Tremella aurantialba. In this study, ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF/MS) was used to screen the chemical components in T. aurantialba. Then, network pharmacology was used to reveal the potential biological activities, active compounds, and therapeutic targets of T. aurantialba. Finally, the potential binding sites of the active compounds of T. aurantialba and key targets were studied by molecular docking. Results showed that 135 chemical components in T. aurantialba, especially linoleic acid, and linolenic acid have significant biological activities in neuroprotective, anticancer, immune, hypoglycemic, and cardiovascular aspects. The existence of these bioactive natural products in T. aurantialba is consistent with the traditional use of T. aurantialba. Moreover, the five diseases have comorbidity molecular mechanisms and therapeutic targets. The molecular docking showed that linolenic acid, adenosine, and vitamin D2 had higher binding energy with RXRA, MAPK1, and JUN, respectively. This study is the first to systematically identify chemical components in T. aurantialba and successfully predict its bioactivity, key active compounds, and drug targets, providing a reliable novel strategy for future research on the bioactivity development and utilization of T. aurantialba.As a kind of medicinal and edible homologous fungus, there is a lack of data on the medicinal value of  Tremella aurantialba is a well-known medicinal and edible plant belonging to fungi, Basidiomycota, Basidiomycotina, Tremellales, Naemateliaceae, Naematelia Fr  grade methanol, acetonitrile, ammonium formate and formic acid were purchased from Sigma-Aldrich . Deionized water (18.3 MΩ) was generated by a Milli-Q water purification system . Tremella aurantialba samples was frozen dried under vacuum condition and crushed into powder through a 100-mesh screen. Then the samples were kept at –80°C for further use. The extraction procedure and conditions were performed as following: 1.0 g of freeze drying Tremella aurantialba powder was weighted and placed in 50 mL centrifuge tube, and then the sample was extracted with 20 mL methanol-water  . After vortexing for 1 min and sonicating at room temperature for 30 min, the mixture was centrifuged at 10 000 r/min at 4°C for 5 min. 1 mL supernatant was filtered with a 0.22 μm nylon membrane before UPLC-Q-TOF/MS analysis.A LC-30AD UPLC system  was used for the chromatographic separation of the samples. Compounds separation was performed on a Waters ACQUITY UPLC HSS T3 column  using a gradient elution consist of 0.01% formic acid + 2 mmol/L ammonium formate (A) and acetonitrile + 0.01% formic acid + 2 mmol/L ammonium formate (B). The gradient program was: 5–12% B at 0–5 min, 12–55% B at 5–7 min, 55–65% B at 7–10 min, 65–98% B at 10–20 min, 98% B at 20–25 min, 98–5% B at 25–25.1 min and 5% B at 25.1–28 min. The flow rate was 0.3 mL/min, the column temperature was maintained at 40°C. The sample injection volume was 2 μL.m/z. Other optimized MS parameters were set as follows: ion spray voltage 5500 V in positive ion mode and 4500 V in negative ion mode; the ion source gas1, 50 psi; the ion source gas 250 psi; the curtain gas, 35 psi; ion source temperature, 500°C; declustering potential, 80 V; collision energy, 60 V.The QTOF MS analysis, controlled by the Sciex OS software , was performed on a hybrid quadrupole time-of-flight tandem mass spectrometer Q-TOF/MS with an electrospray ionization (ESI) source . The ionization of compounds was in the positive or negative mode. Information dependent acquisition method was used for acquiring spectra data with a scan range from 100 to 1000 Tremella aurantialba were screened by SwissADME platform1 and literatures. At SwissADME platform, gastrointestinal absorption, one of the pharmacokinetic parameters, was set as “HIGH” as the condition for drug absorption and active compounds with good oral bioavailability were screened. At SwissADME platform, the screening criteria for bioavailability are lipophilicity: XLOGP3 between –0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Å2, solubility: log S not higher than 6, saturation: fraction of carbons in the sp3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds. Besides, the drug-likeness is also considered. For cosmeceutical parameters , two or more of them with “YES” can be regarded as active components. At the same, the components with significant pharmacological activity reported in the literature are also considered as active ingredients. Secondly, Swiss Target Prediction platform2 was applied to predict the possible targets. Swiss Target Prediction selected the targets whose probability is >0.12 in the prediction results for further analysis. At the same time, experimentally verified targets information was downloaded from NPASS3 and the entries related to the active components of Tremella aurantialba were extracted. Finally, target information was integrated and accumulated to obtain the possible targets of Tremella aurantialba active components.First, the active components of 4), the Online Mendelian Inheritance in Man Database (OMIM5), the GeneCards database,6 the Therapeutic Target Database (TTD7), the Comparative Toxicogenomics Database (CTD8), a database of gene-disease associations (DisGNet9), using “Nervous system disease,” “Immune System Diseases,” “Endocrine System Diseases,” “Neoplasms,” “Cardiovascular Diseases” and their synonyms and descendants in the CTD Database as the keywords, respectively. The above targets were converted and queried into the UniProt ID format with “Homo sapiens” as the qualifying condition in the UniProt database.10 Finally, the gene library of all was established by eliminating repeated targets.Data for all-associated disease targets were acquired from six databases, including the National Center for Biotechnology Information database ”. Input protein-protein interaction (PPI) information into Cytoscape 3.7.113 for visualization and constructs network of potential key targets.The intersection targets above were imported into the STRING databaseThe CytoHubba plugin in Cytoscape was used to identify hub genes. Three critical topological parameters were chosen for screening the core composite targets based on the PPI network: degree (D), betweenness (B) and closeness (C). Values for the three parameters indicated the significance as well as the impact of relevant nodes in the entire network. The top 10 nodes were set as the core targets and the hub gene was obtained through the intersection of the top 10 core targets obtained by different algorithms. The MCODE plug-in in Cytoscape was used to screen PPI network modules using various cut-offs: degree = 2, k-core = 2, node score = 0.2, and max depth = 100.14 with FDR < 0.05 and P < 0.05 as cut-off values. R version 3.4.1 was used to visualize the results.The intersection targets above were subjected to the Gene Ontology (GO) biological process analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the DAVID database,Tremella aurantialba. For visualization, potential active components and matching intersection targets were imported into Cytoscape 3.7.1 software and a network of compound-target network was built. Each component of targets is represented by nodes and the relationship between the components, diseases and the targets are represented by connecting lines.After obtaining the intersection genes, reverse screening the active components in 15 and the PubChem.16 Molecular docking was performed using the AutoDock software. The water molecules and atoms were removed from the target receptors and then the affinities were obtained. Finally, the binding sites of composition and targets were visualized by the PyMOL software.The crystal structures of the targets and the chemical structures of the composition were obtained from the PDBTremella aurantialba and the chromatography condition was optimized to obtained higher peak capacity, shorter retention and better resolution of components in Tremella aurantialba. Subsequently, a total of 135 chemical components were rapidly identified by comparing with TCM MS/MS database, online Chemspider database or inferred through mass spectrometry fragment ion analysis and literature data, including 22 organic acids, 20 amino acids and their derivatives, 12 fatty acids, 13 saccharides, 8 nucleosides, 6 vitamins, 7 alkaloids, 8 esters, 4 amides, 3 terpenoids, 2 phenols, 2 ethers, 1 alcohol, 1 ketone and 26 other classes  database, fragmentation pathway and literature data. We took compound 3 as an example, which showed a protonated ion [M-H]Tremella aurantialba. We took compound 13 as an example. Based on the analysis of its elemental composition and fractional isotope abundance, its molecular formula was predicted to be C6H8O7. The precise molecular weight was 192.0270, and the main fragment ions were analyzed via the MS/MS screening and observed at m/z 129.0115 [M-H-H2O-CO2]–, 111.0018 [M-H-2H2O-CO2]–, 85.0241 [M-H-H2O-2CO2]– and 67.0143 [M-H-2H2O-2CO2]– in the negative ion spectrum. It was identified as citric acid by searching the database, inferred through mass spectrometry fragment ion analysis and literature data  and its receptor RAGE can cause functional disorders of blood vessels and immune cells, changes in programmed cell death signals and so on . EndogenThe ErbB protein family is a family of four structurally related receptor tyrosine kinases, including EGFR1/ErbB1/HER1, EGFR2/ErbB2/HER2, EGFR3/ErbB3/HER3, and EGFR4/ErbB4/HER4. These receptors can form homodimers or heterodimers with each other, and dimers are required for signaling activity. So far, there are many evidences about the importance of ErbB signaling pathway in the development of cancer, nervous system diseases and cardiovascular diseases. For example, CARF promotes colorectal cancer stemness by activating the ErbB signaling pathway . ErbB acTremella aurantialba to fight disease through this pathway.Relaxin (RLN) is a part of the insulin superfamily, including RLN1, RLN2 and RLN3. RLN2 acts as neuropeptides in the nervous system, as vasodilators and cardiac stimulants in the cardiovascular system, and as antifibrotic agents while the roles of RLN1 and RLN3 are unclear. Many of the effects of human gene-2 relaxin (H2 relaxin) are mediated by its homologous G protein-coupled receptor (GPCR) and relaxin family peptide receptor (RXFP1). For example, the activation of RXFP1 by rh-RLN2 can improve mast cell degranulation and neurological function by inhibiting NF-κB of PI3K-AKT/neoplasm necrosis factor-alpha-induced protein 3 (TNFAIP3) signaling pathway, which indicated that rh-RLN2 may be a promising therapeutic agent to reduce neuroinflammation and secondary brain injury in germinal matrix hemorrhage patients . RLN-2 pProtein-protein interaction (PPI) network was used to analyze the interactions between overlapping targets. Among the overlapped 282, 297, 205, 376 and 178 targets, a total of 598, 640, 446, 725 and 343 PPIs were obtained from STRING Database, respectively. By setting a degree value greater than 3, optimized PPI networks were further constructed using Cytoscape software . FurtherTremella aurantialba on five diseases  were obtained, respectively, by CytoHubba plugin analysis and the common core targets of Tremella aurantialba on five diseases were also obtained, such as AKT1, JUN and ESR1 -costunolide, phenylalanine, cinnamic acid, mannitol, adenosine, were obtained by reverse screening based on the above key targets. They were considered key active components that have the activity of exacerbating or fighting five diseases in this study. Except adenosine, which was only selected as a key compound for immune system, the other compounds were speculated to have potential effects on all five diseases. A visual network of key active compounds, key genes and five diseases were shown in antialba . But theTremella aurantialba have been widely recorded in TCM books. However, there are few modern studies on the chemical components and pharmacological activities of Tremella aurantialba, which seriously hinder the development and utilization of Tremella aurantialba. Traditional Chinese medicine emphasizes the holistic concept and its drug use has the characteristics of multi-target and multi-function. However, for TCM with unclear chemical composition, blindly relying on the experience of ancestors will bring some safety risks to patients, and it is impossible to fully explore the pharmacological effects of Tremella aurantialba. In this study, UPLC-Q/TOF-MS was first used to investigate the chemical components of Tremella aurantialba and network pharmacological analysis was used to explore the potential pharmacological activities of Tremella aurantialba. Then, a total of 135 compounds were identified or tentatively characterized. Among them, the enrichment analysis of the targets of the active components of Tremella aurantialba suggests that Tremella aurantialba has potential pharmacological effects on nerve, immune, endocrine, neoplasm and cardiovascular diseases, which is consistent with the traditional Chinese medicine books and related literature reports. Next, the targets of five diseases were collected and collated, and the intersection with the targets of Tremella aurantialba components was obtained. Then, the main targets, biological processes and metabolic pathways of Tremella aurantialba for the intervention of five diseases were also obtained. Simultaneously, fourteen active components and their key targets were first screened by reverse screening to have potential therapeutic effect for nervous system, immune system, endocrine system, neoplasm system, as well as cardiovascular system diseases. Chinese medicine components are the material basis of efficacy. By extrapolating the active components acting on the key intersection targets, we hypothesized that these components might be the important material basis for the pharmacological activities of Tremella aurantialba in five diseases. In this regard, we used molecular docking technology combined with literature review to verify and investigate the above predicted results. Interestingly, we also identified key comorbid mechanisms for Tremella aurantialba to simultaneously intervene in five diseases. In this step, three core targets, seven biological processes and eight metabolic pathways were identified. These results provide reference for the treatment of some disease complications and the exploration of potential disease treatment targets. In conclusion, this study will provide new ideas for further understanding the pharmacological activities of dominant plant resources such as Tremella aurantialba and exploring key functional components (groups), and provide important research strategies for the full development and utilization of dominant plant resources. Of course, the prediction results need to be further verified in vivo and in vitro.As a famous medicinal and edible homologous fungus, the traditional pharmacological effects of The original contributions presented in this study are included in the article/YY: conceptualization, writing—original draft, and supervision. MW: methodology, investigation, and data curation. XG: data curation. XW, YL, and NC: validation. CF: supervision and writing—review and editing. PL: investigation, conceptualization, and funding acquisition. YZ: investigation, conceptualization, and writing—review and editing. All authors read and agreed to the published version of the manuscript.


Eleutherococcus senticosus (Rupr. and Maxim.), Maxim , Lonicera japonica Thunb , Crataegus pinnatifida Bunge , and Auricularia auricula. SEJZ has been designed to treat hyperlipidemia. Despite the therapeutic benefits of SEJZ, its underlying mechanism of action is not known. We explored the efficacy of SEJZ against hyperlipidemia by integrating network pharmacology and 16S rRNA gene sequencing and elucidated its mechanism of action. First, SEJZ targets were found through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and from the literature. Hyperlipidemia-related therapeutic targets were obtained from GeneCards, Online Mendelian Inheritance in Man, and DrugBank databases. Then, Search Tool for the Retrieval of Interacting Genes/Proteins and Cytoscape were applied for the analyses and construction of a protein–protein interaction (PPI) network. The Kyoto Encyclopedia of Genes and Genomes database was employed to identify signaling pathways that were enriched. Second, the therapeutic effects of SEJZ against hyperlipidemia induced by consumption of a high-fat diet in rats were evaluated by measuring body weight changes and biochemical tests. SEJZ treatment was found to alleviate obesity and hyperlipidemia in rats. Finally, 16S rRNA gene sequencing showed that SEJZ could significantly increase the abundance of short-chain fatty acid-producing bacteria, restore the intestinal barrier, and maintain intestinal-flora homeostasis. Using PICRUSt2, six metabolic pathways were found to be consistent with the results of network pharmacology: “African trypanosomiasis”, “amoebiasis”, “arginine and proline metabolism”, “calcium signaling pathway”, “NOD-like receptor signaling pathway”, and “tryptophan metabolism”. These pathways might represent how SEJZ works against hyperlipidemia. Moreover, the “African trypanosomiasis pathway” had the highest association with core genes. These results aid understanding of how SEJZ works against dyslipidemia and provide a reference for further studies.Shenerjiangzhi formulation (SEJZ) is a new traditional Chinese medicine formulation (patent number: CN110680850A). SEJZ contains  Hyperlipidemia is a common disorder of lipid metabolism. It is one of the leading risk factors for cardiovascular disease, atherosclerosis, and other metabolic disorders. Hyperlipidemia is often manifested by obesity . HyperliLipid-lowering medication has undergone a revolutionary development since the introduction of statins . HoweverLachnospira and Haemophilus and low expression of bacteria of the phylum Bacteroidetes and genera Ruminococcus, Blautia, and Roseburia are found, which hinder SCFA production. Increased intestinal permeability, which leads to chronic inflammation and interferes with cholesterol transport, thereby increases susceptibility to certain metabolic disorders, such as diabetes mellitus and obesity (The “second genome” of the human body is the gut microbiota (GM). The latter can extract energy from indigestible dietary components to promote host metabolism and play a part in synthesizing vitamins and producing important bioactive metabolites, such as short-chain fatty acids (SCFAs) . Further obesity .Eleutherococcus senticosus (Rupr. and Maxim.) Maxim , Lonicera japonica Thunb , Crataegus pinnatifida Bunge , and Auricularia auricula. SEJZ has been employed to treat hyperlipidemia caused by stasis due to excessive accumulation of heat toxins, as well as weakness of the spleen and stomach. The polysaccharides in E. senticosus (Rupr. and Maxim.) and Maxim  can improve glucose utilization and regulate blood levels of lipids (E. senticosus (Rupr. and Maxim.) and Maxim.  are closely correlated with lipid levels in the blood, and triterpenoid saponins have been reported to have optimal activity against hyperlipidemia  and C. pinnatifida Bunge  have been demonstrated to prevent and treat hyperlipidemia  is the most commonly used single botanical drug to treat hyperlipidemia  consists of f lipids . The proipidemia . Variousipidemia . C. pinnipidemia . Apigeniipidemia , kaempfeipidemia   as the keywords to harvest the targets of the active ingredients of SEJZ through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (https://old.tcmsp-e.com/tcmsp.php). The other compounds in the botanical drugs in SEJZ were obtained from the literature (www.genecards.org/), DrugBank (https://go.drugbank.com), and Online Mendelian Inheritance in Man (www.omim.org/) databases using “hyperlipemia” as the keyword to search for disease targets. Then, all the target proteins obtained above were converted into standardized gene names through the UniProt database (www.uniprot.org/).We used “terature . This inhttps://string-db.org) and analyzed with organism species limited to Homo sapiens and a high confidence score (>0.9). The obtained PPI data were entered into Cytoscape 3.8.2 (https://cytoscape.org/) for visual analyses, and the topological parameter “Degree” was set to be two times greater than the median value  database  to conduct analyses of signaling pathway enrichment via the KEGG database. Species were limited to Homo sapiens and the diagram for signaling pathway enrichment was obtained using R 3.5.2. .We wished to clarify the potential signaling pathways of SEJZ in the treatment of hyperlipidemia. We used the Metascape database (E. senticosus (Rupr. and Maxim.), Maxim  (lot number Z20180312), C. pinnatifida Bunge.  (Z20180306), and L. japonica Thunb.  (lot# Z20180215) were purchased from Harbin Pharmaceutical Group Shiyitang . A. auricula was obtained from the Mudanjiang planting area and identified by Professor Wang Zhenyue of Heilongjiang University of Traditional Chinese Medicine . Standard chow and a HFD  were provided by the laboratory animal center of HUTCM.E. senticosus (Rupr. and Maxim.), Maxim , L. japonica Thunb. , C. pinnatifida Bunge. , and A. auricula were prepared at a ratio of 6:4:4:3 (w/w/w/w): 265 g of E. senticosus (Rupr. and Maxim.), Maxim. , 177 g of L. japonica Thunb. , 177 g of C. pinnatifida Bunge. , and 133 g of A. auricula. After mixing, all botanical drugs were soaked in 12 volumes of water for 30 min and heated at temperature 100°C to prepare the decoction. The formulation was decocted twice for 3 h, and the two decoctions were condensed and refluxed after combination; the compound extract was concentrated at 55°C and 30 r/min by rotary evaporator, and a 236.1 g of compound extract was prepared by freeze drying. First, the experimental drug solution was prepared according to the concentration required for high-dose administration. Using the weight of 24 Sprague–Dawley (SD) rats in the administration group, we determined that 25 g of the compound extract of the crude drug was required. Subsequently, 40 g of the extract was taken to account for potential experiment error. To meet the high dose (7.32 g/mg), we required 13.3 g of the compound extract. The volume of intragastric administration of the high-dose compound extract was 1.25 ml for each mouse, making the concentration of high dose 1.33g/ml. Therefore, the 40 g of compound extract was resuspended in 30.2 ml of water. To reduce experimental error, the high-dose solution was used as the mother solution. Subsequently, 10 and 5 ml of this high-dose mother solution were diluted with 20 and 19 ml of water, respectively, to prepare medium- and low-dose experimental solutions, respectively. Compound extract ratio and similar basic quality parameters, qualitative and quantitative assessments, prepared ratio and extraction process  the study protocol. Specific pathogen–free male SD rats (180 ± 20 g) were purchased from the Experimental Animal Center of HUTCM [certificate number: SYXK (Heilongjiang) 2018-007]. Rats were maintained in a controlled environment at 22 ± 3°C and humidity of 53 ± 7%. Rats were exposed to natural lighting conditions and allowed to acclimatize to their environment for 3 days. All rats had free access to chow and water.n = 8) and model group (MG) (n = 32). Rats in the BC group were fed normal chow. The remainder of the rats was modeled for 2 weeks with a HFD similar to that employed for the development of hyperlipidemia in humans. Two weeks after modeling, blood was collected from the orbital vein after an overnight fast. If the serum level of TG, TC, and LDL in the MG was significantly higher than that in the BC group, then the model was deemed to have been created (p < 0.01)  group ( created . Thirty- < 0.01) . Then, tg for 10 min at 4°C. We took 0.3 g of large intestine (cecum) contents  to avoid pain before killing. Blood samples were collected from the abdominal aorta and placed into disposable blood-collection tubes. Then, the blood samples were placed at room temperature for 30–60 min and centrifuged at 1788.8 × contents , placed An automated biochemical analyzer  was used to measure the level of TC, TG, LDL, and HDL in the serum of rats in each group.https://github.com/torognes/Vsearch). QIIME 2019.4 (http://qiime.org/) was used to calculate the Chao1 Index, Observed Species Index, Shannon Index, Simpson’s Diversity Index, and Good’s Coverage Index to ascertain α-diversity. The unweighted UniFrac method was employed to carry out principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) for β-diversity analysis. All processes were undertaken on GenesCloud (www.genescloud.cn/). PICRUSt2 (http://picrust.githubio/) was used to predict functionally enriched signaling pathways with regard to gut metabolism  sequenced the bacterial DNA on the MiSeq™ platform . Bioinformatics analysis was carried out on the basis of operational taxonomic units, which were clustered on the basis of sequence similarity of 100% according to Vsearch (tabolism .https://bioinfogp.cnb.csic.es/tools/venny/) to obtain the therapeutic targets of SEJZ against hyperlipidemia , which indicated that the model had been established (p < 0.01). Compared with the MG, the body weight and level of TC, TG, LDL, and HDL in serum were altered significantly in the treatment groups, especially in the HD group (p < 0.01), indicating that SEJZ could improve the hyperlipidemia caused by consumption of a HFD in a dose-dependent manner. The HD group had the best effect for lowering the lipid level in the blood , Maxim. , and L. japonica Thunb  in SEJZ  were selected to evaluate the lipid-lowering effect of SEJZ. TC is used to measure the cholesterol contained in lipoproteins in blood. HDL molecules remove fat and cholesterol from the body and protect the cardiovascular system. LDL molecules move cholesterol around the blood and deposit it on artery walls . TG moleBacteroides is potent, which may be related to SCFA generation . Overexpression of proinflammatory factors can activate overexpression of the suppressor of cytokine signaling-3, increase the production of mature sterol regulatory element-binding protein-1, aggravate TG deposition, and cause abnormal lipid metabolism . In addid plasma . TherefoLactobacillus species can be used as probiotics that can produce aryl hydrocarbon receptor ligands to accelerate tryptophan metabolism, thereby affecting SCFA production and reducing the cholesterol level. Abnormal metabolism of arginine and proline is also common in dyslipidemia  provide energy to colonic epithelial cells and regulate gene expression (Lactobacillus species. Studies have shown that Lactobacillus species can affect cholesterol oxidase and fatty acid synthase, as well as redistribute TG and cholesterol in the liver and blood, thereby improving lipid metabolism disorders and intestinal hepatic circulation of bile acid (Oscillospira species, which degrade glycan components in human intestines (Helicobacter species colonize the human gastric mucosa, can cause gastric cancer and peptic ulcers, and can alter the lipid profile in the body (Helicobacter species was increased significantly in the SEJZ treatment group. Fortunately, the prevalence of Helicobacter species is declining worldwide (The abundance of bacteria from n humans . We showoseburia , Phascolevotella , and Blapression , inhibitpression  (Zhang epression . SEJZ trile acid . In additestines . Helicobthe body . We showorldwide .SEJZ could relieve the hyperlipidemia caused by consumption of a HFD through upregulation of metabolic pathways related to restoration of the intestinal barrier. Several signaling pathways affected by SEJZ in the treatment of hyperlipidemia were identified by network pharmacology and 16S rRNA gene sequencing. Our data provide new insights on the lipid-lowering effect of SEJZ.


Objective: To investigate the effect of Zhilong Huoxue Tongyu capsule (ZLH) in the treatment of cerebral ischemia–reperfusion injury and determine the underlying molecular network mechanism.Methods: The treatment effect of Zhilong Huoxue Tongyu capsule (ZLH) was evaluated for cerebral ischemia–reperfusion injury in middle cerebral artery occlusion (MACO) rat, and the underlying molecular network mechanism was explored by using molecular network analysis based on network pharmacology, bioinformatics including protein–protein interaction (PPI) network, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as molecular docking.Results: The neurological function of rats in the ZLH group was significantly improved compared to those in the NS group (p = 0.000), confirming the positive effect of ZLH for the treatment of brain ischemia. There were 126 intersecting genes screened in ischemia–reperfusion cerebrum that are associated with several important biological processes, such as lipopolysaccharide, and the most important cell component, such as raft, as well as the most important molecular function pointed as cytokine receptor binding. The most important KEGG signaling pathway was the AGE-RAGE signaling pathway in diabetic complications. Moreover, according to the STRING interaction in the PPI network, 10 hub genes including MAPK14, FOS, MAPK1, JUN, MYC, RELA, ESR1, STAT1, AKT1, and IL6 were selected and exhibited in Cytoscape and molecular docking. Lastly, the relation between PPI, GO, and KEGG was analyzed. These findings indicated that multiple hub network genes have been involved in behavior improvement in cerebral ischemia–reperfusion rats subjected to ZLH treatment.Conclusion: Zhilong Huoxue Tongyu capsule improves cerebral ischemia–reperfusion and is associated with multiple network gene expressions. Stroke, one of the three most common causes of death and disability, is increasing with the high-pressure life. About 70% of strokes are derived from ischemic, and the rest are intracerebral or subarachnoid hemorrhages . After sAstragalus, Cassia twig, leech, Panax notoginseng, and chuanxiong, is effective in dispelling wind and dampness and relieving pain for vascular headache and migraine. Among of them, Astragalus can hold the surface for qi and fold sweat; cassia twig can generate muscle, warm the meridians, and pass through Yang Qi; leech can precipitate and break blood clots (Zhilong Huoxue Tongyu Capsule (ZLH) (Sichuan Medicine Z20070528) is an available traditional Chinese medicine (TCM) that could be utilized for the treatment of cerebral stroke . As a cood clots . HoweverNetwork pharmacology is based on core target analysis, network interaction relationship analysis, and description of key biological processes and signaling pathways to explain the disease–target-drug co-association module, which could exhibit the network regulatory relationship between drugs and diseases, so as to uncover the mechanism of pharmaceutical agents , especiaIn this study, we investigated the protective effect and network mechanism of ZLH against cerebral ischemia–reperfusion, by gene network analysis, molecular docking, and animal experiment.n = 12), NS group (n = 10), and ZLH group (n = 11). After 8 h of fasting, the rats were deeply anesthetized with 3% sodium pentobarbital and performed the right common carotid artery occlusion in rats, by inserting a wire into the internal carotid artery. Then, the wound was sutured till the embolization was removed after 1 h  K2020-0004); the animal ethics code is KMMU20220854, and the rats were randomly grouped into the sham group , and 2 ml/day was intragastrically administered to each rat in the ZLH group. The rats in the NS group were given 2 ml/day of saline, and the sham group was fed and watered as usual. All rats in each group were observed until sacrificed at day 5.All rats were assessed 24 h after awakening from anesthesia by using Zea-Longa scores. The scoring criteria were as follows: 1) no signs of nerve damage, 0 points; 2) inability to fully extend the contralateral front paw, 1 point; 3) rotate to the opposite side while crawling, 2 points; 4) falling to the opposite side while standing or crawling, 3 points; 5) inability to walk on their own and loss of consciousness, 4 points. The higher the score indicates the more severe the behavioral disorder of the animal.Astragalus, Cassia twig, leech, Panax notoginseng, and chuanxiong in ZLH were extracted by TCMSP (https://old.tcmsp-e.com/tcmsp.php), according to the condition of oral bioavailability (OB) that was set at > 20 and drug similarity (DL) was at >0.15. After retrieving the active ingredient of the drug, the name of the target protein corresponding to the active ingredient is checked in “related targets” according to the molarity of the active ingredient, and the name of the gene corresponding to the target protein is correspondingly searched in UniProt.The components and related target proteins of https://www.genecards.org/), we downloaded a list of genes related to cerebral ischemia–reperfusion.After entering the keyword “cerebral ischemia-reperfusion” in GeneCards (https://bioinfogp.cnb.csic.es/tools/venny/index.html). We input the gene name using “BI” in List1 and “ZL” in List2 and then constructed the Venny intersection map.The genes involved in cerebral ischemia–reperfusion were screened, and the Venny intersection map with ZLH was constructed  was used to perform the GO enrichment and KEGG pathway analysis of the intersection obtained in Venny. In the Java environment, after the computer was first installed with the activePerl.exe file, org.Hs.eg.db, DOSE, clusterProfiler and pathView Enrichplot, ” we then used the scripts of SymolID, GO, and KEGG file packages to analyze the gene network, which includes the biological processes (BP), cellular components (CC), and molecular functions (MF), and the KEGG signaling pathway that can be obtained.https://cn.string-db.org/) was performed using the intersection derived from the analysis in Venny 2.1.0. The results were then obtained by selecting multiple proteins and entering the intersecting genes in the “list of name” and selecting “Homo sapiens” for analysis.PPI network analysis  type 1, drug mole number and corresponding genes; 2) type 2, disease name and disease gene; 3) type 3, KEGG signaling pathways and corresponding genes; 4) type 4, drug name and mole number; 5) type 5, KEGG signaling pathways. The disease–drug active component–key target–KEGG pathway diagram was constructed rationally by changing its shape, color, and position.A total of 199 genes of components and related target proteins for ZLH were examined , and 1,4A total of 199 genes for ZLH and 1,493 genes for cerebral ischemia–reperfusion were crossed in Venny 2.1.0, and 126 intersecting genes were obtained ; Table 3raft, which plays a regulatory role in the area around cerebral infarction. The most important molecular function is cytokine receptor binding, which activates intracellular enzymes to regulate gene expression , cellular component (CC), and molecular function (MF) were analyzed. The most important biological process was response to lipopolysaccharide; the main component of lipopolysaccharide was the outer membrane of Gram-negative bacteria, leading to the immune reaction. It plays an early warning role in inflammatory response after cerebral ischemia–reperfusion. The most important cell group is pression .The results showed that the top 10 KEGG signaling pathways were as follows: AGE-RAGE signaling pathway in diabetic complications, lipid and atherosclerosis, fluid shear stress and atherosclerosis, IL-17 signaling pathway, TNF signaling pathway, Kaposi sarcoma-associated herpesvirus infection, hepatitis B, HIF-1 signaling pathway, human cytomegalovirus infection, and Chagas disease .In total, 126 intersection genes were obtained from Venny 2.1.0, shown in The top 10 genes were sorted by the degree value, and the circle from the largest to smallest represented the decreasing degree value. As shown, the top 10 hub genes were MAPK14, FOS, MAPK1, JUN, MYC, RELA, ESR1, STAT1, AKT1, and IL6 .Based on the 10 target proteins screened in Cytoscape, molecular docking was carried out with ZLH. The crystal structures of the target proteins were downloaded from the PDB database, formatted by Open Babel, and molecular docking of the target proteins to the ZLH was performed in AutoDock Vina. Finally, PyMOL software was used to process and adjust the color of the large protein, process the ligands and residues, calculate the length of hydrogen bonds, and export the ligands and the overall picture .The disease–drug active component–key target–KEGG signaling pathway diagram was constructed by importing the 44 active ingredients corresponding to ZLH, 126 common cross-targets of cerebral ischemia–reperfusion and ZLH, and the first 10 pathways of KEGG into Cytoscape software. As a result, we constructed the network interaction among all related targets, in which, the closely related pathways were linked in the figure to illustrate the regulatory role of ZLH on cerebral ischemia–reperfusion .To know the relation and link of hub genes with PPI, GO, and KEGG pathway, we compared the position of hub genes in the GO and KEGG pathway. We found that almost all hub gens were involved in BP and selected signal pathways, but only MAPK1 or Mapk14 were involved in CC. These pointed out that 10 hub genes selected from PPI are most importantly related to the findings of GO analysis and signal pathway. In addition, the molecular hub genes like MAPK14, FOS, MAPK1, and JUN may simultaneously locate in different signaling pathways involved in the AGE-RAGE signaling pathway in diabetic complications, lipid and atherosclerosis, fluid shear stress and atherosclerosis, IL-17 signaling pathway, TNF signaling pathway, Kaposi sarcoma-associated herpesvirus infection, hepatitis B, HIF-1 signaling pathway, human cytomegalovirus infection, and Chagas disease, which means that some co-expression genes scattered in different signaling pathways. In turn, the same signaling pathway like the IL-17 signaling pathway includes different genes including MAPK14, FOS, MAPK1, JUN, RELA, and AKT1, simultaneously .p = 0.000, Zea-Longa scores were performed on the sham group, NS group, and ZLH group at 1, 3, and 5 days after surgery. The results showed a significant increase in neurological dysfunction in the NS group compared to the sham group at 5 days after surgery, whereas it reversed in the ZLH-treated group, compared to the NS group (In the present study, we confirmed the effect of ZLH on neurological function repair in brain ischemic rats and determined the gene network in the cerebral ischemia–reperfusion model . We screAfter the brain ischemia model was successfully established and the high-dose treatment of ZLH was used, we found that the score of neurological impairment was higher in brain ischemia, and it is lower in the ZLH group at 5 days. This suggested that the neurological function of cerebral ischemia–reperfusion rats was remarkably improved after high-dose administration of ZLH. Previously, it has been reported that ZLH exhibited a positive effect for the treatment of brain damage , but theNetwork pharmacology can explain the relationship between drugs and gene network of diseases. Through network analysis, different genes regulating the disease and interacting genes on diseases could be found. Network pharmacology also analyzes the mechanism of action of TCM prescriptions on disease, therefore providing a new way to explain the molecular mechanism of TCM efficacy . DespiteThe genes of ZLH were intersected with the genes of cerebral ischemia–reperfusion, and 126 intersection genes were obtained. The GO enrichment and KEGG signaling pathway analysis of the intersection genes showed that these genes were located in lipopolysaccharide as the most important BP. The main component of lipopolysaccharide is the outer membrane of Gram-negative bacteria, which causes immune response and plays an early warning role in the inflammatory reaction after cerebral ischemia–reperfusion . The mosThe top 10 genes with the closest relationship were obtained, and we found that CCND1, ESR1, and JUN were connected with multiple genes in the top 10 pairs . These cAstragalus was docked with MAPK14, AKT1, FOS, IL6, MPK1 MYC, ESR1, JUN, RELA, and STAT1; Dahuang was directly docked with JUN, ESR1, and MYC; Cassia twig was docked with JUN, ESR1, and RELA. However, ESR1 and RELA had no direct interaction with Cassia twig. Therefore, through molecular docking, we provided the vital explanation for the drug directly targeting the molecule (As key nodes, the 10 hub genes were speculated as core genes in cerebral ischemia–reperfusion with ZLH treatment . Each ofmolecule .By constructing a disease–drug active component–key target–KEGG pathway map, it can find the network interactions between the related targets . In thisThe relation of hub genes with PPI, GO, and KEGG pathway was analyzed. Several important genes were linked with the different cellular locations, biological processes, and molecular functions, meanwhile sharing the different signal pathways . MAPK4, In summary, we confirmed the effect of ZLH in protecting the ischemic brain from injury, and by using network pharmacology, molecular docking, and bioinformatics analysis, we explained the net mechanism in cerebral ischemia–reperfusion treated with ZLH. The results revealed that the effect of ZLH in improving neurological function is associated with multiple gene expressions in cerebral ischemia–reperfusion rats. Despite determining the effect of ZLH in treating brain ischemia and explaining the associated network mechanism, we suggest that the follow-up experimental research is absolutely needed to be developed to validate the deep mechanism.


Peri-implants is a chronic disease leads to the bone resorption and loss of implants. Polygoni Cuspidati Rhizoma (PCRER), a traditional Chinese herbal has been used to treat diseases of bone metabolism. However, its mechanism of anti-bone absorption still remains unknown. We aimed to identify its molecular target and the mechanism involved in PCRER potential treatment theory to Peri-implants by network pharmacology. The active ingredients of PCRER and potential disease-related targets were retrieved from TCMSP, Swiss Target Prediction, SEA databases and then combined with the Peri-implants disease differential genes obtained in the GEO microarray database. The crossed genes were used to protein–protein interaction (PPI) construction and Gene Ontology (GO) and KEGG enrichment analysis. Using STRING database and Cytoscape plug-in to build protein interaction network and screen the hub genes and verified through molecular docking by AutoDock vina software. A total of 13 active compounds and 90 cross targets of PCRER were selected for analysis. The GO and KEGG enrichment analysis indicated that the anti-Peri-implants targets of PCRER mainly play a role in the response in IL-17 signaling, Calcium signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway among others. And CytoHubba screened ten hub genes . Finally, the molecular docking results indicated the good binding ability with active compounds and hub genes. PCRER’s core components are expected to be effective drugs to treat Peri-implants by anti-inflammation, promotes bone metabolism. Our study provides new thoughts into the development of natural medicine for the prevention and treatment of Peri-implants. Recent studies have shown that up to 56% of implant patients and even 43% of implant sites are affected by Peri-implants2.Peri-implant, which refers to inflammatory damage to the hard and soft tissues around implants, including peri-implant mucositis and peri-implants inflammation. Peri-implant mucositis is limited to the surrounding soft tissue, rather Peri-implants could penetrate into the implants and cause severe bone resorption, if left untreated, can cause the loss of the implant3. Subgingival plaque is the main pathogenic cause of this disease, and the pathogenic bacteria are mainly anaerobic bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitan, etc. Huge of systemic and local factors, including pathogenic bacteria, poor oral hygiene, smoking and alcohol consumption are associated with the development of Peri-implants4. There are similarities and differences in understanding and treatment of implants in traditional Chinese and Western medicine. In western medicine, both Peri-implants and periodontitis are infectious diseases caused by plaque microorganisms. Therefore, the treatment of Peri-implants mainly involves long-term application of a large number of antibacterial drugs to combat bacterial inflammatory destruction, degradation of collagen fibers and matrix, so as to eliminate periodontal pocket and restore the bone loss5. Minocycline hydrochloride, could inhibit collagen enzyme activity, has the excellent affinity with osseous tissue and has a wide antimicrobial spectrum strong sterilization activity, it also prompts the implant surrounding soft tissue regeneration because of its ideal permeability7. But the use of antibiotics often brings many adverse reactions, such as allergic reactions, gastrointestinal reactions and so on8.Peri-implants is mainly manifested as soft tissue inflammation, abscess and fistula formation10. For a long time, Polygoni Cuspidati Rhizoma Et Radix(PCRER) was considered as an invasive plant in Europe and North America, but its recent inclusion in the European Pharmacopoeia makes it possible to use it as a traditional plant medicine11. PCRER mainly contains anthraquinones, stilbenes and some fatty acid compounds, which has a variety of pharmacological effects, including anti-inflammatory, antiviral, anti-apoptotic, regulating blood lipids, anti-thrombosis, myocardial protection, anti-oxidation, anti-tumor and other pharmacological effects. As a traditional Chinese medicine, PCRER was often used in combination with different TCMs to treat liver injury, chronic pelvic inflammatory disease, acne, menstrual irregularities, burns, and arthritis etc.14. Some studies have reported that extracts of PCRER or its main compounds have antioxidant and antibacterial effects and it was used in Korea, China and Japan to treat osteomyelitis16. It has also been proved having antibacterial activity against Streptococcus mutans and was used by civilian medical organizations to maintain oral hygiene in South Korea18. Hadzik et al. obtained extracts of PCRER with the highest bacteriostatic and bactericidal activities against the caries-pathogens, especially to streptococcus. In addition, the cytotoxicity of PCRER’s extracts to S. mutans was low at antibacterial concentration, and appears to have stimulating effect on normal human fibroblasts, which may accelerate the healing of gingival wounds19. At present, there are amount of experiments on treating peri-implants with Traditional Chinese medicine or PCRER’s compounds such as Mangiferin20, Cranberry21, Quercetin22, Resveratrol23. However, the specific mechanism of treatment of peri-implants with PCRER is still unclear.Traditional Chinese Medicine (TCM) has been used in China for thousand years, which has a multi-target therapeutic effect on a variety of diseases, including complex bone metabolic diseases, such as osteoporosis and periodontitis24. The mode of "network target, multi-component" was taken as the core concept of network pharmacology of TCM25.Bio-information network construction and network topology analysis strategies based on high-throughput data analysis, virtual computing and network database retrieval can systematically clarify the molecular mechanism of TCM treatment of various diseases, and a huge of studies have been published27. Network pharmacology uses artificial intelligence to predict drug targets and binding patterns, identify biomarkers for diseases and syndromes, retarget drugs, and use algorithms and big data at its core to understand the occurrence and progression of disease and syndrome28. Therefore, in this study, we combined the web-based pharmacology approach with the Gene Expression Omnibus Database (GEO), the potential mechanism was explored through GO&KEGG pathway analysis, and the “hub genes” of PCRER treatment of Peri-implants were screened, to clarify the comprehensive mechanism of PCRER against peri-implants.Before the term "network pharmacology" was proposed, the study of TCM and biological network appeared for the first time in 2007 proposed by Shao Li who who laid a foundation for the establishment of new research strategies of biological network and TCMhttp://lsp.nwu.edu.cn/tcmsp.php) database. The TCMSP database provides information on Chinese herbal medicines from the Chinese Pharmacopoeia, as well as drug chemistry, drug similarity, drug target, disease targeted by each active compound, and other relevant information29. OB stands for the efficiency with which bioactive ingredients enter the systemic circulation, while DL represents the qualitative indicator that is applied to drug design to estimate similarities between ingredients and certified drug structures. We selected drug similarity characteristics and oral bioavailability as conditions, where (DL) ≥ 0:18, (OB) ≥ 30%30, and the active components of PCRER reported in literature were also included in the database.The ingredients of PCRER were obtained from TCMSP (http://www.Swisstargetprediction.ch/)31, SEA  was used for target information comparison and gene name standardization. After the targets in the above three databases were combined and deleted the duplicate values, putative targets of PCRER were obtained.Targets for major compounds in PCRER were identified and implemented by the following database TCMSP, Swiss Target Prediction (http://www.ncbi.nlm.nih.gov/geo) to construct our research database by analyzing differentially expressed microarrays. The search strategy (‘Peri-implants’ [All Fields]) AND (‘Homo sapiens’[Organism] AND ‘Expression profiling by array’[Filter]) was adopted. Expression profiling data from GSE178351, GSE57631 and GSE106090 were downloaded from the GEO database based on the microarray platform GPL23159, GPL15034(both from Affymetrix Human Gene Expression Array) and GPL21827(Agilent Human Gene Expression Array). Gene IDs were identified according to the platform annotation probe ID information. DEGs between patients with Peri-implants and healthy individuals were screened using the ‘limma’ package of R software (version 3.6.3) according to P < 0.05, and |log2 fold change (FC)|> 1. Then, the volcano plot and heatmaps of DEGs from three dataset were plotted by the ‘Pheatmap’ and ‘ggplot’ package in the R software. Finally, we combined the differential genes in the three data sets, deleted the duplicate values, and established the gene target database of peri-implantitis after standardization with Uniprot database.Few targets related to Peri-implants can be found in the current epidemic disease database, so we chose the GEO database (https://cytoscape.org/) to conduct network topology analysis on these data and construct P-C-T-P network.We obtained the target genes of the active components of PCRER and the therapeutic targets of Peri-implants from the above four databases and obtained overlapping genes, integrated network information on ingredients, genes and disease targets. Finally, we use Cytoscape software  are set to 0.05. The first 15 enrichment results were output to draw bubble graphs, bar graphs and circos graphs of GO-BP, GO-CC, GO-MF and KEGG regulatory networks. And KEGG pathway enrichment network map with crossover genes was generated by Cytoscape 3.7.2 software.'org.hs.eg. Db', 'stringi', 'ClusterProfiler' and 'ggplot2' of the R package was installed in software R 3.6.3 for enrichment analysis of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG). Go enrichment analysis was carried out with the function of "Enrichment go". KEGG enrichment analysis carried the enrich-KEGG function and the database was KEGG database (http://www.string-db.org/), the target-target interaction network, target interaction in protein–protein interaction (PPI) network diagram  and. tsv data were obtained34. Next, to further identify the core therapeutic targets, Cytoscape plug-in MCODE (Molecular Complex Detection) was used to identify significant modules (MCODE score ≥ 4) and another plug-in Cytohubba was used. MCC algorithm was used to study node degree (score ≥ 10) of key nodes in significant modules35, the hub genes contained in PPI network was screened.Enter overlapping targets of PCRER/Peri-implants into STRING database  and stored in PDB format. Candidate compounds of two-dimensional (2D) structure was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), save in SDF format. Ligands and receptors were prepared with Chem3D using AutoDock Tools (V. 1.5.6). Among them, the preparation of the receptor includes deleting the original ligand and water molecules from the crystal structure of the receptor, adding non-polar hydrogen, and calculating the partial charge of Gasteiger37. The process for handling ligands involves applying energy to minimize and distribute atomic charges and atoms. All prepared receptors and ligands are stored in PDBQT format. Then, Autodock Vina was used for molecular docking, and the docking center was set by the grid box function in the software38. The best docking position was the one with the minimum root mean square deviation (RMSD) predicted by X-ray crystal configuration, and the affinity between ligand and target protein was evaluated to indicate the binding strength. Affinity <  − 5.00 kcal/mol indicates good binding strength, and affinity <  − 7.00 kcal/mol indicates good binding strength. The docking conformation was visualized by Pymol 2.3.Molecular docking refers to process in which two or more molecules identify each other through geometric matching and energy matching, including electrostatic interaction, hydrogen bonding, van der Waals interaction and hydrophobic interaction. In the field of drug design, the purpose of molecular docking was to find the best binding position and binding conformation between small molecule and target enzyme proteinAll data used in this study came from public databases and does not include any studies related to animals or humans.A total of 87 active ingredients were obtained from TSMSP platform and 13 core active compounds were selected according to the screening criteria of ADME model, including 6,8-Dihydroxy-7-methoxyxanthone, Physovenine, Picralinal, Physcion- diglucoside, rhein, Torachrysone-8-O-beta-D-(6'-oxayl)-glucoside, beta-sitosterol, (+)-catechin, luteolin, quercetin, resveratrol, polydatin, emodin. The molecular ID, ingredients names and ADME-related parameters are listed in Table 2 FC|> 1 and P < 0.05. Joint analysis of gene chips in the GEO database  contained 11 samples from healthy individuals and 16 Peri-implants patients which identified 1398 differentially expressed genes related to Peri-implants  was screened including IL1B, IL6, CXCL8, IFNG, MMP9, PTPRC, PECAM1, CXCL2, SELL, MPO .  and the receptor protein biological macromolecules in combination with each other, and calculated its mode and affinity according to the physical and chemical parameters through geometric matching and energy between molecules, looking for the best combination of small molecules (the ligand) and biological macromolecules (receptors) of process.40. According to the results of molecular docking, the binding energy of most receptor-ligand is less than or equal to − 5 kal/mol. At the same time, through conformational analysis of molecular docking structure, all receptors can form good docking pockets, all ligand molecules are located in the corresponding docking pockets, forming hydrogen bonds between receptors and ligands, confirming the high accuracy of drug target prediction in this study from the perspective of molecular docking. The compound-target interactions with the free binding energy scores along with their binding mode were determined using PyMoL-2.3.  entered a new period, using science and technology combined with traditional Chinese medicine theory, the network medicine pharmacology aims to clarify the research method of traditional Chinese medicine effective component and targets in the system of the molecular level to better understand and predict the behavior of cells, tissues or organs of the body function of phenotypic effects which provides a new perspective method to analyze drug effects. The research mode of "one drug, one target" is transformed into the research concept of "multiple approaches and multiple targets"44. Yu Wei et al. found that quercetin increased the antioxidant capacity of PDLCs by activating NRF2 signaling pathway, alleviated oxidative stress damage, and alleviated alveolar bone loss in periodontitis45. Luteolin, flavonoid plant, has potent anti-inflammatory effects both in vitro and in vivo that can effectively inhibit the production of TNF-α, IL-6 and NO in LPS induced macrophage-like cell lines, and luteolin's inhibition of inflammatory cytokines and/or ROS production may lead to the inhibition of osteoclast differentiation46. Kim found that luteolin also reduced the absorption activity of mature osteoclasts. In addition, it prevented the loss of bone mass, especially trabecular bone that occur after ovaries removal by inhibiting bone turnover47. In the experiment of luteolin, Hatice found that luteolin significantly reduced alveolar bone loss by decreasing MMP-8 and RANKL expression, increasing osteoblast activity and upregulation of TIMP-1, BMP-2 and OPG expression48. Resveratrol can inhibit the expression of Toll-like receptor (TLR) and pro-inflammatory genes, activate Sirt1 and then inhibit the expression of inflammatory factors such as TNF-α, IL-1, IL-6, MMP-1, MMP3 and COX-2 induced by NF-KB, and play a double blocking role in NF-KB signaling pathway49. In addition, resveratrol regulates immunity by interfering with immune cell regulation, proinflammatory cytokine synthesis and gene expression and plays a beneficial role in the prevention of chronic diseases related to inflammation. Resveratrol has also been proved to inhibit osteoclast differentiation and induce bone formation potential. Ribeiro found resveratrol could improve bone repair around titanium implants in rats, reverse the negative effects of implants and reduce the expression of RANKL/OPG in peri-implant tissues during bone repair50. Hua Y's study found that resveratrol treatment could improve osseointegration of implants and promote bone formation by reducing bone loss damage caused by AGE’s deposition23. Therefore, resveratrol may be the key component of polygonum cuspidate in the treatment of peri-implantitis.Due to the lack of corresponding disease target data in Peri-implants, we combined GEO database to conduct network pharmacology analysis which was also the first article about Peri-implants combined with network drugs. We used TCMSP database to identify the active components of PCRER. A total of 13 core active components were identified. Among them, beta-sitosterol, Luteolin, Quercetin and Resveratrol can match more than 20 targets. The pathogenesis of Peri-implants was complex and believed to be caused by a series of interactions such as inflammation, oxidative stress and bacterial infection. The osteoprotective effect of Quercetin has been confirmed by a large number of in vitro and in vivo experiments. Studies have reported the activation of Quercetin on osteoblast formation, as well as stimulating matrix mineralization, calcium deposition, and the expression of ALP, COL1, RUNx-2 and other osteogenic genes51. There is evidence that IL-17 is involved in the pathogenesis of periodontal diseases, and the level of IL-17 in peri-implant sulcular fluid (PISF) increases during peri-implant inflammation52. Calcium (Ca2+) is essential for bone homeostasis. Ca2+ signaling regulates proliferation, differentiation and apoptosis of osteocytes. RANKL induces Ca2+ signaling in osteoclasts through calmodulin. Ca2+ could bind to CaM and stimulates Ca2+/ CAM-dependent kinase (CaMK) and calcineurin, leading to induction and activation of NFATc1 and (PGC1β). PGC1β regulates mitochondrial biogenesis and plays an important role in the terminal differentiation of osteoclasts54. As mentioned above, T cell signaling pathways are hypothesized to be key mediators of persistent infection-induced chronic inflammatory processes in periodontitis and periapical periodontitis which is also influenced by Ca2+ signaling pathways and Ca2+ channel regulation. With advances in the study of Ca2+ signaling pathways in T cell pathogenicity and homeostasis, oral barrier immune cells may be affected by CCB and may lead to inflammatory gingival enlargement55. As you can imagine, the susceptibility of periodontitis or Peri-implants are directly related to calcium signaling or dysfunction. Toll-like receptor (TLR) is a class of pattern recognition receptors that can recognize microbial components. LPS can interact with TLR2, a major member of the TLR family, to activate the downstream protein nuclear factor-κB (NF-κB), P38 mitogen-activated protein kinase (MAPK) and C-Jun N-terminal kinase (JNK) of TLR2 and regulate the production of LPS-induced pro-inflammatory cytokines such as IL656. TLR2 initiates intracellular signaling cascades through cytoplasmic intermediates including Myd88, ultimately leading to activation of NF-κB and MAPK, thereby enhance transcription of inflammatory cytokines. A recent study also confirmed that TLR2 signaling activation plays a key role in bone loss and increased inflammatory infiltration in peri-implant inflammation57. Therefore, inhibition of TLR2 signal activation may be an effective strategy for the treatment of Peri-implants. Currently, TNF-α was considered to mediate bone resorption mainly by promoting osteoclast differentiation and inhibiting osteoblast differentiation58. Darabi found that TNF-α content was positively correlated with periodontal depth (PD)59. The increase of PD suggested that the binding between implant and surrounding tissue was damaged, and the degree of inflammation around implant increased, indicating that TNF-α expression level was closely related to the severity of implant inflammation and could indirectly reflect the health status of surrounding tissue60. TNF-α promoted osteoclast synthesis, reduced bone matrix calcification and promoted bone resorption, so it was speculated that TNF-α may be involved in the reconstruction of implant bone tissue.According to the active ingredients of the drugs mentioned above, we used related database to screen the putative targets and obtained 930 targets of PCRER. Then, we integrated three gene microarray chips of GEO, finally obtaining 1399 disease target genes. Peri-implants is a chronic inflammatory disease associated with a variety of inflammation pathways and cell phenotypes. To explore the PCRER 's potential mechanism, we conducted GO and KEGG enrichment analysis to explore possible regulating network. The results showed that the mapped targets were enriched to 29 items in biological process, which were mainly related to the regulation of membrane function included raft, microdomain, region, organelle outer membrane and caveola among others. It also enriched to 51 items in the cell composition, 13 items in biological process, and 328 terms in molecular functions. In addition, we observed 20 KEGG pathways related to Peri-implants and constructed a “Targets-Pathways” network, which involved IL-17 signaling pathway, Calcium signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway. IL-17 signaling pathway plays an important role in maintaining the balance between Th17 cells and Treg cells, promoting the differentiation of Th17 cells and the secretion of IL-17, triggering the immune response of the body, thus activating osteoclasts and secreting MMP to cause the degradation of type II collagen61 and primarily secreted by neutrophils and macrophages, regulates inflammation in tissues and diseases62. MMP9’s expression is significantly increased in chronic periapical infection area and overexpression of MMP-9 attenuates osteoclast formation and inhibits secretion of pro-inflammatory cytokines63. MMP9 initiates osteoclasts by removing collagen from demineralized bone, which is essential64. Shimada found that titanium stimulated the expression of MMP-9 mRNA in osteoblasts cultured in vitro, and zirconia inhibited the expression of MMP-9 mRNA65. Meanwhile, Degidi et al. also confirmed that the level of MMP9 around the healing cap was increased66. However, more research is needed on the regulatory mechanism of MMP-9.In this study, STRING database was used to calculate the degree of PCRER anti-Peri-implants targets (90 genes), MCODE and CytoHubba plug-in in Cytoscape software was used for screening the top 10 hub genes . MMP-9 ,an inflammatory marker of peri-implant inflammation, mainly present in oral fluid and inflamed gingival tissue in this specimen67, regulates immune response, stimulates the mononuclear macrophage and produce IL-6, which can increase the activity of osteoclast. At the same time, IL-1 can also inhibit the synthesis of osteoblast calcium cord and destroy normal bone metabolism, so it is called osteoclast activating factor68. In addition, IL-1β also interact with other inflammatory mediators, promots the expression of cytokines such as IL-6, TNF-α and intercellular adhesion molecules, and cascades the inflammatory effect to amplify the inflammatory response, resulting in aggravated tissue damage69. Studies have shown that the expression level of IL-1β at peri-implantitis sites was significantly higher than healthy implant sites. IL-6 is produced by mononuclear macrophages under the induction of IL-1. The level of IL-6 is related to the active stage of the disease, which is consistent with the detection results of gingival crevicular fluid in patients with periodontal disease. IL-6 stimulates the growth and differentiation of osteoclast precursors. Promoting alveolar bone absorption which is thought to amplify the biological effects of IL-170. Sakai et al. found that IL-1β concentration was correlated with bone tissue absorption around implants, which could be used as a sensitive indicator to detect bone absorption at peri-implant inflammatory edge70. These results suggest that the pro-inflammatory cytokine IL-1β was involved in the occurrence and development of peri-implant inflammation, which could be used to distinguish the peri-implant health from the inflammatory state and a standard tool for the evaluation of peri-implant tissue health and treatment of Peri-implants.Peri-implants is mainly caused by bacterial infection of the implant-confined tissues and the destruction of the soft tissue closure of the cuff of the implant. As a result, the inflammation of the body tissues is the result of the interaction between the pathogenic agent and the immune system of the host. Cytokines are involved in the inflammation and immune response of the body. IL-1β is closely associated with implant inflammation around the important inflammatory factor which inhibits the expression of alkaline phosphatase, coordinates to polyclonal activators to stimulate proliferation of T cells and B cells growth and differentiation71. However, IFNG is more complex in vivo, in cell culture, IFNG can inhibit the function of osteoblasts and effectively inhibit the formation of osteoclasts. In bone explants, it inhibits osteoclast differentiation72. IFNG together with interleukin 1 (IL-1) stimulates high levels of NO production in bone, and these early studies support the role of IFNG in bone formation73. In addition, IFNG induces the expression of Best5 gene expressed during bone formation in rats74. Gustavo found that the addition of low doses of IFNG in ovariectomized mice reversed the phenotype of previous osteoporosis, which proved that the beneficial effect of low doses of IFNG on bone formation, and IFNG's effect on bone resorption was dominant75. Although the role of IFNG in osteoclast differentiation and activity has been extensively studied, little is known about its role in periodontitis and peri-implants. However, it remains to be determined whether IFNG indirectly regulates osteoclast activity mainly through RANKL expression in osteoblasts. Chemokines are proteins  with low molecular weight  that attract white blood cells to migrate to the site of infection and play an important role in inflammatory response. CxCL8/IL-8 could induce neutrophils to produce chondrodegrading enzymes, resulting in joint tissue damage. Elevated IL-8 levels often associated with locally infiltrated monocytes and neutrophils76. CXCL2 as a subtype of chemokine, has been widely expressed in RANKL-induced osteoclast precursor cells and plays an important role in the formation, migration and differentiation of osteoclasts. The study of Ha et al. showed that RANKL could promote CXCL2 expression of osteoclasts in vitro, so as to enhance their proliferation and differentiation ability, which might have a positive effect on bone resorption. Thus, CXCL2 is indeed closely associated with bone remodeling77. Previous studies by Gamonal et al. found that the level of IL-8 in the gingival of patients with periodontitis was higher than that of healthy subjects, but decreased significantly after periodontal treatment, suggested that IL-8 was involved in the inflammatory process of periodontitis78. Pietruski et al. observed that IL-8 level in gingival crevicular fluid increased significantly 24 h after implant implantation, indicated that there was local inflammation on the day after surgery, and surgical trauma inevitably led to regeneration and repair of local body tissues and IL-8 level decreased 4 months after surgery79.Among cytokines regulating bone metabolism, interferon G (IFNG) has been shown to play an important role in the regulation of osteoporosis. In vitro, IFNG inhibit osteoclast formation by stimulating bone marrow monocyte precursors with receptor-activated nuclear factor-κB ligand (RANKL)80. Liskmann et al. concluded that elevated MPO levels were associated with detecting bleeding and pocket depth around diseased and healthy implants in the same individual. Specifically, it was found that only 9.4% of healthy implants had MPO levels above 25 ng/mg, while 96.9% of diseased implants had MPO levels above 25 ng/mg81, Montero found that the level of Myeloperoxidase was in direct proportion to the risk of peri-implants in beagles (odds ratio: 1.1)82. Quantitative measurement of MPO can be used as an adjunct to traditional clinical parameters83. SELL (Lymphocyte homing receptor) belongs to the lymphocyte homing receptor (LHR) family, which is one of the family members of cell adhesion molecules. SELL is involved in cell extension and movement, cell signal transduction and inflammatory response, immune response, thrombosis, wound healing and other physiological and pathological processes84. Seidelin et al.85 showed that the serum SL-selectin level of patients with severe infectious diseases was significantly higher than that of normal people. Asami et al. also screened SELL as its hub gene after bioinformatics analysis of the GEO database of periodontitis86, but there is still a lack of basic research on this gene in periodontitis and peri-implantitis.Myeloperoxidase (MPO), the most abundant protein in neutrophils which is a powerful oxidant and is involved in defense mechanisms against infectious agents; However, when it is uncontrollable or over-activated, it can act on host cells and inactivate humoral factors87 and atherosclerosis88. A previous report by Cheng et al.89 suggested that PECAM1 was critical in the inflammatory response and apoptosis of hepatitis liver. Meanwhile, Liu et al. found that PECAM1 could interact with CXCR4 in experimental pulpitis in mice, which lead to inflammatory response and increased apoptosis of human pulp fibroblasts by activating the NF-KB signaling pathway90. Wu found that PECAM-1 was found to be a negative regulator of monocyte derived osteoclast formation in PECAM-1 knockout mice91. Therefore, we speculate that PECAM-1’s deficiency may have a direct and significant effect on osteoclast formation and indirectly affect osteoblast function. PTPRC encodes protein Tyrosine Phosphatase (PTP), a signaling molecule known as CD45 that regulates a variety of cellular processes and plays a key role in the immune system92. In addition, PTPRC can negatively regulate cytokine receptor signaling by inhibiting JAK signaling93. PECAM-1 and PTPRC have not been reported in relation to periodontitis or peri-implantitis. It is worth noting that molecular docking simulation is an important method for structural molecular biology and computer-aided drug design, and the results of molecular docking also show that PCRER’s components have good binding performance with the Hub genes.Platelet endothelial cell adhesion molecule-1 (PECAM1), also known as CD31, is a type I transmembrane adhesion protein, it has been shown that inhibition of PECAM1 can reduce inflammatory responses in various human diseases, such as arthritis42. In terms of data collection, the current prediction platform lacks information on active ingredient activation or inhibition targets and signaling pathways, which is the deficiency of this paper. If we can constantly improve the above shortcomings, we will be able to provide more reliable theoretical basis for the study of traditional Chinese medicine.In this study, network pharmacology and molecular docking methods were used to predict the mechanism of PCRER in the treatment of peri-implantitis. At the same time, the direct intersection targets were analyzed by GO annotation and KEGG enrichment, and the Hub targets were screened from the direct targets by PPI network and Cytoscape intersection analysis, revealing the possible physiological and pathological process of PCRER intervention in peri-implantitis. At the same time, because the traditional Chinese medicine may play a role in treating diseases through the synergistic effect of multiple components, how to predict and evaluate the synergistic effect of multiple compounds become a challenge we are facing at present. As for the limitations of this paper and aspects that need further study, first of all, we can use liquid chromatography-mass spectrometry to verify and supplement the effective compounds of PCRER, animal and cell experiments and clinical samples were also needed to detect the corresponding gene and pathway levels and conduct corresponding pharmacokinetic and metabonomics studiesIn summary, the potential molecular mechanism and target gene of PCRER treat Peri-implants were elucidated by network pharmacology method that beta-sitosterol, luteolin, quercetin, resveratrol could be the vital ingredients for PCRER. PCRER’s core components are expected to be effective drugs to treat Peri-implants by anti-inflammation, promotes bone metabolism. However, whether it is suitable for long-term maintenance treatment of Peri-implants still needs to be determined according to the future basic experiments. In addition, clinical trials are needed to elucidate the mechanism of action.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Legends.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Legends.


Objective: Colon cancer is a malignant neoplastic disease that seriously endangers the health of patients. Pulsatilla decoction (PD) has some therapeutic effects on colon cancer. This study is based on the analytical methods of network pharmacology and molecular docking to study the mechanism of PD in the treatment of colon cancer.Methods: Based on the Traditional Chinese Medicine Systems Pharmacology Database, the main targets and active ingredients in PD were filtered, and then, the colon cancer-related targets were screened using Genecards, OMIM, PharmGKB, and Drugbank databases. Then, the screened drug and disease targets were Venn analyzed to obtain the intersection targets. Cytoscape software was used to construct the “Components–Targets–Pathway” map, and the String database was used to analyze the protein interaction network of the intersecting targets and screen the core targets, and then, the core targets were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Molecular docking was implemented using AutoDockTools to predict the binding capacity for the core targets and the active components in PD.Results: Sixty-five ingredients containing 188 nonrepetitive targets were screened and 180 potential targets of PD anticolon cancer were identified, including 10 core targets, namely, MAPK1, JUN, AKT1, TP53, TNF, RELA, MAPK14, CXCL8, ESR1, and FOS. The results of GO analysis showed that PD anticolon cancer may be related to cell proliferation, apoptosis, energy metabolism, immune regulation, signal transduction, and other biological processes. The results of KEGG analysis indicated that the PI3K-Akt signaling pathway, MAPK signaling pathway, proteoglycans in cancer, IL-17 signaling pathway, cellular senescence, and TNF signaling pathway were mainly involved in the regulation of tumor cells. We further selected core targets with high degree values as receptor proteins for molecular docking with the main active ingredients of the drug, including MAPK1, JUN, and AKT1. The docking results showed good affinity, especially quercetin.Conclusion: This study preliminarily verified that PD may exert its effect on the treatment of colon cancer through multi-ingredients, multitargets, and multipathways. This will deepen our understanding of the potential mechanisms of PD anticolon cancer and establish a foundation for further basic experimental research. Colon cancer is a common malignant tumor of the gastrointestinal tract that originates in the mucosal epithelium of the colon. In recent years, the incidence rate of colon cancer has been increasing year by year, and the incidence rate in young people has increased rapidly . AccordiLocked Anus Hemorrhoids.” Before treating colon cancer, TCM physicians will comprehensively understand the patient’s physical condition according to the patient’s pulse, tongue, symptoms, signs, and relevant modern laboratory examination. The treatment scheme varies for patients with different physical statuses, but the overall treatment is based on clearing heat and dampness, cooling the blood, and detoxification. Pulsatilla decoction (PD) comes from the classical Chinese medicine book “Shang Han Treatise.” It is a herbal formula composed of four herbs, namely, Phellodendron, Pulsatilla, Coptis Chinensis, and Cortex Fraxini, which have the effect of clearing heat and dampness, cooling the blood, and detoxification. Contemporary pharmacological research has proven that PD has immune function regulation, bactericidal, anti-inflammatory, antitumor, and other effects , colon cancer is called “ effects . It is c effects . In recevia the P53 signaling pathway  enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and molecular docking techniques to explore the potential mechanisms of PD anticolon cancer. The flow chart for this study is shown in https://lsp.nwu.edu.cn/tcmsp.php) (https://www.uniprot.org/)  (msp.php)  to obtaiot.org/)  was usedhttps://www.genecards.org/) (https://www.omim.org/) (https://www.pharmgkb.org/) (https://go.drugbank.com/) (Colon cancer was searched in the GeneCards (ds.org/) , OMIM (him.org/) , PharmGkkb.org/) , and Drunk.com/)  databaseThe compound targets of drugs and diseases are the potential targets of drugs acting on diseases . In thisThe Cytoscape software (version 3.8.2) was used to correlate the drugs, active components, overlapping targets, pathways, and diseases and construct the interactive network diagram between the components and the targets, in which the edge represents the interrelationship between nodes and the node stands for the active ingredients and targets; then, the main active ingredients of drugs were analyzed.https://string-db.org/) (The overlapping targets of colon cancer and PD were imported to the String database (db.org/) , and thep < 0.05 to obtain the enrichment analysis results of GO and KEGG; the results were displayed in barplot or bubble.To investigate the biological process and signaling pathway transduction process of PD anticolon cancer, the R language and colorspace, stringi, ggplot2, ClusterProfiler, enrichplot, DOSE, and pathview packages in Bioconductor software were used, and the filtering condition was set to http://www.rcsb.org/pdb/)  (rg/pdb/) , and theA total of 65 active ingredients of four drugs in PD were identified by searching the TCMSP under the conditions of OB ≥ 30% and DL ≥ 18%, including 37 Phellodendron, 11 Pulsatilla, 14 Coptis Chinensis, and 3 Cortex Fraxini. Afterward, 53 active ingredients of PD were obtained after removing the repeated ingredients , cellular component (CC), and molecular function (MF) for visual analysis . In the p-value from large to small, so the larger the red node indicates the higher the significance of the signaling pathway and the more important the role, which mainly includes lipid and atherosclerosis, PI3K-Akt signaling pathway, fluid shear stress and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, hepatitis B, cellular senescence, MAPK signaling pathway, pathways of neurodegeneration-multiple diseases, human cytomegalovirus infection, Epstein–Barr virus infection, proteoglycans in cancer, human T-cell leukemia virus 1 infection, human papillomavirus infection, prostate cancer, IL-17 signaling pathway, hepatocellular carcinoma, TNF signaling pathway, influenza A, Kaposi sarcoma-associated herpesvirus infection, and hepatitis C. We found that many enrichment pathways are associated with additional pathological effects, possibly caused by homologous molecular targets in various diseases. Among them, the PI3K-Akt signaling pathway, MAPK signaling pathway, IL-17 signaling pathway, TNF signaling pathway, cellular senescence, and proteoglycans in cancer were most closely associated with the mechanism of PD anticolon cancer (In the KEGG pathway analysis , the sizn cancer .The “Components–Targets–Pathway” network diagram revealed that the quercetin node is the main active component of PD. Topological analysis shows that MAPK1, JUN, and AKT1 were the main targets. Therefore, the molecular docking of MAPK1, JUN, and AKT1 with quercetin was carried out by using Auto Dock Vina software. The results are shown in Colon cancer is a common malignant neoplastic disease that often occurs at the intersection between the sigmoid colon and colon. Its pathogenesis is complex. Smoking, alcohol consumption, red meat consumption, and family history of colon cancer are all considered to be the risk factors for this cancer . With thWith the advancement of modern science and technology, medical research is becoming more and more important with the help of computer technology and other research tools. As new research technology means integrating system biology, bioinformatics, and network science, network pharmacology can analyze the molecular association between drugs and therapeutic objects and reveal the systemic pharmacological mechanism of drugs from the overall perspective of systemic level and biological network. Its characteristics of multichannel and multitarget are very suitable for research with this kind of complex component of TCM . TherefoAccording to the “Components–Targets–Pathway” network, quercetin, isorhamnetin, and beta-sitosterol may be the most important potential components of PD anticolon cancer. It has been reported that quercetin can bind to kinases such as PI3K-Akt/PKB, SEK1, and MAPK to induce tumor cell cycle arrest and inhibit growth and metastasis, antioxidant replication, and angiogenesis by participating in the induction and expression of phosphorylation of key intracellular targets . Cosan eThe PPI network analysis of the duplicate targets of PD and colon cancer revealed that the core targets were MAPK1, JUN, AKT1, TP53, TNF, RELA, MAPK14, CXCL8, ESR1, and FOS. There are abundant interactions with other targets, indicating a higher degree of hub, to complete specific molecular functions. It was demonstrated that MARK1 and MAPK14 can affect the multiplication and apoptosis in cancer cells . Yong Y The results of GO enrichment analysis showed that the anticolon cancer effect of PD may be related to a variety of biological processes, mainly including response to molecule of bacterial origin, response to oxidative stress, response to reactive oxygen species, response to lipopolysaccharide, cellular response to chemical stress, G protein-coupled amine receptor activity, membrane raft, drug binding, steroid hormone receptor activity, ubiquitin-like protein ligase binding. Moreover, the results reflect the involvement of multiple changes in cell proliferation, apoptosis, energy metabolism, signal transduction, and immune regulation in the process of PD against colon cancer.The results of KEGG enrichment analysis indicated that numerous different illnesses were also enriched besides colorectal cancer, probably because of the existence of the same molecular targets in different disease pathologies processes. We selected signaling pathways closely connected with colorectal cancer for the analysis. We found that “PI3K-Akt signaling pathway,” “MAPK signaling pathway,” “Proteoglycans in cancer,” “IL-17 signaling pathway,” “Cellular senescence,” and “TNF signaling pathway” may be the potentially critical mechanism of PD anticolon cancer. The PI3K-Akt signaling pathway is an important signal transduction pathway for colon tumors. PI3K activation produces phosphatidylinositol triphosphate, which then leads to the activation of Akt through a series of signaling cascades. Akt activation can inhibit or activate a series of downstream substrates through phosphorylation, further regulating cell differentiation, proliferation, migration, and apoptosis . ActivatOur study utilized network pharmacology theory and relevant software to systematically elaborate the potential mechanism of PD anticolon cancer. A variety of components of PD could exert an effective role in the treatment of colon cancer through multiple pathways and multiple targets. The accuracy of the outcomes was further verified using subsequent molecular docking. The results showed that PD could regulate the proliferation, metastasis, differentiation, senescence, apoptosis, and other biological processes of tumor cells by regulating the expression of related genes in colon cancer cells, reflecting the anticolon cancer effect of PD. However, the research results still need to be further corroborated using animal experiments and other relevant experiments to ensure the reliability of the study results. In summary, our research provides a new basis for further exploration and subsequent experimental verification of PD in the treatment of colon cancer.


Experimental and theoretical studies have shown that ephaptic coupling leads to the synchronisation and slowing down of spikes propagating along the axons within peripheral nerve bundles. However, the main focus thus far has been on a small number of identical axons, whereas realistic peripheral nerve bundles contain numerous axons with different diameters. Here, we present a computationally efficient spike propagation model, which captures the essential features of propagating spikes and their ephaptic interaction, and facilitates the theoretical investigation of spike volleys in large, heterogeneous fibre bundles. We first lay out the theoretical basis to describe how the spike in an active axon changes the membrane potential of a passive axon. These insights are then incorporated into the spike propagation model, which is calibrated with a biophysically realistic model based on Hodgkin–Huxley dynamics. The fully calibrated model is then applied to fibre bundles with a large number of axons and different types of axon diameter distributions. One key insight of this study is that the heterogeneity of the axonal diameters has a dispersive effect, and that a higher level of heterogeneity requires stronger ephaptic coupling to achieve full synchronisation between spikes. The SPM is inspired by phase-coupled oscillators in the sense that each spike is endowed with an intrinsic propagation velocity based on the morphology of the associated axon. Furthermore, each spike can be ascribed a coupling function that characterises the spatiotemporal profile of ephaptic coupling with spikes in nearby axons. The collective ephaptic coupling generated by all spikes in a fibre bundle then perturbs the intrinsic velocity of a single spike, which decelerates if the axonal membrane is hyperpolarised, and vice versa. In the resulting numerical scheme, the position of each spike is propagated based on its perturbed velocity. In turn, the positions and velocities of the spikes determine their mutual ephaptic coupling effects.We begin our investigation by considering an exemplary bundle containing two axons, for which we develop the theoretical basis that can be extended to larger fibre bundles. First, we compute the effect that the spike in an active axon has on a nearby passive axon, which we do analytically under the assumption that the axons are homogeneous and that the perturbation of the passive axon can be captured by the linear terms of the cable equation. As most axons in peripheral nerves are myelinated, the assumption of homogeneity implies that the specific location of nodes of Ranvier is irrelevant. We calibrate the proposed SPM using a biophysically realistic model based on Hodgkin–Huxley dynamics.The calibrated SPM is then used to study large axon bundles systematically, especially the interplay between axon diameter distribution and the strength of ephaptic coupling, with the latter having a synchronising effect, and the former a dispersive effect. The coupling strength is determined by the fibre density (i.e. relative volume occupied by nerve fibres), and the resistivity of the extracellular medium. The aim here is to identify conditions under which synchronisation can occur given different axon diameter distributions and structural parameters, especially fibre density.In this section, we lay the theoretical basis of the SPM by considering fibre bundles with a small number of axons. First, we derive an analytical expression for the extracellular potential generated by a spike in an active axon. This is then used to compute the perturbation of the membrane potential of a passive axon (an axon that does not carry a spike) generated by a spike in a nearby active axon. The perturbation can be regarded as the ephaptic coupling between two nerve fibres. The resulting mathematical framework is then extended to the interaction between multiple axons. To reduce the computational load, we introduce a piecewise quadratic approximation of the spatial spike profile, which allows us to compute the ephaptic coupling function analytically. Lastly, we introduce the SPM and calibrate it by fitting free parameters to a biophysically realistic model based on the cable equation and Hodgkin–Huxley dynamics.V is defined as the difference between intracellular and extracellular potential, Axons may be regarded as core conductors Rall , wherebyN being the total number of axons. It is easy to see from Eq. .V. Here, we are interested in recovering a relationship between the extracellular potential V. Combining Eq. . The perturbation of the extracellular medium by a spike in the Typically, spikes are measured as the spatiotemporal profile of the depolarisation of the membrane potential ning Eq.  and Eq. mentclass2pt{minimA spike can be regarded as a travelling wave along the axon. If the axon is sufficiently homogeneous, then this wave appears stationary in the co-moving frame. For simplicity, we assume here that the axons under consideration are either homogeneous, which would be the case for unmyelinated axons, or homogenised in the case of periodically myelinated axons. The term homogenised refers to the technique proposed by Basser , which yc is the propagation velocity of the spike, which results inL, or nodes of Ranvier with length l. The node and myelin-specific parameters were chosen to be d being the axon diameter. These parameters are the same as in Schmidt and Knösche :The core concept of the SPM is that a spike can be represented by its position on the axon, and its velocity. We remark here that the number of spikes in the SPM is preserved, i.e. that spikes are neither created (‘ectopic spikes’) nor annihilated by the ephaptic coupling. The velocity is considered constant along the axon in the unperturbed case. Perturbations of the membrane potential caused by spikes in other, contiguous axons, however, have an effect the propagation velocity, which is modelled in linearised form by:In total, the SPM has three free parameters, which are the shape parameter We first fit the shape parameter mentclass2pt{minimase Fig. b.To identify realistic values for To identify the best-fitting set of parameters, we define the following cost function that is to be minimised:In Fig. To further illustrate the match between the SPM and the biophysical model, we plot spike trajectories generated with the SPM Fig. d for seldel Fig. e.nth axon, and we insert Eq. . The novelty here lies in the fact that the SPM reduces the computational effort as compared to the biophysical representation of the axon bundle, which allows us to model the interaction within spike volleys in large bundles. The numerical efficiency allows us to perform parameter screenings to describe the behaviour of the SPM in detail. Specifically, we vary the relative amount of extracellular space, which scales inversely with the amount of ephaptic coupling, and we vary the width of the distribution of axon diameters. The fibre bundles are 100 mm long and we record the axonal delays, which are measured as the time difference between spike initiation and spike termination. We compare two types of distributions: a uniform distribution, and a shifted alpha distribution.The uniform distribution is set up as follows:In Fig. N.To test whether these results depend on the type of diameter distribution, we perform the same analysis for a shifted alpha distribution:Once more, we find a parameter regime at small values of ise Fig. a, b. At We have identified parameters at which the spike volleys undergo complete synchronisation. While we have used the biophysical model to calibrate the SPM, that was done for a relatively small number of axons  necessary to produce full synchronisation within a spike volley also increases. This effect is independent of the specific type of fibre distribution chosen. At sufficiently high levels of heterogeneity, spike volleys no longer synchronise, even if the nerve bundle is completely filled with nerve fibre  that was previously devised to simulate spike volleys in white-matter fibre bundles  spikes, or extinguishing them. In the present study, we only consider spike volleys that engage all axons, therefore only the latter would be a possibility. A mechanism of spike generation and extinction could be incorporated into the SPM by defining conditions involving the perturbation of the membrane potential. More precisely, if the membrane of a passive axon is sufficiently depolarised, then a spike is generated there; conversely, if the membrane is sufficiently hyperpolarised, a spike is extinguished. A previous study, based on a spatially extended FitzHugh–Nagumo model, has demonstrated the possibility of emerging ectopic spikes in ephaptically coupled axons (Sheheitli and Jirsa Another limitation is that our study is based on a homogenised formulation of axonal morphology, which discounts the precise location and alignment of nodes of Ranvier. While a previous study has shown that the effect of ephaptic coupling was stronger when nodes are aligned, a staggered arrangement of nodes yielded the same qualitative results (Binczak et al. Our study considers relatively narrow axon diameter distributions to illustrate the effect of ephaptic coupling up to complete synchronisation. Peripheral nerves typically have a wide range of axon diameters, typically Finally, we would like to contemplate the possibility to confirm our results experimentally. While it is difficult to record spikes directly in peripheral nerves, it is easy to record the EP generated by nervous activity using surface electrodes. The EP of a spike volley can be computed using the line-source approximation (Holt and Koch