05). No statistical difference was found between AH Plus and TFHF according to the quadrants in the middle portion (p > 0.05). In the coronal portion, AH Plus showed significantly deeper penetration in the buccal, oral and distal section (p < 0.05)  . At all The penetration depth of endodontic sealers was evaluated by SEM investigation when a warm vertical compaction technique was used. AH Plus was found to penetrate deeper into dentinal tubules than TFHF in parts of the apical and coronal portion of the root. Therefore, the null hypothesis was rejected.Sealer penetration depth is commonly investigated using CLSM. Sealers are mixed with organic dyes such as rhodamine B and indirectly detected by fluorescence of the organic dye . It has Overall, AH Plus showed significantly higher penetration depth values than TFHF at all root levels investigated. According to the quadrants at each level, significant differences did not occur in all levels. Penetration depths in mesial and distal quadrants were generally lower than in buccal or oral quadrants, which correlate with the number and size of the dentinal tubules . In caseComparing the present results to previous studies, it is apparent that penetration depth was found only in a small extent . PenetraSealer penetration deep into dentinal tubules is implausible according to the results of the present study. Though the penetration of fluorescent dyes that were added to the sealer in several studies was reported ,20,23 noPenetration of endodontic sealers deep into dentinal tubules of the root dentin is unlikely. Even high pressure on the root canal filling material is not able to press sealer deep into dentinal tubules. AH Plus penetrated deeper into dentinal tubules than TFHF in parts of the apical and coronal portion of the root.


To provide a basis for the diagnosis and treatment of acalculous biliary pancreatitis, this study investigated the impact of serum metabolites on the pancreatic transcriptome in acute acalculous cholecystitis (AAC). 1H NMR. Two pancreatic tissue chips of the AAC group and the normal control group were prepared and sequenced. We utilized the limma package of R software, the DAVID database, the STRING database, Cytoscape software, and the CFinder analysis tool to perform differential expression gene analysis, gene function enrichment analysis, protein interaction network (PPI) construction, and network module mining, and we performed gene enrichment analysis in each module.  Fourteen rabbits were randomly divided into two groups , blood was collected from the 14 rabbits, and metabolomic analysis was performed through κB pathway, and the cancer signaling pathway.  Serum metabolism analysis showed that in AAC, the metabolism of sugar, lipids, and protein, that is, the three major nutrients, was affected to varying degrees, and levels of serum trimethylamine N-oxide (TMAO) increased. Bioinformatic methods were utilized to identify a total of 183 differentially expressed genes and 3 key genes. Enrichment analysis showed that differentially expressed genes were significantly enriched in cation transport, the inflammatory response, the NF- Metabolomic analysis and functional analysis of 3 key genes demonstrated that abnormal serum metabolites affected the pancreatic transcriptome and induced a sensitive state of inflammation in the pancreas. These metabolites may represent important targets for future research on the pathogenesis, clinical diagnosis, and treatment of noncalculous biliary pancreatitis. Acute pancreatitis (AP) is a conditions marked by acute pain in the abdomen; this disease not only affects the pancreas but also invades multiple organs. AP exhibits high morbidity and mortality. AP is primarily caused by cholelithiasis, excessive alcohol intake, hyperlipidemia, endoscopic retrograde cholangiopancreatography, and various drugs. These factors cause dysfunctional cell pathways and organelle pathology followed by the activation of pancreatic enzymes in the pancreas, which further leads to systemic inflammatory response syndrome , 2. Biliad libitum, and given at least a week to acclimate. The rabbits were fasted overnight with free access to water before experiments. The study was designed in accordance with the guidelines for the care and use of laboratory animals in research and was approved by the Ethics and Research Committee of Harbin Medical University.To study the serum metabolites in rabbits, we followed the methods of Li et al. . Female Escherichia coli was injected with a 1 ml syringe . The rabbits in the control group were injected with an identical volume of normal saline into the gallbladder after laparotomy.Fourteen healthy rabbits were randomly divided into two groups. The rabbits in the AAC group were anesthetized with 3% pentobarbital sodium, administered at a dose of 30 mg/kg by ear vein injection. A vertical incision of 4-5 cm was made in the middle of the abdomen to expose the gallbladder, and 0.3 ml of μl serum superstratum was transferred into a 5 mm nuclear magnetic tube. Next, 100 μl D2O (-for lock) was added, and the solution was vibrated and fully mixed. 1H NMR experiments were performed on a Varian 600 spectrometer with a 599.925 Hz 1H resonance frequency. One-dimensional spectra were recorded by suppressing the strong water signal using a standard NOESY1DPR pulse sequence (RD-90°-t1-90°-tm-90°-ACQ). The relaxation delay (RD) was 2.5 s, the time for mixing (tm) was 100 ms, and the cumulative frequency was 64. Saturated irradiation was performed on the water at the RD and tm. A Carr-Purcell-Meiboom-Gill (CPMG) sequence was used for the transverse (T2) relaxation weighing experiment, for which the RD was 4 s, the total echo time was 100 ms, and the cumulative frequency was 256.Three-milliliter blood samples were collected from the rabbits in both groups 72 h after the operation, and the samples were centrifuged at 1500 rpm for 5 min. The supernatants were collected and stored in a -20°C refrigerator for metabolomic analysis. The frozen serum sample was thawed at room temperature, and 400 δ 0-10. The method was as follows. First, the full spectrum of each sample (δ 0-10) was divided into 1000 sections with 0.01 ppm intervals, the integral value of the water signal (δ 4.71-5.20) was removed, the remaining 950 section integral values were added and normalized, and SIMCA-13.0 software was used to perform principal component analysis (PCA) in center-scaling mode.The range of the spectrum interval selected by principal component analysis was the signal with a chemical shift within T-test between the AAC group and the control group was employed, and the difference peak was selected with a P value < 0.05.According to the results of the PCA between the groups, the signal peaks of the above metabolites were selected, the After the pancreatic tissue was isolated, it was placed in a cryotube, marked, quickly frozen, and stored in liquid nitrogen. For RNA extraction, the pancreatic tissue samples were removed from the liquid nitrogen tank, placed in a precooled mortar, and ground for 8-10 min; next, 2-3 ml TRIzol reagent  was added to each mortar and grinding continued until the temperature of the mortar returned to room temperature; after that step, TRIzol reagent was transferred to a glass homogenizer, and samples were homogenized for 3-5 min and subsequently transferred to a 1.5 ml centrifuge tube, and total RNA was extracted according to the standard procedure for TRIzol reagent. Total RNA was purified by column with a NucleoSpin® RNA clean-up kit  and was finally quantified by a spectrophotometer and subjected to quality inspection through formaldehyde denaturing gel electrophoresis.We started with total RNA and the T7 Oligo (dT) Promoter Primer Kit containing the T7 promoter sequence as primer, and we used the CbcScript enzyme to synthesize first-strand cDNA. RNase H was used to cut the RNA in the hybrid strand into short fragments. DNA polymerase uses short RNA fragments as primers to synthesize second-strand cDNA and purify double-stranded cDNA. Using cDNA as a template, T7 Enzyme Mix was used to synthesize cRNA, and an RNA clean-up kit (MN) was used for purification. Five micrograms of cRNA, CbcScript II enzyme, and random primer were used for reverse transcription, and a PCR NucleoSpin Extract II Kit (MN) was used to purify the reverse transcription product. Taking the above reverse transcription product, we used Random Primers as the primer for KLENOW enzyme labeling, and we used the PCR NucleoSpin Extract II Kit (MN) to purify the labeled product and then drain it after purification (Cy5-dCTP or Cy3-dCTP) . The labeled DNA was dissolved in hybridization solution (2X GEx Hyb Buffer (HI-RPM), 25% formamide) and hybridized at 45°C overnight. After hybridization, the cells were first washed with 0.2% SDS and 2x SSC at 42°C for 5 min and subsequently washed in 0.2x SSC at room temperature for 5 min. After the slides were dried, an Agilent rabbit gene expression microarray was used to scan the slides and obtain the hybridization image. Feature extraction image analysis software was used to analyze the chip image and convert the image signal into a digital signal.Oryctolagus cuniculus species. We converted the gene ID in the expression matrix to a gene symbol. Finally, 898 gene expression values were obtained.The limma package of R software was used to read and preprocess the data of 4 sample chips under the Agilent platform. The preprocessing process included background correction and data normalization. We downloaded the corresponding platform annotation file from NCBI GEO , and theT-test provided by limma was used to calculate the significance P value of the differentially expressed genes. For each significantly differentially expressed gene, the differential expression P value < 0.05 and the fold change (FC) difference between the sample groups were not less than 1.5-fold . We downloaded the pancreatic gene expression dataset GSE119844 of mouse acute pancreatitis, and the pancreatic gene expression dataset GSE143754 of human chronic pancreatitis, from the NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/), and then, we used limma package of R software (version 4.03) to analyze the differential expression of genes in the two datasets, respectively.Next, we continued to use the limma package to analyze the differentially expressed genes in the AAC group and the control group. In the analysis process, the corrected P value < 0.05.For the differential expression genes in the rabbit pancreas, we used the commonly used enrichment analysis tool DAVID  to analyOryctolagus cuniculus. The parameter PPI score was set to 0.4 (indicating medium confidence), and the protein nodes that required interaction were all differentially expressed genes. Cytoscape [The STRING  databaseytoscape  was usedWe combined four calculation methods of network topology properties to analyze the importance of nodes in the network. The four methods, that is, degree centrality, betweenness centrality, subgraph centrality, and closeness centrality, were employed to analyze the scores of nodes in the network. The tool used to calculate the four centrality scores of the network was the Cytoscape plugin CytoNCA   of each network module and screened out significantly enriched results.Based on the analysis tool CFinder , subnetwY variable) into PCA, orthogonalize the previous variable matrix (X variable), and eliminate the irrelevant discriminant analysis results, as performed by X orthogonal and retained related variables. From PCA , glucose , lactic acid , glycoprotein (δ 2.04), and trimethylamine N-oxide (TMAO) (δ 3.31).From the load diagram , it coulP value < 0.05 serving as the threshold. The screening showed that there were significant differences in the changes in serum glucose , fat , glycoprotein (δ 2.04), and TMAO (δ 3.31). In this study, we primarily investigated the changes in serum TMAO  is an inflammation of the gallbladder not associated with the presence of gallstones. The main mechanisms of ACC are cholestasis  and gallκB pathway, and the tumor signaling pathway. Among these genes, the expression of TNF and TGFB1 and the downregulation of NOS3 may be affected by serum metabolites and may play important roles in the pathophysiological process of acalculous biliary pancreatitis.This study showed that during AAC, the serum TMAO content was increased, and the differentially expressed genes in pancreatic tissue were enriched in such pathways as cation transport, inflammatory responses, the NF-κB to induce inflammatory stimuli, thereby leading to increased production of inflammatory mediators and exacerbating peripheral tissue inflammation and inflammatory hyperalgesia. NF-κB is responsible for the expression of many proinflammatory factors and chemokines, and the pathways from pathogen recognition to proinflammatory cytokine production have been demonstrated to be particularly dependent on NF-κB [β, IL-6, and TNF-α by activating the p65 NF-κB pathway which, in turn, causes inflammatory diseases [κB/MAPK signaling pathway [2+ concentration in pancreatic acinar cells was the trigger point of acute pancreatitis [in vitro. After acute treatment with TMAO, calcium fluorescence in myocardial cells was increased, and the increase in intracellular calcium ions was observed when TMAO influenced cell strength [κB signaling to promote calcium/phosphorus-induced calcification of rat and human vascular smooth muscle cells in a dose-dependent manner and promoted vascular calcification [κB activation [κB can increase intracellular Ca2+ levels, thereby leading to Ca2+ overload [β, IL-6, and TNF-α to increase significantly by activating the p65 NF-κB pathway, which increases the level of Ca2+ in pancreatic acinar cells, inducing a sensitive state of pancreatitis.TMAO can be obtained directly from such foods as fish and shellfish , and choon NF-κB . Many otdiseases –30, whil pathway . The patreatitis . Studiesreatitis . High costrength . TMAO acfication . In the tivation , 37. Theoverload  and induβ, IL-6, and TNF-α in vitro [κB pathway is necessary for TMAO to induce bone marrow mesenchymal stem cells to produce proinflammatory cytokines and release active oxygen [α can directly induce premature activation and necrosis of pancreatic acinar cell proteases, which depend on the activity of calcium ions and cathepsin-B [α can inhibit the release of inflammatory factors and plays a role in the treatment of acute pancreatitis [γ and TNF-α. TMAO is related to TGFB/SmAD signal transduction [α and induces a sensitive state of pancreatitis.The TNF gene encodes a multifunctional proinflammatory cytokine involved in the regulation of various biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and blood coagulation. It has been proven that TMAO can promote the production of the proinflammatory cytokines IL-1in vitro , 40, andhepsin-B . Downregreatitis , 43. Thesduction . TMAO casduction . The NOSsduction . In cerusduction . TherefoκB pathway, and the TNF gene directly participates in the NF-κB pathway. Therefore, we surmised that elevated TMAO may increase the expression of inflammatory factors, such as TNF, through the NF-κB pathway, and these inflammatory factors can participate in the NF-κB signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, TGFB signaling pathway, and other pathways, in turn. Therefore, a positive feedback loop was formed, which led to an increasing level of Ca2+ in pancreatic acinar cells, inducing a sensitive state of inflammation and increasing the risk of pancreatitis.We obtained five subnetwork modules through protein interaction network module mining. The KEGG functional enrichment of the module 2 subnetwork module showed that node genes were mainly involved in such pathways as the T cell receptor signaling pathway, TNF signaling pathway, and TGFB signaling pathway. In the KEGG database, we found that the TNF gene directly participates in the T cell receptor signaling pathway, TNF signaling pathway, and TGFB signaling pathway, while these pathways are indirectly connected with the NF-κB pathway, and vascular smooth muscle contraction. Based on the PPI network of differentially expressed genes, three key genes, TNF, NOS3, and TGFB1, were screened out. Through metabolomic analysis and functional analysis of differentially expressed genes, we realized that there was an association between serum metabolites and pancreatic gene expression during AAC, that is, TMAO activated NLRP3 inflammasome and NF-κB signaling to promote calcium/phosphorus-induced calcification of rat and human vascular smooth muscle cells in a dose-dependent manner and promoted vascular calcification [β, IL-6, and TNF-α by activating the p65 NF-κB pathway [In summary, this study analyzed the serum metabolomics of rabbits with AAC and found that the serum TMAO content of the AAC group rabbits increased, and the change of TMAO content may facilitate the diagnosis of acalculous biliary pancreatitis. Furthermore, a total of 183 common differentially expressed genes were screened from the pancreatic tissue microarray data of the rabbit AAC model. The enrichment analysis results showed that differentially expressed genes were significantly enriched in such pathways as cation transport, the inflammatory response, the NF-fication , and TMA pathway –30, whic


Gelsemium elegans (Gardner and Champ.) Benth. (Gelsemiaceae) (GEB) is a toxic plant indigenous to Southeast Asia especially China, and has long been used as Chinese folk medicine for the treatment of various types of pain, including neuropathic pain (NPP). Nevertheless, limited data are available on the understanding of the interactions between ingredients-targets-pathways. The present study integrated network pharmacology and experimental evidence to decipher molecular mechanisms of GEB against NPP. The candidate ingredients of GEB were collected from the published literature and online databases. Potentially active targets of GEB were predicted using the SwissTargetPrediction database. NPP-associated targets were retrieved from GeneCards, Therapeutic Target database, and DrugBank. Then the protein-protein interaction network was constructed. The DAVID database was applied to Gene Ontology and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis. Molecular docking was employed to validate the interaction between ingredients and targets. Subsequently, a 50 ns molecular dynamics simulation was performed to analyze the conformational stability of the protein-ligand complex. Furthermore, the potential anti-NPP mechanisms of GEB were evaluated in the rat chronic constriction injury model. A total of 47 alkaloids and 52 core targets were successfully identified for GEB in the treatment of NPP. Functional enrichment analysis showed that GEB was mainly involved in phosphorylation reactions and nitric oxide synthesis processes. It also participated in 73 pathways in the pathogenesis of NPP, including the neuroactive ligand-receptor interaction signaling pathway, calcium signaling pathway, and MAPK signaling pathway. Interestingly, 11-Hydroxyrankinidin well matched the active pockets of crucial targets, such as EGFR, JAK1, and AKT1. The 11-hydroxyrankinidin-EGFR complex was stable throughout the entire molecular dynamics simulation. Besides, the expression of EGFR and JAK1 could be regulated by koumine to achieve the anti-NPP action. These findings revealed the complex network relationship of GEB in the “multi-ingredient, multi-target, multi-pathway” mode, and explained the synergistic regulatory effect of each complex ingredient of GEB based on the holistic view of traditional Chinese medicine. The present study would provide a scientific approach and strategy for further studies of GEB in the treatment of NPP in the future. Chronic pain condition is a major health issue that comprises five of the 11 top-ranking conditions lived with disability and is responsible for economic burden worldwide . The preGelsemium elegans (Gardner and Champ.) Benth. (Gelsemiaceae) (GEB) is a toxic plant indigenous to Southeast Asia especially China, which has long been used as Chinese folk medicine for the treatment of various types of pain, such as neuralgia, sciatica, rheumatoid arthritis, and acute pain  is an abundant resource for drug development and provides innovative insight into therapeutic approaches. ute pain . Phytochute pain . These aute pain . As an iactivity  in the sactivity , and inhactivity . Other aactivity . HoweverNetwork pharmacology is mostly used to screen the active ingredients, predict the corresponding target, and explore the comprehensive molecular mechanisms of TCM. The key ideas of network pharmacology are based on the theory of system biology and multi-direction pharmacology, which are consistent with the holistic philosophy of TCM . Moleculhttp://www.megabionet.org/tcmid/) (http://bionet.ncpsb.org/batman-tcm/) (http://tcm.cmu.edu.tw/zh-tw/) (http://www.swissadme.chwebsite)  , Bioinfoan-tcm/) , and Tra/zh-tw/) . Active website) . The finhttp://www.swisstargetprediction.ch), a popular online server that could accurately predict the targets of bioactive molecules with known ligands (https://pubchem.ncbi.nlm.nih.gov/) and imported into SwissTargetPrediction for identification of potential drug targets in humans. After removing duplicate targets, the targets of ingredients with SwissTargetPrediction probability ≥0.1 were chosen as potential targets, and compounds without target information were excluded.Targets of the active ingredients were predicted using SwissTargetPrediction ( ligands . 3D struhttps://www.genecards.org/) (https://db.idrblab.org/ttd/) (https://go.drugbank.com) (The disease-associated targets of NPP were collected from GeneCards (ds.org/) , the Therg/ttd/) , and Druank.com) . “Neuralhttps://stringdb.org/) to explore the core regulatory genes (http://cytoscape.org/.ver.3.7.2). NetworkAnalyzer analysis was used to screen key targets according to the degree value. The top 15 important proteins with a higher level of degrees in the interaction network were considered as the key targets for GEB in the treatment for NPP. Furthermore, the Molecular Complex Detection (MCODE) plugin was used to detect cluster modules from the complex network with the node score cutoff of 0.2, K-core of 2, and degree cutoff of 2.The intersection of ingredients-related targets and disease-associated targets was visualized by overlapping with a Venn diagram. Then, a PPI network was constructed through the String database (ry genes . PPI infhttps://david.ncifcrf.gov/.ver.6.8) was applied to conduct the enrichment analysis , and the crystal structures of the target proteins were downloaded from the PDB database (https://www.rcsb.org/) with a crystal resolution of less than 2 Å. Molecular docking was performed by importing the crystal structure into the Pymol 2.4.1 Software (https://pymol.org/2/) for dehydration, hydrogenation, and ligand separation. Thereafter, Autodock Vina 1.1.2 software was used to construct a crystal structure docking grid box for each target. Then the molecules with the lowest binding energy for each active compound in the docking conformation were allowed for semi-flexible docking by comparing with the original ligands and intermolecular interactions . Box center coordinates and size of the box were determined for evaluating the interaction. The results were analyzed and visualized using Pymol, and the numbers of grid points in the three dimensions used in this study were 40 40 40 0.375.Molecular docking was used to predict the interactions between core active ingredients of GEB and proteins selected from the center targets from a molecular perspective. 3D structures of active ingredients in SDF (.sdf) format were selected from the PubChem database  heating to 300 K, followed by 100 ps for constant NPT  with a time step of 2 fs. The bonds of atoms were restrained by recruiting the LINCS algorithm. After the processes of energy minimization and equilibration, the molecular dynamics simulation was conducted the leap-frog algorithm for 100 ns with a time step of 2 fs. The geometrical parameters of the systems, such as root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were determined and compared with the primitive ligand complex.The molecular dynamics simulation study is employed to assess the stability and interaction between the protein and ligands after docking. The simulation run was performed for 100 ns using the NVIDIA RTX 1060 GPU accelerated GROMACS 2020 software molecular dynamics package. In the preliminary stage, the Charmm36 force field was used for the protein parameters. The CGenFF server was used for the ligand topology, and a TIP3P water model with appropriate Naw/v NaCl).Koumine (99% purity) was isolated from GEB as described by Su et al. , and it Male Sprague–Dawley rats (180–200 g) were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The rats were adapted in the condition of 25 ± 2°C with a 12-h light/dark cycle (lights on at 8:00 am) and free access to standard laboratory food and water. The experiments met the requirements of guidelines for animal care and the use of Fujian Medical University. The experimental protocols were reviewed and approved by the Committee of Ethics of the Fujian Medical University . Animals were assigned into different groups: the sham control group , the CCI model group . The dose used in the experimental assay was based on the published literature , and no Behavior tests consist of thermal hyperalgesia and mechanical allodynia tests. The thermal hyperalgesia test using a commercial thermal paw stimulator  was evaluated before operation (baseline), drug administration (pre-dosing), and 6, 8, 10, 12, and 14-days after drug administration (post-dosing), and paw thermal withdrawal latency (TWL) was calculated as described by Hargreaves et al. . The mecThen, rats were anesthetized by chloral hydrate, and the lumber segments (L5-L6) of the spinal cord were dissected, weighed, and stored at −80°C. Then, the lumber segments were homogenized for 30 min in an ice bath with RIPA lysis buffer  containing phosphatase inhibitor  after ultrasonic crushing. Protein concentrations were determined using an enhanced BCA protein assay kit , and the protein samples were stored at −80°C until use. Total protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto the nitrocellulose membrane. The membrane was blocked with 5% non-fat dried milk in tris buffer for 1 h at room temperature and washed with tris-buffered saline and tween 20 every 10 minutes for three times. Then, the membrane was incubated with antibodies  overnight at 4°C. After incubation with the appropriate secondary antibodies  at room temperature for 1 h, the protein blots were visualized in the ChemiDoc XRS imaging system .A total of 98 compounds in GEB were retrieved from published literature and online databases, and 57 potentially active ingredients were filtered by OB and DL provided by SwissADME . 679 potPPI analysis was performed on 52 putative common targets with String database as illustrated in The GO and KEGG pathway analysis were performed on the 52 common targets to further explore the possible mechanisms of GEB against NPP. A total of 184 BPs, 34 CCs, and 35 MFs were obtained in GO analysis . The topp-values were selected to generate a bubble chart for visualization . Koumine attenuated the CCI-induced NPP effect in the dose-dependent and time-dependent manner  synthesis processes, which indicated that a potentially novel mechanism for pharmacological intervention of GEB against NPP. Protein phosphorylation plays a key role in the cellular regulatory mechanism of enzymes and receptors. Most of the above-mentioned target activation is involved in the phosphorylation process. For example, phosphorylation of AKT at Thr308 and Ser473 mediate pain behavior through the PI3K/AKT signal pathway . Notably pathway , which i pathway . These f pathway . Neuroac pathway . As conf pathway . Mirtaza pathway . Based o pathway .Inflammatory mediator regulation is another interesting pathway described by KEGG. An increasing number of studies have indicated that inflammatory responses play a key role in the development of NPP . Cytokinin vitro and in vivo, though the current data on 11-hydroxyrankinidine is extremely limited. Furthermore, traditional TCM usually is thought to act in synergy to achieve a holistic therapeutic outcome, suggesting the understanding of the synergistic action of these alkaloids with a holistic view.Based on the “ingredient-target-pathway” network, 47 pivotal active ingredients related to NPP were obtained, such as 11-hydroxyrankinidine, 11-hydroxyhumantenine, gelsamydine, koumidine, and gelebolines C. These ingredients might play a synergistic role in the treatment of NPP. Structurally, the 47 phytochemicals have exhibited diversity. Molecular docking results have disclosed that 11-hydroxyrankinidine, 11-hydroxyhumantenine, gelsamydine, and koumine could bind autonomously with the active pocket of EGFR, JAK1, and AKT1 to form a complex with a relatively stable structure through hydrogen bonds and other interactions. Among them, 11-hydroxyrankinidine has the lowest binding energy and the highest affinity to EGFR. Encouragingly, it was observed that the RSMD and RSMF profiles of the 11-hydroxyrankinidine-EGFR complex were relatively stable, which indicated 11-hydroxyrankinidine showed promising inhibitory activity in NPP. Due to the accessibility, the extracts and monomers derived from GEB have been found to possess anti-NPP biological activity, especially some of the active ingredients, such as koumine , gelseniThis present study also has several limitations. Firstly, given the limitations of network pharmacology, the public databases investigated in the study would be constantly updated so some ingredients and targets information has partly lagged. The chemical fingerprint may be a better choice for network construction and mechanism exploration. Secondly, SwissTargetPrediction is a ligand-based tool for predicting the interacting targets of small molecules, which is useful for understanding the molecular mechanisms underlying a specific phenotype or bioactivity, as well as assessing the possibility of repurposing therapeutically-relevant compounds. It is based on the so-called “similarity principle,” which states that two similar molecules are likely to have similar properties. However, when there are few (or no) known active ligands for a target of interest, their predictive reliability suffers. Furthermore, when molecules with high structural similarities but different biological activities for the same target coexist, a limitation may also exist. Thirdly, the validation experiment in the current study only provided limited protein, and more targets and pathways may be studied further, especially the possibility of influence and correlations with cytokines. Besides, although 11-hydroxyrankinidine showed promising inhibitory activity in network pharmacology, it is not the most abundant ingredient in GEB. Due its inaccessibility, there has been no report on its pharmacological activities to date. Therefore, koumine was selected in validation in the experiment as a substitute, based on the fact that it is a high-content active alkaloid of GEB that is easily obtained. Furthermore, molecular docking results demonstrated that koumine bonded to the ligand stably. For further study, we can also use pH-zone refining counter-current chromatography to purify the active monomer , for exaIn the present study, the active ingredient and anti-NPP mechanism of GEB were mapped using network pharmacology, molecular docking, molecular dynamics simulation, and bioinformatics approach. It could promote the understanding of the synergistic action of GEB with a holistic view to explore the key ingredients, targets, and pathways. Different from the current mode of single-target pharmacology in TCM, network pharmacology based on computational prediction could provide broad ideas and be a useful supplement for the mechanism exploration of TCM. In addition, the new potential lead compounds screened in our study, for example, 11-hydroxyrankinidine, may provide a rational direction for future drug discovery and development. With the development of monomer purification technology , it willThis study revealed the underlying pharmacological mechanisms of GEB on NPP based on network pharmacology and experimental evidence. Forty-seven active alkaloids might play a synergistic role in the treatment of NPP, and 11-hydroxyrankinidin had excellent stability in the active site pocket of EGFR, JAK1, and AKT1, the core targets in network pharmacology. Meanwhile, GEB participates in the regulation of 73 pathways including neuroactive ligand-receptor interaction in the pathogenesis of NPP concentrated mainly on phosphorylation reactions and nitric oxide synthesis processes. Experimental evidence also proved that GEB may regulate the expression of EGFR and JAK1 after the formation of ligand-receptor complexes. In the future, network pharmacology based on computational prediction may provide broad ideas for TCM mechanism exploration as well as cost-effective strategies during the drug discovery and development process.


These data provide a scientific basis for future investigations into the clinical application of CUR against CP-AKI.Cisplatin-induced acute kidney injury (CP-AKI) is a severe complication in patients receiving CP chemotherapy. However, effective therapies for CP-AKI are currently lacking. Curcumin (CUR), a natural polyphenol, is extracted from the rhizome of turmeric and has been reported to have nephroprotective activity. However, the role of CUR in CP-AKI remains unclear. This study aimed to explore the mechanism of CUR in CP-AKI by combining a network pharmacology approach with experimental validations. The analysis revealed 176 potential targets of CUR based on the HERB database and 1,286 related targets of CP-AKI from the GeneCards, DrugBank, and OMIM databases. Further, 106 common targets of CUR against CP-AKI were obtained, and these common targets constructed a protein-protein interaction (PPI) network. In addition, the core targets were screened from the PPI network using Cytoscape. Molecular docking revealed that CUR displayed the best binding to AKT1. Gene Ontology (GO) analysis indicated that the primary biological processes of CUR against CP-AKI included cellular response to chemical stress and apoptotic regulation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis suggested that the PI3K-Akt signaling pathway was most significantly enriched in CUR against CP-AKI. Western blotting and flow cytometry showed that CUR inhibited apoptosis induced by CP by activating the Akt signaling pathway in human kidney tubular epithelial cells (HK-2). Altogether, our findings demonstrated that CUR alleviated apoptosis by activating the Akt signaling pathway in CP-AKI  ClinicaCurcuma longa and is known for its multiple pharmacological activities, including anti-inflammatory, antioxidant, and apoptotic regulation properties [Curcumin (CUR) is a phenolic compound isolated from operties . Extensioperties . Furtheroperties . AlthougNetwork pharmacology is an emerging and promising approach. It can integrate bioinformatics with pharmacology to provide persuading evidence for drugs on disease through visualization . This st2.The protocol of this study is shown in 2.1.http://herb.ac.cn/, accessed on 24 August 2021) [https://pubchem.ncbi.nlm.nih.gov/, accessed on 24 August 2021) [The target proteins of CUR were collected from the HERB Database st 2021) . This da21 . This2.2.www.genecards.org, accessed on 24 August 2021) [https://www.drugbank.ca/, accessed on 24 August 2021) [https://omim.org/, accessed on 24 August 2021) [Disease-related targets were obtained from the GeneCards database (https:// st 2021) , and thest 2021)  using th2.3.https://bioinfogp.cnb.csic.es/tools/venny/, accessed on 24 August 2021) [The common targets between CUR and CP-AKI were intersected using the VENNY 2.1 online platform  [Homo sapiens; confidence scores were larger than 0.4, and the other variables remained as the default. Thereafter, the PPI network was exported in TSV format and analyzed using Cytoscape 3.7.2 (https://cytoscape.org/) [The PPI network of common targets was constructed using the STRING database (st 2021) . The orgpe.org/) . PPI net2.5https://www.rcsb.org/, accessed on 25 August 2021) [The 3D structures of the top four proteins by the degree value of core targets were achieved from the Protein Data Bank database (st 2021) , AKT1 (Pst 2021) . The recst 2021) . Dockingst 2021) . The 2D st 2021) . Affinit2.6.https://david.ncifcrf.gov/, accessed on 25 August 2021) [p < 0.05 and FDR < 0.05 were considered significant. The top 20 enriched terms were visualized using an online tool  [Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for common targets were performed using DAVID 6.8 (st 2021) . The enrst 2021) . A targe2.7.Human kidney tubular epithelial cells (HK-2) with normal biochemical features and proximal tubular cell morphology were purchased from the American Type Culture Collection  and were cultured in a DMEM medium at a 37°C incubator in 5% CO2. The DMEM medium was supplemented with 10% FBS , streptomycin (100 mg/mL), and penicillin (100 IU/mL) .The cells were starved of serum for 12 h before treatment during the experiments. CP was purchased from Sigma  and was administered to HK-2 cells at a concentration of 20 µM for 24 h to establish the CP-AKI cell model . CUR was2.8.3/well in 100 μL of medium and treated with four concentrations  of CUR for 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance at 450 nm was measured, indicating cell viability. All experiments were performed in triplicate. All assays were repeated seven times. Wells without cells served as blanks.According to the manufacturer’s instructions, viability was assessed using CCK-8 kits . Cells were cultured in 96-well microplates at a density of 5 × 102.9.An Annexin V-FITC/Propidium Iodide apoptosis kit  was used to evaluate cell apoptosis. Treated HK-2 cells were cultured to a density of 80–90% in 6-well plates and collected following trypsin digestion. Cultured cells were washed with PBS and then were collected for detection. Apoptosis was detected by resuspending the cells in 500 μL of binding buffer. A mixture of 5 μL PI and 5 μL of annexin V-FITC was added to the binding buffer and incubated for 30 min in the dark at room temperature. Subsequently, cell apoptosis was examined using flow cytometry. Apoptosis in the early stage was indicated by PI-negative/Annexin V-positive staining, while more advanced apoptosis was indicated by PI-positive/annexin V-positive staining. The cell experiments were repeated in triplicate. The apoptotic data of flow cytometry was analyzed using FlowJo 10 software.2.10.HK-2 cells were lysed with the lysis buffer of the radioimmunoprecipitation assay (RIPA)  in accordance with standard procedures. The BCA protein assay kit  was used to calculate the protein concentration. The proteins were separated by SDS-polyacrylamide gel electrophoresis (8–10%) and transferred to polyvinylidene fluoride membranes . The membranes were blocked with 5% skimmed milk  for 2 h and then incubated with primary antibodies against β-actin , Bcl2, Bax, cleaved caspase-3, p-Akt (Ser-473) and Akt .2.11.p < 0.05.Statistical analysis was performed using SPSS 20.0 software . All data were expressed as mean ± SD. All experiments were repeated in triplicate. The normality of the data was examined using Shapiro–Wilk test. For data variables with normal distribution and independent samples, Student’s unpaired t-test was used to compare the two groups , while o3.The protective effects of CUR against CP-AKI and the possible mechanisms of action have not been fully elucidated. Here, we used a network pharmacology approach to predict the potential targets and candidate pathways of CUR involvement in CP-AKI. CP-induced HK-2 cells were used as a cellular model to validate the role and mechanism of CUR against CP-AKI.3.1.http://www.tcmip.cn/ ETCM/). In general, a drug-likeness weight ≥ 0.18 indicates good drug-likeness. Collectively, these results suggest that CUR has good pharmacokinetic properties.We obtained the 2D structure of CUR from the PubChem database ) and ass(c), 2(d)). These core targets formed another PPI network consisting of 10 nodes and 36 edges with an average degree of 8 . To invFigures 2ree of 8 ). These 3.2.To verify the binding activity of CUR to the targets, we selected the top four core targets by degree value for molecular docking, including AKT1, TP53, CASP3, and HIF1A. We summarized the affinities of CUR with the interactive residues of the selected targets and predicted the number of hydrogen bonds formed between interactive residues. The docking results are shown in 3.2.1.The binding energy of CUR on AKT1 was – 9.5 kcal/mol. As shown in 3.2.2.The binding energy of CUR on TP53 was – 6.6 kcal/mol. As shown in 3.2.3.The binding energy of CUR on CASP3 was – 6.7 kcal/mol. As shown in 3.2.4.The binding energy of CUR on HIF1A was – 8.9 kcal/mol. As shown in With respect to the interaction between the molecule and target, the binding energy indicates the affinities. The more negative the binding energy, the stronger the interaction . A bindi3.3.To explore the underlying mechanism of CUR against CP-AKI, GO and KEGG enrichment analysis were carried out using the DAVID database. The main GO biological processes (BP) included cellular response to chemical stress, cellular response to oxidative stress, regulation of apoptotic signaling pathway, among others. For cell composition (CC), the membrane raft, membrane microdomain, and membrane region were the main classifications of these targets. Molecular function (MF) category included cytokine receptor binding, phosphatase binding, and ubiquitin-protein ligase binding ). The pr3.4.To clarify the relationships between the targets and pathways, the CUR, targets, and critical pathways were used to construct the compound target pathway (CTP) network ). As the3.5.in vitro, HK-2 cells were treated with 20 µM CP for 24 h to establish the CP-AKI cell model [in vitro , human kidney tubular cells (HK-2) were treated with various concentrations of CUR for 24 h. The CCK-8 assay showed that CUR concentrations up to 20 μM caused a significant decrease in cell viability, and CUR concentrations of 0–10 μM had no cytotoxic effect on HK-2 cells ). Thus, ll model . In addill model . In thisll model . CASP3 win vitro ). These 3.6.(b), 4(c)). The molecular docking results also showed that CUR had the best binding affinity to AKT1. AKT1, the major Akt isoform, is a hub member of the PI3K-Akt signaling pathway [The KEGG enrichment analysis results revealed that the PI3K-Akt signaling pathway was the most significantly enriched and included 24 target proteins . There are three Akt isoforms (AKT1–3), with AKT1 being the essential subtype. AKT1 is a hub member of the PI3K-Akt signaling pathway . CUR exel injury . In addil injury . CASP3 (l injury . Consistin vitro. According to previous reports, the PI3K-Akt signaling pathway plays a significant role in promoting cell survival by inhibiting the intrinsic apoptotic pathway [in vitro experiments coincide with the literature.CUR has been reported to significantly alleviate CP-induced nephrotoxicity in rats at the ultrastructural and molecular levels. CUR reduced tubular necrosis and inflammation in the peritubular area, while in tubular epithelial cells, CUR prevented oxidative stress and apoptosis ,55. Cons pathway ,57. Akt  pathway . Dexmede pathway . Undoubtin vitro and verified the accuracy of network pharmacology analysis. 2. Distinct from other CP-AKI-related studies focused on the validations of some exact signaling pathways [Although many studies have reported the possible mechanisms and intervention measures in CP-AKI, our findings are still important: 1. Based on the exploration of the pharmacology network, we associated the PI3K-Akt signaling pathway with CUR treatment in CP-AKI. Further, we successfully demonstrated that CUR may be used as an Akt activator to treat CP-AKI pathways ,60. Our Altogether, these results demonstrate that CUR may play a renoprotective role by inhibiting TEC apoptosis via the activation of the Akt signaling pathway in CP-AKI. This study had some limitations as multiple complicated pathological pathways are involved in the mechanism of CUR against CP-AKI. In addition, apart from the severe damage to the proximal tubules, cisplatin can cause an injury in the distal tubules, which is also involved in the development of AKI . Further5.In conclusion, we identified the core targets and critical pathways of CUR against CP-AKI using a pharmacological approach. Further, we confirmed that CUR inhibits cell apoptosis by activating Akt signaling in CP-AKI cell models. These results suggest that CUR is a promising and effective multitarget medication for CP-AKI.


Background: The purpose of the research is to identify the main active ingredients in Coptidis Rhizoma (CR) and explore the possible molecular mechanisms in the treatment of Kawasaki disease (KD).Materials and Methods: A total of 58 children with KD were randomly divided into a control group and a Berberine treatment group. The therapeutic indicators of the two groups before and after treatment were compared. Then, compounds and drug targets of CR from the TCMSP, SWISS, SEA, and the STITCH were collected, and targeted KD genes were retrieved from the DisGeNET, DrugBank, and GeneCards databases. The network pharmacology approach involved network construction, target prediction, and module analysis. GO and KEGG enrichment analysis were performed to investigate the possible pathways related to CR for KD treatments. Finally, protein expression was determined to verify the core targets using Western blotting in the cell experiment.Results: In total, nine compounds, 369 relative drug targets, and 624 KD target genes were collected in the above database. The network analysis revealed that 41 targets might be the therapeutic targets of CR on KD. GO and KEGG enrichment analysis revealed that the biological processes, namely, response to hormone, response to inorganic substance, and enzyme-linked receptor protein signaling pathway, and Pathways in cancer, Toll-like receptor signaling pathway, and Pancreatic cancer are the most significant. Protein expression of CASP3, PTGS2, and SRC was upregulated and AKT1 and ERK were downregulated.Conclusion: We provided useful resources to understand the molecular mechanism and the potential targets for novel therapy of KD. Kawasaki disease (KD) is a systemic inflammatory vasculitis predominantly affecting children younger than 5 years of age  and is nRanunculaceae. The Isoquinoline Alkaloids in Coptis Chinensis, namely Berberine (BBR), Coptis Chinensis, and Rhizopine, are the main effective components of CR. Data have proved that CR has anti-inflammatory (via the Phosphatidyl Inositol 3-kinase (PI3K)/serine/threonine protein kinase B/endothelial nitric oxide synthase signaling pathway (Coptidis Rhizoma (CR), called Huang Lian (HL) in Chinese, belongs to perennial herbs of ammatory , antibacammatory , antitumammatory , antidiaammatory , and pha pathway . In addi pathway .Network pharmacology is a part of bioinformatics based on the concept of “disease–gene–target–drug” , which iTo investigate the clinical effects of BBR on patients with KD and its pharmacological mechanisms, a randomized and case–control study was performed in Shenzhen Children's Hospital and a comprehensive network pharmacology approach was established to probe the appliances of CR on KD by network pharmacology analysis.g for 10 min and aliquots were stored at −80°C.The sample group of this study is formed by 20 children with KD  admitted to the Department of Cardiology and 20 healthy children  from Child Health Section, Shenzhen Children's Hospital from October 2018 to May 2019. They have randomly divided them into the routine treatment group and the routine treatment + BBR group. All children were typical KD patients and the diagnostic criteria were in line with the AHA and American Academy of Pediatrics 2004 guidelines . The KD http://lsp.nwu.edu.cn/tcmsp.php)  Database (msp.php) , a systemsp.php) . In addihttps://www.drugbank.ca/) (https://www.genecards.org/) (http://www.disgenet.org/home/)  , GeneCards.org/)  and DisGg/home/) . It is nvia targets. The target fishing was used to search for or predict the potential targets of small molecules and the small molecular structure information of the active ingredients in CR by TCMS could also be predicted by applying the Similarity Ensemble Approach (http://sea.bkslab.org/), STITCH (http://stitch.embl.de/), and Swiss Target Prediction (http://www.swisstargetprediction.ch/).The active ingredients of drugs play an important role in related biological functions Approach   to draw the Venny map and also to obtain the common target genes of the intersection. Then, the online tool STRING (http://string-db.org) was used to construct the target protein–protein interaction (PPI) network of CR acting on KD. The network construction was established, first, with a network between active compounds and targets of CR and also with PPI network of compounds and targets developed by linking the compound targets and predicted targets of other human proteins. Another PPI network of KD targets was also constructed by linking the known KD-related target.In this phase, the CR targets and the acquired KD targets were screened by the online tools Venny2.1.0  and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched by GSEA  with FDRg for 10 min and aliquots were stored at −80°C.In our cell experiment, a total of 28 subjects, including 14 normal children and 14 KD patients, were recruited from Shenzhen Children's Hospital. Serum samples were separated by centrifugation at 1,000 × 2. Cells were then stimulated with culture medium containing 15% serum [15% (V/V)] from KD patients or healthy donors for 24 h. The cells were then used in subsequent studies. Each measurement was performed in triplicate (n = 3). The BBR was dissolved in sterile ultrapure water. The stock solution of BBR was 40 μm, which can be diluted into final concentration (20 μm) with cell culture medium.HCAECs were brought form ScienCell  and cultured with endothelial cell growth medium containing growth factors, supplements, and 10% fetal bovine serum at 37°C in 5%COThe protein samples of the three groups were extracted and measured through a BCA protein assay. We loaded and separated the proteins through 4 and 10% SDS-PAGE and then transferred them onto PVDF membranes. The membranes were washed and then blocked using 5% skimmed milk powder blocking solution for 2 h. The antibody  was diluted at 1:1,000, diluted with 5%BSA TBST, and incubated overnight at 4°C. After washing three times with TBST for 4 min each, the membranes were incubated with secondary antibodies  for 2 h at room temperature. Then, the membranes were washed four times with TBST for 4 min each. Finally, the image is collected and analyzed.p-values were calculated by t-test and chi-square, as appropriate. p < 0.05 was regarded as having statistical significance.Here, all quantitative data are presented as means ± SEM. For pairwise comparison of two or more groups of quantitative data, One of the first things to be observed in this phase is that no statistically significant differences were found in baseline data and hematologic indicators between the two groups. The BBR group was able to reduce the values of C-reactive protein (CRP), neutrophils/lymphocytes (NLR), and platelets/lymphocytes (PLR) shown in , and theOverall, nine target compounds were collected from TCMSP and 369 target genes in the whole formula in total were retrieved from TCMSP, SWISS, SEA, and STITCH database . The valTo further determine the target of CR on KD, a compound-target network was built with an online tool Cytoscape3.6, representing a compound-target gene network including nine compounds and 41 target genes shown in . CompounTo investigate the biological functions of the direct targets of CR for KD, the target genes GO and KEGG pathway function enrichment was performed by GSEA. As shown in The KEGG pathway enrichment is shown in Cytoscape3.6.0 software was used to demonstrate the enrichment of the 21 proteins of the above five pathways shown in . Most ofFor this part of the study, the String online server was the main tool that helped to build the PPI network, containing 41 nodes in total shown in . The sigWe selected the top 5 genes according to the degree of PPI, including PTGS2, CASP3, ERK, AKT1, and SRC. After the treatment of berberine, the expressions of CAPS3 and PTGS2 were extremely significantly decreased, and the difference was statistically significant. While the expression of SRC was decreased, the expression of ERK and AKT1 was increased, and there was no statistically significant difference .KD is a systemic inflammatory syndrome of small and medium vessels. In recent years, more and more evidence has shown that the pathogenesis of this condition is related to infectious factors, susceptibility genes and autoimmune activation. Traditional Chinese medicine is suitable for treating diseases with complex mechanisms because of its multiple curative effects and minimal side effects. The effects of BBR on KD in clinical practice were tested based on some previous findings regarding this topic . The preIn the clinical trial stage, C-reactive protein (CRP) and peripheral blood cell parameters were also used as markers of systemic inflammation. Among them, CRP plays a role in promoting phagocytosis and immune regulation. Data have shown that during the course of KD, the increase of CRP level is related to coronary artery dilatation and is a high-risk factor of complication with coronary artery injury for KD . NLR andAKT1, CASP3, TP53, MAPK3/8/14, PTGS2, SRC, RELA, and MTOR were found and set as top 10 target genes as well.To investigate the mechanisms of BBR on KD, network pharmacology was used. Nine target compounds of CR were collected from TCMSP; 369 target genes of CR were collected from TCMSP, SWISS, SEA, and STITCH database; and a total of 624 target genes of CR were obtained by searching related databases together with 41 target genes obtained by the intersection of CR and KD. By further screening this sample of genes, AKT1, a serine/threonine protein kinase, is widely expressed in various tissues. It is known that activated AKT plays a regulatory role in the cell cycle, apoptosis, and proliferation by activating downstream factors. Also, the specific activation of AKT1 in vascular endothelial cells can alleviate the injury after carotid artery ligation by increasing the expression of nitric oxide and protecting the function of the endovascular cortex . By initMAPK, an intracellular serine/threonine protein kinase, is an important signaling system for cell-mediated extracellular signals to intracellular responses and plays a key role in cell proliferation, apoptosis, inflammation, immunity, and angiogenesis . StudiesSRC encodes tyrosine protein kinase also has to be mentioned since it is a member of the non-receptor protein tyrosine kinase family. This protein is constantly associated with multiple signaling pathways in cells and its related genes are involved in important biological processes such as growth, differentiation, adhesion, and transcription. Studies have confirmed that the SRC-1 gene is related to the susceptibility to coronary artery aneurysm of KD complications , indicatAdditionally, CASP3 is a key apoptotic protease in the final pathway of apoptotic cell death, mediating exogenous and endogenous cell death signaling pathways. CASP3 leads to transcriptional activation of inflammatory genes by activating the NF-κB pathway in the mechanism of KD. As a genetic variation of genes may also cause damage and remodeling of vascular structures, TP53 is considered to be an important tumor suppressor gene that can affect cell cycle, DNA repair, apoptosis, signal transduction, transcription, and autophagy, as well as regulate the growth, differentiation, and senescence of cells. PTGS2 contains 10 exons and 9 introns, encoding cyclooxygenase 2 (Cox-2). Cox-2, in this case, is expressed in vascular smooth muscle, monocytes, and fibroblasts, and is a cardinal inflammatory mediator in the process of atherosclerosis . In addiRELA is a member of the NF-κB family. NF-κB plays a key role in inflammatory and immune responses in cells. Studies have shown that in the pathological process of KD, the NF-κB signaling system regulates transcription of almost all genes involved in inflammatory mediators and cell proliferation and activation. Activation of the NF-κB signaling pathway is linked to the occurrence of KD vasculitis in the acute phase, which is likely to aggravate KD vasculitis response and participate in the formation of coronary artery injury . ICAM-1 In addition, the GO enrichment analysis in this study showed that response to hormone, response to inorganic substance, enzyme-linked receptor protein signaling pathway, and immune effector process are the major biological processes for CR treatment of KD. The disturbance of these biological processes is likely the cause of KD vasculitis. Accordingly, CR may be important in the regulation of different biological functions of KD.Finally, the enrichment analysis of the KEGG pathway found that the five signaling pathways closely related to CR and KD prevention and treatment included Pathways in cancer, Toll-like receptor signaling pathway, Pancreatic cancer, Neurotrophin signaling pathway, and MAPK signaling pathway. The Toll-like receptor signaling pathway is a family of receptors composed of members of multiple receptors and has a key function when it comes to inflammatory and immune injuries of endothelial cells caused by pathogen infection and immune response . ClinicaThis study systematically explored the putative bioactive compounds in CR and pharmacological targets of CR for KD prevention and treatment through network pharmacology and Western blotting. All the ingredients of CR were extracted from edible plants, and they were all claimed safe in previous applications. Therefore, this study provides a new way to explore the mechanism of CR in treating KD. However, the examinations here conducted have certain limitations. It is suggested for the future that relevant experiments are carried out to better explain the mechanisms of CR in the treatment of KD.After conducting this examination, we postulated that BBR is effective in the treatment of young children with KD and can be considered as an alternative treatment for KD. This study has preliminarily revealed the possible mechanism of CR in the treatment of this disease by regulating multi-targets with multi-components. Furthermore, the outcomes demonstrated that a network pharmacology-based approach was useful for elucidation of the interrelationship between complex diseases and interventions of Chinese herbal medicines, potentially providing references for further research on its mechanisms of action in the future.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Shenzhen CHildren Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MX conceived and designed the research, and reviewed and revised the manuscript. XF wrote the manuscript. XG and YL performed a preliminary analysis. All authors read and approved the final version of the manuscript.This study was supported by the National Nature Science Foundation of China (81870364), Shenzhen Sanming Project (SZSM20162057), Shenzhen Scientific Plan (JCYJ20190809164004023), and Guangdong Province High Level Clinical Key Specialties (SZGSP012) .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.


Caenorhabditis elegans and its relationships with gene expression, physiology, and the response to therapeutic drugs. Visualization of the metabolic pathways that comprise the metabolic network is extremely useful for interpreting a wide variety of experiments. Detailed annotated metabolic pathway maps for C. elegans are mostly limited to pan-organismal maps, many with incomplete or inaccurate pathway and enzyme annotations. Here, we present WormPaths, which is composed of two parts: (1) the careful manual annotation of metabolic genes into pathways, categories, and levels, and (2) 62 pathway maps that include metabolites, metabolite structures, genes, reactions, and pathway connections between maps. These maps are available on the WormFlux website. We show that WormPaths provides easy-to-navigate maps and that the different levels in WormPaths can be used for metabolic pathway enrichment analysis of transcriptomic data. In the future, we envision further developing these maps to be more interactive, analogous to road maps that are available on mobile devices.In our group, we aim to understand metabolism in the nematode  Metabolism can be broadly defined as the total complement of reactions that degrade and synthesize biomolecules to produce the biomass and generate the energy organisms need to grow, function, and reproduce. Metabolic reactions function in metabolic pathways that are interconnected to form the metabolic network. In metabolic networks, the nodes are metabolites and the edges are conversion and transport reactions carried out by metabolic enzymes and transporters. Caenorhabditis elegans, and humans , a platform that provides pan-organism annotations and metabolic pathway maps .Over the last five decades or so, the free-living nematode nd aging . More re the lab . Numerourk model . The recC. elegans metabolism on paper to help us interpret our data. In KEGG, enzymes are indicated by Enzyme Commission numbers and maps are colored with those enzymes predicted to be present in an organism of interest; however, organism-specific pathways cannot be extracted. Furthermore, many of these maps contain incorrect or partially correct reactions for C. elegans. We found that redrawing pathway maps that contain information about metabolites, genes encoding the proteins that catalyze metabolic reactions or transport metabolites between cells or cellular compartments, molecular structures, and cofactors was very helpful to our studies , amino acid metabolism, and pathways fundamental to C. elegans physiology . Each map connects to other pathways, thereby covering the entire iCEL1314 network. Importantly, the network was expanded by adding reactions and genes found in the literature that were heretofore missed. Maps were carefully curated, hand-drawn, and then visualized in a standardized Scalable Vector Graphics (SVG) format, which allows interactive usage in web applications. WormPaths annotations and maps are publicly available on the WormFlux website (http://wormflux.umassmed.edu). Our careful gene-to-pathway annotations at different levels (see Results) enable statistical enrichment analyses. Finally, our maps may provide a useful format for the drawing of metabolic pathway maps in other organisms. In the future, we envision further refining the maps through detailed literature reviews and experiments.Here, we present WormPaths, a web-based collection of standardized metabolic pathway maps for i.e., RM01859 in iCEL1314 and R01859 in KEGG) was removed from valine degradation and isoleucine degradation maps to avoid a redundant overlap with propionate degradation, where this reaction defines a critical step. In KEGG, R01859 is associated with glyoxylate and dicarboxylate metabolism in addition to valine, leucine, and isoleucine degradation and propionate metabolism, thus appearing in three places. However, propionyl-CoA to methlymalonyl-CoA conversion is clearly the first step of canonical propionate degradation.The design of pathway maps aimed at capturing and visualizing metabolic functions in such a way that would be broadly useful for both statistical analyses and navigation purposes. The starting point for pathway definitions was the pathway annotations of reactions and genes of iCEL1314 in WormFlux and in KEGG. Existing pathways were then split and/or modified such that the functional resolution of pathways was increased without disrupting the coherence of reactions, while the number of overlapping reactions was minimized. For example, the valine, leucine, and isoleucine degradation pathway (one map in KEGG) was first divided into three maps to increase pathway resolution: valine degradation, leucine degradation, and isoleucine degradation. Then, a reaction that existed in the original pathway that converts propionyl-CoA to methylmalonyl-CoA , (ii) a connection to other pathways , and (iii) an endpoint that can be transported to or from extracellular space . The connections of terminal metabolites to other pathways are indicated in maps by clickable pathway boxes as in KEGG, unless the metabolite is associated with more than two other pathways. When a terminal metabolite is not associated with any other pathway, a proper transport that explains the source or fate of the metabolite is included. If a transport is not available either, then it follows that the metabolite is associated with reactions not included in WormPaths maps yet, which is indicated by a box labeled “other”. In any case, the number of pathways and the types of transports (cytosol-extracellular space or mitochondria-cytosol) a metabolite is associated with are indicated by colored squares and circles, respectively, as shown by a legend appended to every map. Furthermore, clicking a metabolite brings the page of that metabolite in WormFlux, which shows all pathways and reactions it is associated with. Thus, information about the pathway associations and transportability of, not just terminals, but every metabolite in a pathway, is reachable from the pathway map.Typically, pathways were designed to start or end with three types of metabolites: (i) the main substrate or product by definition (http://inkscape.org) following a template . Maps were blended with WormFlux pages and made interactive using PHP language for server-side processes  and JavaScripting language for the client-side actions .Draft maps were drawn as SVG files in the open-source vector graphics editor Inkscape . This tool takes a list of genes from the user as the input. First, the overlap between the input and the background  set, i.e., the entire gene list of iCEL1314, is determined. This overlap defines the metabolic hits. For each gene category at each level, the strength and significance of the intersection between category genes and metabolic hits are evaluated. The strength is the enrichment score calculated as k/n, where k is the number of genes in the intersection set, and n stands for the total number of genes in that category. The significance was based on the hypergeometric test performed using the hypergeom function in scipy package of Python 2.7. Briefly, a hypergeometric distribution is derived for the size of the intersection set between the category genes and the metabolic hits using hypergeom, where M is the size of the universal set, which is 1314, and N is the number of metabolic hits. Then, the probability mass function of this distribution is used to calculate the enrichment p-value as the sum of probabilities for k and greater integers, which corresponds to the total probability of having k or more genes in the intersection. Similarly, the depletion p-value is calculated as the total probability of having k or less genes in the intersection. The PEA tool provides an interactive color-coded table that illustrates enrichment scores and enrichment or depletion p-values for every category at every level. The results are also provided in greater detail as a downloadable tab-separated text file. Importantly, a multiple hypothesis testing correction is not done by PEA to avoid the underestimation of enrichment strength. The users are encouraged to apply Bonferroni correction when searching enrichments or depletions at a significance level of α, such that p < α/n_test must be satisfied, where n_test is the number of tests . A workflow is shown in To facilitate pathway enrichment analysis (PEA), we developed an interactive webtool in the WormFlux website . All pathway maps are available in WormFlux (http://wormflux.umassmed.edu/WormPaths/wormpaths.php) and can be used interactively to visualize information related to metabolites, genes, reactions, and pathways in this website. Each pathway map can be downloaded in PNG, PDF, and SVG formats from the corresponding pathway page.All pathway annotations from this study are available in the GENETICS online.C. elegans metabolic pathways at different levels. Categorizing genes into pathways at different levels enables enrichment analyses at different levels of resolution (see below). Level 1 includes the broadest assignment to ten annotations: amino acids, carbohydrates, cofactors and vitamins, energy, lipids, nucleotides, one-carbon cycle, reactive oxygen species, other amino acids, and other -based vector image format for general useability on the Internet by both individual users and computer programs. Metabolite structure images for all products and reactants were downloaded from KEGG and other resources  and some that were not available were hand drawn. All reactions on the SVG maps were manually verified and checked for errors. Maps were then uploaded to the WormFlux webpage, where they are available in a drop-down list. All maps are searchable and clickable. For example, a search for the gene hlighted . In reveC. elegans metabolic pathways that connect into the larger iCEL1314 network. pck-1, pck-2, or pck-3 (pck-3) was undetectable  into acetyl-CoA (accoa)  .fat-1 was missing from the model even though this reaction is described in the literature . Altogether, WormPaths identifies four pathways for alh-2, and all are shared with KEGG (In KEGG, genes are associated with any pathway assigned to that gene by gene-protein-reaction associations. However, sometimes these reactions can be isolated because surrounding reactions are not found in the organism of interest, thus the isolated reaction does not connect to the larger pathway or network of said organism. The isolated reactions may be incorrect annotations that are not likely to exist in the organism, or they may have been incorrectly inserted into the pathway based on homology to another organism . For inspathways . HoweveriCEL1314 . This caith KEGG . Refininp-adjusted value of ≤0.05 and a fold change of ±1.5 and performed PEA using the PEA tool in WormFlux (see Materials and methods and To demonstrate how the levels in WormPaths can be used to identify high-resolution metabolic pathway enrichment in transcriptomic data, we analyzed a previously published RNA-seq dataset measuring the transcriptomes of untreated animals, animals treated with 20 nM vitamin B12, or 20 nM vitamin B12 and 40 mM propionate (In collaboration with the Walker lab, we previously developed WormCat, an online tool for identifying genome-scale coexpressed gene sets . In WormC. elegans metabolic pathway maps and gene annotations. Our overall annotations predict a total of more than 3000 metabolic genes in C. elegans, based on homologies with metabolic enzymes or protein domains (C. elegans researchers to contact us and help with updates and additions, and to point out any errors they may find. We expect that new metabolic reactions and metabolites will continue to be added to future versions of iCEL as they are discovered. For instance, the iCEL1314 model incorporates the relatively recently discovered ascaroside biosynthesis pathway (We have developed WormPaths, an expandable online catalog of  domains . Therefoiyab089_Supplementary_DataClick here for additional data file.


Purpose: Osteoporosis, a common disorder especially prevalent in the postmenopausal women and the elderly, is becoming a worldwide public health problem. Osteoporosis can cause severe joint pain, fragility fractures, and other symptoms, which can seriously impair the daily lives of affected patients. Currently, no gold-standard drug is available that can completely cure osteoporosis. Tanshinone is a traditional Chinese medicine, which can exhibit multiple biological activities. It might also display a protective effect on osteoporosis. However, the molecular mechanism through which tanshinone can improve osteoporosis remain unclear. The objective of our study is to explore the underlying mechanism behind the protective actions of tanshinone.Methods: The common KEGG pathways of tanshinone-targeted genes and osteoporosis were analyzed by using bioinformatics analysis. The bioinformatics analysis results were further validated both by in vitro and in vivo experiments.Results: 21 common KEGG pathways were identified between osteoporosis and tanshinone-targeted genes. It was further found that tanshinone could induce expression of AKT1, promote the proliferation of MSCs, and ultimately suppress their apoptosis.Conclusion: Taken together, our findings indicate that tanshinone can alleviate osteoporosis, its effect was potentially mediated through modulating AKT1 expression. Thus, tanshinone could serve as a promising treatment option for osteoporosis. Osteoporosis is a major bone-turnover disorder which can commonly affect the elderly and postmenopausal women . AlthougOsteoporosis is primarily an age-related bone disease, which has emerged as a worldwide public health problem . It has There are currently no treatment modalities that can be used for the complete cure of osteoporosis. There are mainly two types of drugs used for the management of osteoporosis: bone formation promotors and resorption inhibitors. Most of the existing clinical drugs are bone resorption inhibitors. Hence, we need to identify and develop novel drugs, which can promote bone formation. Tanshinone, a traditional Chinese medicine, has been used clinically as a drug for the treatment of various chronic diseases . MoreoveTanshinone-targeted genes were obtained from Search Tool for Interacting Chemicals (STITCH). The interaction network of tanshinone and its targeted genes were constructed using STITCH online tool. The closeness, betweenness, and the degree of the network genes were analyzed by Cytoscape 3.7.2. .p < 0.05 (http://bioinformatics.psb.ugent.be/webtools/Venn/).Human osteoporosis KEGG pathways were retrieved from miRWalk2.0 . Databasp < 0.05 . ThereafThe enrichment result of the top five KEGG pathways were generated and visualized in GO plot . The comp values were analyzed. We used Pathway Builder Tool 20 (www.proteinlounge.com) to retrieve the pathway part related to the tanshinone-targeted genes.The top five common KEGG pathways with the smallest in vivo study was reviewed and approved by The Honghui Hospital, Xi’an Jiao Tong University Standing Committee on Animals. For our study, 30 female C57BL/6 mice (6-week-old) were obtained from the Department of Research of Honghui Hospital, Xi’an Jiao Tong University. To generate the murine osteoporosis model, eight-week-old male C57BL/6 mice were treated with a daily intragastric administration of prednisone acetate (2.1 mg/kg/d), and then, the 30 osteoporosis mice were distributed equally into the PBS treated group (n = 10), 10 mg/kg Tanshinone treated group (n = 10), and 30 mg/kg Tanshinone treated group (n = 10). We administered the treatment for 8 weeks.The 2 incubator. Then, 1 μM dexamethasone was added to produce the in vitro osteoporosis model. After which they were designated as osteoporosis group, 0.2 nM Tanshinone group, and 0.6 nM Tanshinone group for 7 days.MSCs were kindly donated by the Xi’an Jiao Tong University, Xi’an, China. Cells were cultured in a specific osteogenic differentiation medium at 37°C in a 5% COThe micro-CT device used for analysis was BRUKER SkyScan 1276 scanner. The trabecular bone from 0.2 to 0.5 mm below the proximal tibia growth plate was measured. Finally, the total volume (TV), bone volume (BV), BV/TV, and bone mineral density (BMD) were calculated and analyzed.Thermal Cycler C-1000 Touch system  was used to perform qRT-PCR reactions after the total RNA extraction. The primers used in this study have been listed in GraphPad Prism 8.0 was used for the statistical analysis. Statistical analysis was performed using Student’s t test (two groups) or ANOVA with Tukey’s post hoc test (over two groups).CCND1, CYP2C19, ENSGGOG00000022765, TMPRSS11D, CYP2C9, CYP1A1, CASP3, CYP1A2, NOS3, CYP1B1 were shown in the first shell which indicated that tanshinone potentially has a direct effect on these diverse genes. CDK6, AKT1, BIRC2, XIAP, arginine, CDKN1A, CDK4, NG-hydroxy-L-a, CDKN1B, calcium ions were shown in the second shell and BIRC3, citruline, CDK2, caffeine, melatonin, CDKN2A, estradiol, RB1, PCNA, nicotinami.e p. were shown in the third shell, which indicated that tanshinone might have a secondary effect on these genes. According to our analysis, AKT1 displayed the highest weight in the interactive network (We retrieved 21 tanshinone-targeted genes using STITCH with the limitation of three shells. Thereafter, the interaction of tanshinone-targeted genes was established in STITCH . CCND1,  network .p values were the small cell lung cancer pathway, Pathway in cancer, Chronic myeloid leukemia pathway, cell cycle pathway, and glioma pathway (36 KEGG pathways significantly enriched by tanshinone-targeted genes were obtained in DAVID. With miRWalk2.0, 110 human osteoporosis KEGG pathways were retrieved. Finally, 21 common KEGG pathways were identified . The top pathway .AKT1, CDKN1A, CDK6, CDK4, CDK2, CASP3, RB1, CDKN2A, CCND1, CDKN1B, XIAP, BIRC3 and BIRC2 were involved in the top five common KEGG pathways (CND1, CDK6, RB1, CDK4 were observed to be involved in every common KEGG pathway of the top five. Therefore, they were identified as hub genes. The top three genes with the highest degrees were AKT1, CDKN1A, and CDK6. The relative positions on the chromosome and degree centrality information of tanshinone-target genes have been shown in AKT1 displayed the greatest degree, betweenness, and closeness.Among the 21 tanshinone-targeted genes, pathways . CCND1, The part of the top 5 shared KEGG pathways that can be targeted by tanshinone have been shown in AKT1 was measured by qRT-PCR, and the result indicated that tanshinone could promote the expression of AKT1 (OCN, Col-1, and Runx2 were analyzed in the tanshinone-treated cells, and the result indicated a positive impact of the drug on the osteogenesis (via activation of AKT1.First, the levels of  of AKT1 . Next, togenesis . Taken tin vivo mouse model. Consistent with the findings of in vitro experiment, BMD, BV/TV, BV, and TV were significantly increased in tanshinone treated group as compared to the osteoporosis group (The effect of tanshinone on osteoporosis was also validated in an is group .Bones play an important role in the protection of internal organs with both rigidity and toughness. The repeated bone reconstruction process primarily maintains integrity and mechanical stress of bone. Bone reconstruction includes resorption of old bone and formation of new bone, which are generally coupled in time and space. Bone reconstruction occurs every moment, throughout the life of a mammal, and the whole body bones are renewed every 10 years .Bone reconstruction is orchestrated by osteoblast, osteocyte and osteoclast mutually . Osteoblβ-catenin pathways (AKT1 gene expression, promote the MSCs proliferation, and suppress their apoptosis. These findings indicated that tanshinone might improve osteoporosis by targeting AKT1.The bone reconstruction is in a dynamic equilibrium state under the action of the various systemic or local bone growth regulators. A variety of signal transduction pathways have been reported to be involved in regulating bone reconstruction, such as Hedgehog, MAPK, Notch, RANKL/RANK/OPG, PI3K/Akt, and Wnt/pathways . In our in vivo experiments were not performed in this study. Secondly, the enrichment test of AKT1 in different osteoporosis subtypes was not studied.There are some limitations associated with this study. Firstly, Taken together, our findings indicate that tanshinone can alleviate osteoporosis, which was potentially mediated through modulation of the AKT1 expression. Tanshinone could thus serve as a promising agent for the treatment of osteoporosis.


Heart failure (HF) is a serious manifestation or advanced stage of various cardiovascular diseases, and its mortality and rehospitalization rate are still on the rise in China. Based on the network pharmacology method, 59 components of Zhen Wu decoction (ZWD) and 83 target genes related to HF were obtained. Through the PPI network, four potential therapeutic targets were identified: AKT1, IL6, JUN, and MAPK8. The beneficial components of ZWD might intervene HF through the AGE-RAGE signalling pathway in the diabetes component, fluid shear stress and atherosclerosis, the TNF signalling pathway, TB, and Kaposi sarcoma related herpesvirus infection, according to a KEGG enrichment study. The protein interaction network of candidate targets was constructed by the STRING database, and the protein interaction network was clustered by MEODE software. GO and KEGG enrichment analyses were performed on the core modules obtained by clustering. Finally, AutoDock Vina software was used for molecular docking verification of key targets and active ingredients. The result was that 75 active ingredients and 109 genes were screened as potential active ingredients and potential targets of Shengjie Tongyu decoction for CHF treatment. The main active components were quercetin, luteolin, kaempferol, dehydrated icariin, isorhamnetin, formononetin, and other flavonoids. Il-6, MAPK1, MAPK8, AKT1, VEGFA, and JUN were selected as the core targets. Molecular docking showed that the key components were well connected with the target. GO enrichment analysis showed that Shengjie Tongyu decoction could play a role through multiple biological pathways including angiogenesis, regulation of endothelial cell proliferation, binding of cytokine receptors, negative regulation of apoptotic signalling pathways, regulation of nitric oxide synthase activity, and reactive oxygen metabolism. Key pathways mainly focus on the toll-like receptor signalling pathway, nod-like receptor signalling pathway, MAPK signalling pathway, mTOR signalling pathway, JAK-STAT signalling pathway, VEGF signalling pathway, and other pathways. Through molecular docking technology, it was found that a variety of effective components in ZWD, such as kaempferol. Molecular docking technology has preliminatively verified the network pharmacology and laid a foundation for the follow-up pharmacological research. Heart failure (HF) refers to a variety of heart function or organic diseases caused by cardiac insufficiency or dysfunction, resulting in cardiac output which cannot meet the metabolic needs of the disease . After tZhen Wu decoction (ZWD) comes from the treatise on febrile and miscellaneous diseases by Zhang Zhongjing. Modern research shows that it has a therapeutic effect on HF and other diseases . Traditihttps://tcms-pw.com/tcmsp.php). Oral bioavailability is determined by the same principle [https://www.UniProt.org/). Gene mapping is performed towards obtaining gene symbols to the corresponding target.Input each medica of ZWD into TCMSP  data sterinciple  ≥ 30%, drinciple  website https://www.malecards.org/) were used. To obtain the target genes of coronary heart disease, we searched with the keyword “hyperlipidemia” in the abovementioned drug target genes, and disease target genes were introduced into the Venn diagram web (http://bioinformatics.PSB.ugent.be/webtools/Venn/). The common illness and therapeutic target genes were discovered.In the human gene database, GeneCards (https://genecards.Weizmann.ac.il/v3/) and MalaCards  in the PPI network were determined based on the degree values.The suitable target genes of the disorders and medications mentioned above have been entered into the STRING website . The speHomo sapiens” was chosen for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analyses [The illness medication commonality genes were added to the David database, and the species “analyses , 17. AndPotential targets are imported into the David6.8  gene function analysis tool. The identifier is selected. The list type were set to official gene symbol, gene list, and species is limited to Homo sapiens. Submit List is clicked for the GO function and KEGG enrichment analysis. At the same time, the ENSG encoding corresponding to potential genes is obtained by gene ID conversion. The whole human genome is taken as the background gene. The ENSG codes obtained were imported into the OmicShare (WWW. omicshare.com/tools) website for KEGG pathway annotation number analysis. To create the network design of active ingredient, target, and signal pathways, the active ingredient, target, and signal routes were loaded into the Cytoscape 3.6.0 programme.https://www.rcsb.org/search). The abovementioned compounds were entered into the DS programme and prepared using the “prepare ligands” feature. To locate the probable active surface area in the protein cavities, remove water, ligand classes, and irrelevant side chains from the receptors and click on receptor-ligand interactions > define and edit the binding > from receptor cavities. (3) CDOCKER INTERACTION ENERGY (molecular docking binding energy) was utilised to identify and rank the optimum molecular conformations of every docking molecule. The findings reveal that the smaller the binding energy of the docking study conformation, the more stable the binding notion is and the more likely the reactive receptor molecule and ligand will bind.The PDB structure of hub protein was acquired by researching hub genes on the RCSB PDB database  Wolf, 21 active ingredients in Aconiti Lateralis Radix Praeparata, and 5 active ingredients in Zingiber officinale Roscoe, with 59 constituents . The abo13194 genes targets of hyperlipidemia were obtained since the GeneCards website, and disease and drug boards were imported to draw Venn diagram website for Wayne map analysis, and 83 common target genes of illness and drugs were obtained, as shown in As shown in As shown in To more clearly show the relationship between components, targets, and pathways of ZWD in the treatment of HF, the network of pathways, active ingredients, and core targets was constructed using Cytoscape software . A netwoAccording to the method in 1.6 for molecular verification, the docking results of each group are shown by the heat map in HF is a range of complicated clinical syndromes, a significant manifestation or end phase of several heart illnesses . It is pThe compensations and characteristics of old Chinese medicine diagnosis and management of HF are obvious, and rich clinical experience has been accumulated. The combination of Chinese and Western medicine allows patients to get comprehensive treatment in all directions . ZWD is β. The apoptosis of the signal transduction pathway of p38 mitogen-activated protein kinase/extracellular signal-regulated kinase can inhibit apoptosis, thus improving the cardiac protection of KF. Kaempferol is a kind of flavonoid compound, which prevents the cardiac remodelling induced by angiotensin II by reducing the inflammation and oxidative stress induced by angiotensin II [κ B (NF- κB) and nuclear translocation, and the protective action of DCM is mediated by nuclear factors-like erythrocytes 2 P45 related factor 2 (Nrf-2). Kaempferol reduced diabetes-induced cardiac fibrosis and apoptosis in type 1 diabetic mice generated by streptozotocin. These alterations were similarly associated with decreased inflammation and oxidative stress in diabetic mice's hearts [β. The pathway abridged the programmed cell death of myocardial cells induced by H/R [β, CTGF, Iα, and IIIα. The expression of collagen and phosphorylation of Smad2 and Smad3. Eonoside can inhibit the apoptosis of myocardial cells, and the introduction of AB by the cleavage of Bax and caspase 3 reduces cardiac hypertrophy, fibrosis, apoptosis, and inflammation in stress overload mice and improves left ventricular function [β. The expression of Smad3 and 1 suggests that the potential mechanism of cardiac protection may be related to TGF- β 1/the down-down height correlation of the Smad signal path [In the current research, 59 dynamic apparatuses of ZWD was found, including kaempferol, paeoniflorin, (+) -catechin, 6-methylgingediacetate 2, and (R) -norcoclaurine. Kaempferol blocks Akt/glycogen kinase (GSK)-3ensin II , 23. Kaes hearts . Sinapins hearts . Catechis hearts . Catechid by H/R . Paeoniffunction . Paeoniffunction . Paeonifnal path .We also got 83 common target genes of disease drugs. Through PPI network analysis, we got the hub genes with the highest degree: AKT1, IL6, Jun, and MAPK8. AKT1 is a critical gene in the PI3k/Akt signalling pathway. AKT1 is involved in cell functions such as apoptosis, glucose metabolism, and cell proliferation and growth . Akt actβ, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, and IL-18 were downregulated, while IL-5, IL-7, or IL-33 were downregulated [Many cardiovascular diseases, such as atherosclerosis, cardiac hypertrophy, and vascular remodeling, are linked to changes in Akt signal transduction . In the egulated . The conegulated .Response to the drug, lipopolysaccharide, bacterial molecule response, and other BPs; membrane raft, membrane microdomain, plasma membrane raft, caveola, and other MFs; nuclear receptor activity, live activated transcription factor activity, peptide binding, G protein-coupled amine receptor activity, and other MFs. The AGE-RAGE signalling pathway in diabetes composition, fluid shear stress and atherosclerosis, TNF signalling pathway, TB, and Kaposi sarcoma linked herpesvirus infection are all included in the KEGG enrichment study. It is known that the formation and accumulation of advanced glycation end products (ages) are accelerated in diabetes. In addition, even with improved glycemic control, ages hardly degraded and remained in diabetic vessels for a long time. Therefore, aging can explain why previously accumulated diabetic exposure may lead to the current progression of diabetic vascular complications . Kaempfeα the myocardial effect of CPB also depends on the duration of exposure and its location [In contrast, lower levels of TNF can result in systolic dysfunction, hypertrophy, fibrosis, and cell death. In type 2 cardiomyocytes, the presence of TNF and subsequent activation of the TNF receptor produced protective benefits. In addition to its concentration and receptor subtypes, TNFlocation .Discovery Studio  is a younger breed of molecular modeling software that uses the method of the CDOCKER module to randomise search the conformational changes of molecules and then optimises each shape in the receptor active site region using the stochastic optimization method , 41. We Based on the network pharmacology method, 59 components of ZWD and 83 target genes related to HF were obtained. Through the PPI network, four potential therapeutic targets were identified: AKT1, IL6, Jun, and mapk8. The beneficial components of ZWD might intervene HF through the AGE-RAGE signalling pathway in diabetic composition, fluid shear stress and atherosclerosis, the TNF signalling pathway, TB, and Kaposi sarcoma related herpesvirus infection, according to a KEGG enrichment study. Through molecular docking technology, it was found that a variety of effective components in ZWD, such as kaempferol, could combine with hub protein, which preliminarily verified network pharmacology and laid a foundation for subsequent pharmacological research.