Perilla frutescens (L.) Britt (PF) has been reported to negatively affect adipocyte formation, inhibit body-fat formation, and lower body weight. However, its effect on adipocyte browning remains unknown. Thus, the mechanism of PF in promoting adipocyte browning was investigated. The ingredients of PF were acquired from the online database and filtered with oral bioavailability and drug-likeness criteria. The browning-related target genes were obtained from the Gene Card database. A Venn diagram was employed to obtain the overlapped genes that may play a part in PF promoting adipocyte browning, and an enrichment was analysis conducted based on these overlapped genes. A total of 17 active ingredients of PF were filtered, which may regulate intracellular receptor-signaling pathways, the activation of protein kinase activity, and other pathways through 56 targets. In vitro validation showed that PF promotes mitochondrial biogenesis and upregulates brite adipocyte-related gene expression. The browning effect of PF can be mediated by the p38 MAPK pathway as well as PI3K-AKT pathway. The study revealed that PF could promote adipocyte browning through multitargets and multipathways. An in vitro study validated that the browning effect of PF can be mediated by both the P38 MAPK pathway and the PI3K-AKT pathway.The leaf of  Obesity is mainly caused by an imbalance of energy intake and consumption, which is characterized by a large number of lipids accumulated in white adipocytes . Long-teSeveral important molecules have been reported to play an important role in adipogenesis and browning regulation. Sakaguchi et al.  revealedCinnamomum genus, has been proven to induce browning in the subcutaneous adipocytes of obese mice. Oral administration of cinnamon extract can promote UCP1 expression in the subcutaneous adipose tissue and reduce the body weight of obese mice [Perilla frutescens (L.) Britt (PF) is also a member of the Pungent-Warm Exterior-Releasing medicinal family. Thomas et al. [P. frutescens var. acuta) leaf extract has anti-obesity effects in rodents and can be effective in obesity management due to its ability to reduce lipid accumulation in differentiated adipocytes and prevent an increased body weight in C57BL/6J mice fed high-fat diets. Feng et al. [Traditional Chinese Medicine (TCM) is a rich resource to inspire novel insights into therapeutic approaches. Several herbs from TCM have been investigated for their pharmacological function in increasing calorie expenditure and reducing fat-tissue formation in mice. Cinnamon, the bark of trees of the ese mice . The β3-ese mice  found ths et al.  found thg et al.  found thg et al. . ConsequPF extract comprises more than one component ingredient, indicating complicated pharmacological targets and mechanisms. So far, most existing studies on herb extracts have concentrated on single or limited ingredients and targets, lacking an integral exploration of the pharmacological mechanisms of the herb ,13. As aIn the present study, network pharmacology methods were used in combination with molecular docking to reveal the potential molecular mechanisms of PF promoting adipocyte browning, and results were validated by experimental evidence. The overall workflow is shown in http://www. swissadme.ch, accessed on10 August 2022) [The ingredients in PF were obtained through the symMap database, the Traditional Chinese Medicines Integrated Database (TCMID), and the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) . The chest 2022) .Swiss-Target-Prediction , a websiAdipocyte browning-related genes were found in the GeneCards database; “adipocyte browning” and “adipose browning” were used as search keywords. Protein-encoding genes with a disease relevance score ≥3 were chosen for further investigation.The overlapped genes of both PF-related genes and browning-associated targets were filtered by a Venn diagram. Based on the aforementioned overlapped genes, a topological interaction network was created after inputting the list of genes into the String database, intending to screen out the hub genes. For the topological network establishment, the interaction score was set as 0.4. Further topological analysis was performed using the MCODE plugin of Cytoscape, which was used to screen out the core clusters from the entire network.p-value < 0.01 were set as the cutoff values.The potential pathways and biological functions of the overlapped genes were predicted using enrichment analysis. The R package “cluster profile” was used to execute the analysis. The enriched items with an adjusted To further elucidate the correspondence between the active ingredient and overlapped target proteins, the ingredient–target network was constructed. Furthermore, to study the connection between protein targets and the 15 top pathways, the targets–pathway correspondence was also added, and the ingredient–target–pathway (I-T-P) network was established. To characterize the role of active ingredients in the network, three topological parameters were measured, namely degree, betweenness, and closeness .The top seven active ingredients filtered from the I-T-P network and the top seven targets screened based on the PPI network were paired for a molecular docking simulation, which was conducted using the AutoDock software package . For the+/Cl− was added to the system to balance the system charge, and finally, the topology and parameter files for the simulation were output. The system first underwent energy optimization and was then heated to 298.15 K. A 500 ps NVT  phylogenetic simulation was then conducted and an equilibrium simulation of 500 ps was performed. At the final stage, 100 ns NPT  phylogenetic simulations were performed. The geometrical parameters of the systems, such as the root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were determined and compared with the primitive ligand complex system.The binding of docked ligand–receptor complex systems was further validated by molecular dynamics simulations using the AMBER 18 software . For thePerilla frutescens (L.) Britt leaves were purchased in the local market, and then dried and crushed into powder. Dried PF powder was dissolved in 70% ethanol in a 1:30 (g/mL) ratio [The L) ratio ,30. The L) ratio ,31.Primary mouse-bone-marrow mesenchymal stem cells (BMSCs)  were culAfter 2 days, the ADP1 medium was changed to a maintenance medium , including 10 μg/mL insulin only, until Day 10. PF was added to the BMSCs starting from Day 0 and the medium changed every three days. For the inhibitor test, BMSCs were pretreated with AKT inhibitor SC394003  and p38 inhibitor (SB203580)  for 24 h and then simulated with the same fresh medium for 30 min before sample collection.The Cell Counting Kit-8  assay was employed to determine cell viability. BMSCs were seeded and incubated with PF . After 10 days of induction, the cells were washed with PBS, and 10% CCK-8 solution diluted with alpha-MEM medium was added to each well; then, they were incubated for an hour, and the absorbance at 450 nm was measured by a microplate reader .Real-time quantitative polymerase chain reaction (RT-qPCR)  was usedCells were lysed to obtain the total protein content using freshly prepared radioimmunoprecipitation assay buffer (RIPA) at 4 °C . ProteinThe Oil Red O (ORO) staining method  was emplThe BMSCs that were treated with PF or left untreated for 10 days were washed with PBS and incubated with DMEM containing 100 nM MitoTracker Green FM  for 30 min at 37 °C . Cells wp < 0.05.Statistical analysis was conducted using Prism 8 software. Data were expressed as the mean ± SD and analyzed using one-way ANOVA. Differences between groups were considered to be statistically significant if values of There were 396 compounds in PF obtained from those the aforementioned databases. After screening by oral bioavailability and drug-likeness criteria, 49 compounds were found. A total of 19 compounds were excluded, as no target could be predicted with a prediction probability of more than 0.1. Overall, 427 PF-predicted genes were finally generated from 30 compounds, and 1493 browning-related genes were found using the GeneCards database, based on which 342 genes were selected with a relevance score of more than 3. Eventually, 56 overlapped genes predicted by 17 compounds from both the PF-predicted genes and browning-related genes were selected using Venn diagrams, as shown in The enrichment analysis was performed based on the 56 overlapped targets to predict the potential mechanisms of PF promoting adipocyte browning. Overall, 1735 biological process (BPs), 27 (cell components) CCs, and 82 molecular functions (MFs) were enriched in the GO analysis. The biological process enrichment conducted by the Glue GO plugin is shown in PPI analysis, as plotted in The I-T-P network was established to illustrate the ingredients and their correspondence to the receptors and pathway involved, including 88 nodes and 312 edges, as shown in The top five compounds with the highest edge numbers were luteolin (C8), nerolidyl acetate (C11), 1- propane-2-amine (C1), isoeugenol (C7), and dibutyl phthalate (C4). The mean topological parameters of the top five compounds were 12 degrees, 0.0728 of node betweenness, and 0.3506 of closeness. The top five target nodes with the most connections were AR, ESR1, PTGS2, PTPN1, and SRC. The mean value of topological parameters was 8 degrees, 0.0398 of node betweenness, and 0.3442 of closeness, respectively. The 17 compounds of PF are listed in To further elucidate the combination of compound and predicted proteins, a molecular docking simulation was performed. As the lowest binding energy indicated the most stable binding modality, the binding pattern with the lowest binding energy was simulated. Based on the PPI and enrichment results, eight key browning-related targets  and seven compounds with top-degree values in the I-T-P network were paired for molecular docking. The binding energy results are summarized in the heat map shown in It is generally accepted that a binding energy less than −4.25 kcal/mol indicates specific binding activity of the ligand to the receptor, while a binding energy less than −5.0 kcal/mol indicates better binding activity . AccordiAccording to the I-T-P network, AKT1/C11, MAPK14/C1, and MAPK14/C4 are the possible ligand–receptor complexes for combining AKT1 and MAPK14. The molecular binding was also simulated through molecular dynamics simulation. As shown in For AKT1/C11, the protein–ligand complex part converged at 40 ns and was very stable in the subsequent simulations. The RMSD of the C11 simulation was within 2 Å throughout, with only weak fluctuations occurring around 67 ns. For MAPK14/C1, the RMSD of the protein–ligand complex was found to converge at 15 ns and fluctuate around 3 Å in the later part of the simulation, while the RMSD of the C1 remained stable throughout the simulation, with an RMSD within 1 Å. In the case of MAPK14/C4, the RMSD of the complex system was larger and less stable than MAPK14/C1. Nevertheless, the RMSD of the small molecule fluctuated within 2 Å, indicating that the small molecule can still bind stably in the active site. The RMSF characterizes the rigidity of the ligand–receptor complex. Higher rigidity indicates more stable binding and superior enzymatic activity. As shown in Hydrogen bonding is one of the strongest noncovalent interactions; the higher the number of hydrogen bonds, the more favorable the binding of small molecules and proteins. The number of hydrogen bonds formed between ligands and proteins during the simulation was ranked as MAPK14/C1>AKT1/C11>MAPK14/C4. MAPK14/C1 formed the largest number of hydrogen bonds, which may be the reason that MAPK14/C1 had the strongest stability of the abovementioned complexes, as shown in BMSCs were co-cultured with PF at different concentrations, as shown in ORO staining revealed a notable dose-dependent decrease in lipid accumulation, which indicated the inhibition of adipogenesis in PF-treated adipocytes, as shown in Mito-tracker staining showed increased mitochondrial activity when treated with 200 μg/mL PF, as shown in The expression of core genes screened was validated by Western blot, as shown in The network pharmacology prediction showed that the p38 MAPK and PI3K-AKT pathways could be the potential pathways involved in adipocyte browning. As a result, the expression of p38 MAPK and its phosphorylation was examined, and an increased expression was observed after treatment with PF. The phosphorylation of downstream transcription factor CREB was also found to have an increased expression, as shown in To further confirm the role of the p38 MAPK and PI3K-AKT pathway in adipocyte browning, the expression of browning-specific marker UCP1 and the phosphorylation of p38 were examined after intervention with p38 MAPK inhibitor SB203580. As shown in Similarly, the UCP1 expression and the phosphorylation of AKT1 was examined after being treated with AKT inhibitor SC394003, which inhibited the increased expression of UCP1 as well p-AKT1, as shown in Due to changes in diet and genetic factors, the number of obese people is increasing year by year . When enCurrently, obesity is mainly treated with surgery or drugs to reduce the body’s energy intake. Orlistat is an intestinal fat inhibitor, which can achieve weight loss by reducing the absorption of fat in food, but resulting in a deficiency of fat-soluble vitamins in the long term . LiragluP. frutescens) has various effects, such as regulating glucose and lipid metabolism, antioxidation, antidepression, and relieving cough and asthma [Perilla leaves can regulate glucose and lipid metabolism disorders in diabetic model mice and has a good antidiabetic effect [White adipocytes can be transformed into brown-like adipocytes. This process is called “Browning” . Currentd asthma ,45,46. Sc effect . In thisThe GO enrichment analysis demonstrated that the top enriched biological processes were the intracellular receptor-signaling pathway, activation of protein kinase activity, response to peptide hormone, regulation of lipid metabolic process, and peptidyl-serine phosphorylation, which are responsible for signal transduction. The CC enrichment showed that these proteins were mainly located on the membrane raft, membrane microdomain, vesicle lumen, transferase complex, and transcription regulator complex, indicating that they are mostly located within the cells. The main functions of these potential target genes are related to nuclear receptor activity, ligand-activated transcription factor activity, protein serine/threonine kinase activity, DNA-binding transcription-factor binding, and phosphatase binding, which are mostly related to the signal-transduction cascade. In all, the GO enrichment results indicate that the target genes enriched are mostly responsible for intracellular signal transduction within the cell.A total of 56 target genes of PF were overlapped and selected as potential targets for the promotion of adipocyte browning. Core targets were screened out by PPI network analysis and the MCODE plugin. It was interesting to note that AKT- and MAPK-related targets  were shown in cluster 1, indicating that they may play an important role in the PPI network. MAPK14 belongs to the p38 MAPK family, whose function is to initiate signal-transduction cascades that can finally activate transcription factors when cells are simulated by external stimulation, such as stress or proinflammatory cytokines .The KEGG pathway analysis indicated that lipid and atherosclerosis, the PI3K-Akt signaling pathway, the estrogen signaling pathway, and the p38 MAPK signaling pathway might be involved in the browning process, stimulated by PF. P38 MAPKs initiate downstream signal transduction via phosphorylation. It has been reported that there are more than 200 substrates, including some kinases, leading to phosphorylation cascades, such as CREB1, ATF1, STAT1, and STAT3 . The p38Several studies have also shown that the PI3K-Akt signaling pathway can play a part during the browning process. Zhao et al. showed that Vitamin D3 could inactivate the PI3K/Akt/mTOR/p53 signaling pathway and inhibit the expression of browning-specific markers but could promote autophagy . MoleculThe in vitro experiments were conducted to validate the browning effect of PF based on the BMSCs. The browning process is accompanied by an increase in browning markers, including uncoupling protein-1 (UCP-1), pparγ-assisted activator 1α (PGC-1α), and positive regulatory domain containing 16 (PRDM16) . The resAs for the limitation of the present study, only a small part of the mechanisms or pathways by which PF promotes adipocyte browning were analyzed and discussed. More browning- or adipogenesis-related genes can be examined by RT-qPCR to explore more possible mechanisms. Further studies are needed to validate the potential role of more pathways, such as estrogen signal pathways.In conclusion, the study revealed the therapeutic function of PF and possible mechanisms involved in the promotion of adipocyte browning. It was demonstrated that PF can promote adipocyte browning by the P38 MAPK pathway and the PI3K-AKT pathway. The study showed great potential in combing in silicon methods with experimental validation to investigate the biological function of herb extracts.


Dictamnus dasycarpus Turcz is a traditional Chinese medicine, Dictamni Cortex (DC), which is mainly used in the clinical treatment of skin inflammation, eczema, rubella, rheumatism, and gynecological inflammation. Unexpectedly, there are some cases of liver injury after the administration of DC. However, the mechanism of hepatotoxicity remains ambiguous. The aim of this study was to explore the mechanism and substance bases of DC hepatotoxicity based on network pharmacology and molecular docking, verified through pharmacological experiments. Partial prototype components and metabolites in vivo of quinoline alkaloids from DC were selected as candidate compounds, whose targets were collected from databases. Network pharmacology was applied to study the potential hepatotoxic mechanism after correlating the targets of candidate compounds with the targets of hepatotoxicity. Molecular docking was simulated to uncover the molecular mechanism. Furthermore, the hepatotoxicity of the extract and its constituents from DC was evaluated in vivo and in vitro. We constructed the “potential toxic components-toxic target-toxic pathway” network. Our results showed that the targets of DC included CYP1A2 and GSR, participating in heterologous steroid metabolism, REDOX metabolism, drug metabolism, heterocyclic metabolic processes, the synthesis of steroid hormone, cytochrome P450 metabolism, chemical carcinogens and bile secretion pathways. In vitro and in vivo experiments displayed that DC could result in a decrease in GSH-Px and oxidative stress, simultaneously inhibiting the expression of CYP1A2 and inducing hepatotoxicity. These results further indicated the mechanism of hepatotoxicity induced by Dictamnus dasycarpus, providing a basic theory to explore and prevent hepatotoxicity in the clinical usage of Dictamnus dasycarpus.The root bark of  Herbal medicines have been used to treat various diseases in China for thousands of years. At present, herbal medicines are increasingly available for clinical treatment worldwide, but many herbal medicines can induce toxicity, especially hepatotoxicity. A study performed in China found that of all drug-induced liver injuries (DILI), herb-induced liver injury (HILI) accounts for one-quarter, which should be given more attention .Dictamnus dasycarpus Turcz is a traditional Chinese medicine, Dictamni Cortex (DC), which is widely used in the treatment of skin inflammation, eczema, rubella, rheumatism and gynecological inflammation [Polygoni multiflori and Dictamnus dasycarpus were the most frequent causative agents inducing liver injury identified in a Korean study [The root bark of ammation . Unexpecammation . In Chinammation ,5. Polygan study . The hepan study . Limonoian study , and resan study ,9. Howevan study , construIt is a great challenge to research the pharmacological mechanism of hepatotoxicity induced by herbal medicines due to their complex and various constituents. In addition, constituents from herbal medicines will be metabolized into more complex metabolites, making it more difficult to elucidate the pharmacological mechanism of hepatotoxicity in herbal medicines. As an important branch of network pharmacology, network toxicology has developed with the development of systems biology. Network toxicology provides a new strategy to quickly discover toxic components and explore the mechanism of toxicity by predicting and analyzing the toxic components and the targets of toxicity, aiming to construct the network of ‘toxicity-toxic components-toxic targets-toxic pathways’ ,12. ManyDictamnus dasycarpus.In this study, we selected partial prototype components and metabolites of quinoline alkaloids from DC in vivo as candidate compounds. We applied network pharmacology to study the potential hepatotoxic mechanism of candidate compounds after collecting their targets and hepatotoxicity from databases. Molecular docking was simulated to uncover the molecular mechanism. Furthermore, we evaluated the hepatotoxicity of the compounds and DC extracts in vitro and in vivo. By combining multiple approaches, we systematically studied the pharmacological mechanism and toxic substance basis of hepatotoxicity induced by A total of 561 targets of 36 candidate compounds were retrieved and predicted using TCMSP, Pharmapper, SwisstargetPrediction and SEA databases. The “candidate compounds-targets” network was constructed, and the degree values of nodes were calculated in Cytoscape 3.7.2 . A higheA total of 35 overlapping targets were included after comparing candidate compounds-targets and hepatotoxicity targets A, the dep < 0.05 as the screening condition, a total of 66 pathways were obtained, including 42 biological processes, 17 molecular functions and 7 cellular components . The results of female mice are shown in To estimate the hepatotoxicity of DC in vivo, mice were treated with DC for two weeks. The body weight change during treatment and relative liver weight at sacrifice were recorded. As displayed in The results of H&E staining indicated that DC can lead to hepatocyte injury. Compared with the control group, the administration of DC induced nuclear swelling in the liver pathological sections, whereas partial nuclear dissolution and chromatin deepening existed in the middle and high groups A. No appp < 0.05) and decreased ALP activity (p < 0.05). All doses significantly increased the content of serum TP (p < 0.01). The middle- and high-dose groups  had significantly decreased serum ALB (p < 0.01). No dose had a significant effect on the content of D-Bil and T-Bil. The results of females are shown in The results in p < 0.05), whereas the activities of T-SOD and GSH-Px were remarkably decreased (p < 0.01) compared to those in the normal group  and explore the toxic mechanism, providing a new perspective and new ideas for toxicological research on TCM. Zhang et al. used serum pharmacochemistry and network toxicology to screen the potentially toxic components of  in rats . Jiang elication .In this study, we first constructed the ‘toxicity-toxic components-toxic targets-toxic pathways’ network and researched the potential hepatotoxic mechanism of DC by collecting targets, pathway analysis and molecular docking. The network of ‘toxicity-toxic components-toxic targets-toxic pathways’ revealed that DC produced hepatotoxicity by participating in heterologous metabolism, REDOX metabolism, steroid metabolism, drug metabolism, heterocyclic metabolism, the synthesis of steroid hormones, cytochrome P450 metabolism, chemical carcinogenesis and bile secretion. Although there were many overlapping targets between compounds-targets and hepatotoxicity targets, the key targets were CYP1A2 and GSR according to the results of molecular docking. The results of the experiments on these cells indicated that alkaloids induced cytotoxicity more strongly than limonin. In vivo, the results of the staining and biochemical analysis indicated that DC can lead to hepatocyte injury and oxidation stress.Dictamnus dasycarpus can inhibit the activity of CYP1A2, even more strongly than α-naphthoflavone according to docking. In addition, CYP1A2 can protect against reactive oxygen production in the liver [CYP1A2 is one of the most important CYP enzymes in the liver, accounting for 13% to 15% of hepatic CYP enzymes, and is responsible for the metabolism of polycyclic aromatic hydrocarbon and many clinical drugs . If the he liver . In our Dictamnus dasycarpus, furoquinoline alkaloids can be metabolized to dihydrofuroquinoline alkaloids by CYP3A4, consuming GSH simultaneously [Glutathione reductase (GSR) is a key enzyme in the GSH redox cycle, reducing oxidized glutathione (GSSG) to reduce GSH , which caneously ,32.Our research first constructed the network of ‘toxicity-toxic components-toxic targets-toxic pathways’ of DC, providing further evidence for its hepatotoxicity. In our study, we found that GSR and CYP1A2 were key targets of hepatotoxicity induced by dihydrofuroquinoline alkaloids using network pharmacology. For verifying these results, we proved the cytotoxicity of the furoquinoline alkaloids in vitro. Furthermore, in vivo, we confirmed that DC can decrease the activity of GSH-Px, resulting in oxidative stress injury and inhibiting the expression of CYP1A2, which can induce hepatotoxicity. In other words, we drew a conclusion that the main toxic substances in DC were furoquinoline alkaloids, whose targets were CYP1A2 and GSR.However, there are several questions worth exploring. The cause of the difference in toxicity between the aqueous extract and ethanol extract remains obscure. The compounds were so minor that we could not conduct further research on their deeper mechanism. Further investigation should be carried out on these compounds to determine whether we can obtain sufficient amounts via chemical biosynthesis. In addition to docking, it is more rigorous and reliable to verify the binding abilities of compounds and targets in vitro using molecular biological approaches, such as surface plasmon resonance (SPR) and biofilm interference (BLI) technology. It would be interesting and meaningful to explain the mechanism of CYP1A2-induced hepatotoxicity.Dictamnus dasycarpus Turcz. (Rutaceae) was supplied by the Anguo medicine market, Hebei province, China, and further identified by Professor Peng-Fei Tu, School of Pharmaceutical Sciences, Peking University, Beijing, China. A specimen has been deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine (No. 201508150324).The root bark of v/v) and water by using the reflux extraction method for 1 h each. The extraction solutions were combined, filtered, evaporated and freeze-dried to obtain aqueous extract (DCA) and ethanol extract (DCE). The extraction efficiencies of DCA and DCE were 25% and 3.75%, respectively. DCA and DCE were suspended in 0.5% CMC-Na.DC was pulverized into fine powders (50 mesh) using a medicinal material grinder and then extracted three times with 95% ethanol-water (https://tcmspw.com/tcmsp.php (accessed on 11 March 2021)), PharmMapper (http://lilab-ecust.cn/pharmmapper/index.html(accessed on 11 March 2021)), SwissTargetPrediction (http://www.swisstargetprediction.ch/(accessed on 11 March 2021)) and SEA (https://sea.bkslab.org/(accessed on 11 March 2021)). “Liver Injury” and “hepatotoxicity” were used as keywords to search for hepatotoxicity targets in CTD (http://ctdbase.org/(accessed on 11 March 2021)), and “marker/mechanism” was used as a limited property.On the basis of the results of in vivo metabolite identification in rats dosed with DC , 36 expop < 0.05.The hepatotoxicity targets of the candidate compounds were obtained by comparing the targets of the candidate compounds and hepatotoxicity targets. The hepatotoxicity targets of the candidate compounds were imported into the STRING database, the species was selected as “Homo sapiens”, and the scoring condition was set as >0.40. Discrete targets were removed, and the protein interaction network was obtained and saved as TSV format files. The TSV format file was imported into Cytoscape 3.7.2 software to conduct a visual analysis of the network. The hepatotoxicity targets of the candidate compounds were imported into the David online analysis platform for GO and KEGG enrichment analyses, and the results of GO and KEGG enrichment analyses were selected and sorted under the condition of The data of “DC-candidate compounds”, “candidate compounds-targets”, “targets-pathways”, and “pathways-hepatotoxicity” were collected in Excel and imported into Cytoscape 3.7.2 software to construct the network of “potential toxic components-toxic targets-toxic pathways” of DC-hepatotoxicity. The network characteristics were analyzed using “Network analysis”.Schrodinger 11.8 software was used to construct the molecular docking model of the key targets for molecular docking with the components of DC exposed in vivo. First, the protein structures of the key targets of DC hepatotoxicity were downloaded from the PDB database, and complex crystal structures with small ligand molecules and resolutions of less than 3 Å were selected. At the same time, the related best ligand structure was downloaded as the positive control. Schrodinger 11.8 software was used to conduct dehydration, hydrogenation, residue completion and pocket generation of the protein. The structure of the candidate compounds was optimized, and then the docking experiment was carried out.2 incubator under 95% absolute humidity.The HepG2 cell line was purchased from the Cell Bank of Peking Union Medical College in China. The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin. The cell culture conditions were maintained at 37 °C in a 5% COCell viability was assessed using a 3--2,5-diphenyl tetrazolium bromide (MTT) solution . Adriamycin was selected as the positive control and 1% DMSO was the negative control. The compounds were prepared in a stock solution of 20 mM. HepG2 cells were treated for 48 h, the culture supernatant was removed, and a staining solution containing MTT (5 mg/mL) was added to each well. The cells were then incubated for 4–6 h in the dark at 37 °C. Thereafter, dimethyl sulfoxide (DMSO) was used to dissolve formazan crystals, and the absorbance at 570 nm was determined spectrophotometrically on an ELX800 UV universal microplate reader . Data were graphically displayed using GraphPad Prism (version 6.0).Male and female ICR mice  were obtained from the Laboratory Animal Center of the Peking University Health Science Center . The mice were housed at 25 °C and 60 ± 5% humidity, with a 12 h dark–light cycle. They were fed standard laboratory food and water ad libitum. All mice were fasted for 12 h but had free access to water prior to the sacrifice. The animal facilities and protocols were approved by the Animal Care and Use Committee of the Peking University Health Science Center (LA2015061). All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals .n = 10): normal group, DCE high-dose group (DCEH), DCE middle-dose group (DCEM), DCE low-dose group (DCEL), DCA high-dose group (DCAH), DCA middle-dose group (DCAM) and DCA low-dose group (DCAL). DCEL, DCEM and DCEH were orally dosed with 235 mg/kg/d, 470 mg/kg/d and 940 mg/kg/d DCE for two weeks, respectively. DCAL, DCAM, and DCAH were orally dosed with 1.56 g/kg/d, 3.12 g/kg/d and 6.24 g/kg/d of DCA for two weeks, respectively. All the mice were weighed and recorded every 3 to 4 days.After acclimatization for one week, 70 male mice and 70 female mice were randomly divided into seven groups  of liver tissues were stained with routine hematoxylin/eosin (H&E) staining. All histological images were obtained using an Eclipse Ti-SR fluorescence microscope  and Pannoramic MIDI  and analyzed using Image J software version 1.52a .Frozen liver tissue (0.1 g) was added to 1 mL of normal saline, and a 10% tissue homogenate was obtained using a homogenizer . The homogenate was centrifuged at 12,000 rpm at 4 °C for 15 min, and the supernatant was taken for protein quantification and detection of the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the levels of malondialdehyde (MDA), which were examined with detection kits following the Jiancheng Bioengineering Institute’s protocol .The activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was determined using an ELISA kit according to the manufacturer’s instructions . The concentrations of total protein (TP), albumin (ALB), alkaline phosphatase (ALP), total bilirubin (T-Bil) and direct bilirubin (D-Bil) in the serum samples were measured following the Jiancheng Bioengineering Institute’s protocol .The 10% tissue homogenate of livers was added to ice-cold RIPA buffer containing 1 mM PMSF  to obtain the tissue lysate. Whole proteins were collected by centrifugation at 13,000 rpm at 4 °C for 20 min. The concentration of the total proteins was detected using a protein assay kit . Equal concentrations of protein solutions were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Next, the PVDF membranes were blocked with 5% skim milk at 25 °C for 60 min. Subsequently, the membranes were incubated with primary antibodies against GAPDH , CYP1A2 , CYP2E1  and CYP3A11  at 4 °C for 12 h. After incubation with a second antibody at room temperature for 1 h, the membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate. The protein bands were imaged using a Tanon 5200 Imaging Analysis System . The ImageJ software was used to calculate the relative gray of bands.−ΔΔCT method.The liver tissues were processed for the isolation of total RNA by using TRIzol reagent  according to the manufacturer’s protocol. The RNA concentration was determined, and the quality of the isolated RNA was assessed using the 260/280 nm absorbance ratio (1.8–2.0 indicates a highly pure sample). The RNA pellet was stored at −80 °C until further use. According to the instructions of the supplier, we carried out inverse transcription utilizing MMLV reverse transcriptase . The resulting reverse transcription products were stored at −80 °C until the assay. The polymerase chain reaction was carried out using an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 65 °C for 30 s using a qPCR instrument of Agilent. The sequences of the forward and reverse primers are summarized in t-test was used without correction for multiple comparisons. A p value < 0.05 was considered to be significant.All values were expressed as mean ± SD (standard deviation). Statistical analyses were performed using a one-way analysis of variance with GraphPad Prism 6.0 software. Multiple comparisons were performed by comparing the mean of each column with the mean of every other column. Student’s Dictamnus dasycarpus by molecular docking and animal experiments. Our results showed that the targets of DC included CYP1A2 and GSR, participating in heterologous steroid metabolism, REDOX metabolism, drug metabolism, heterocyclic metabolic process, the synthesis of steroid hormone, cytochrome P450 metabolism, chemical carcinogens and bile secretion pathways. Dictamni Cortex can cause oxidative stress and a decrease in CYP1A2, inducing liver injury. These results further explain the mechanism of hepatotoxicity induced by Dictamnus dasycarpus, providing a basic theory to explore and prevent hepatotoxicity in the clinical usage of Dictamnus dasycarpus.In conclusion, we constructed a “potential toxic components-toxic target-toxic pathway” using a network toxicology method and verified the potential mechanism of the hepatotoxicity of


Objective: We aimed to reveal the potential active ingredients, targets and pathways of Shiwei Hezi pill (SHP) in the treatment of nephritis based on systematic network pharmacology.Methods: The online database was used to screen the common targets of SHP and nephritis, and the interaction between targets was analyzed. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the Bioinformatics website. Molecular docking was carried out to verify the correlation between core ingredients and key targets. Cytoscape 3.6.1 was applied to perform protein-protein interactions (PPT) network construction and data visualization.Results: A total of 82 active ingredients in SHP were screened, and 140 common targets of SHP and nephritis were obtained. Our results demonstrated that TNF, AKT1 and PTGS2 might be the key targets of SHP in the treatment of nephritis. GO enrichment analysis yielded 2163 GO entries (p < 0.05), including 2,014 entries of the biological process (BP) category, 61 entries of the cell composition (CC) category and 143 entries of the molecular function (MF) category. KEGG pathway enrichment analysis produced 186 signaling pathways (p < 0.05), involving the AGE-RAGE, IL-17and TNF signaling pathways. The results of molecular docking showed that three active ingredients in SHP  could effectively bind to the TNF, AKT1 and PTGS2 targets.Conclusion: The effective active ingredients in SHP may regulate multiple signaling pathways through multiple targets, thereby exhibiting a therapeutic effect on nephritis. Nephritis is an immunity-mediated inflammatory response that has a great impact on human health . It beloTripterygium wilfordii combined with Salvia miltiorrhiza on children with allergic purpura nephritis  is becoming increasingly recognized for its potential in treating chronic and complex diseases both at home and abroad . In 2010ephritis . Wan Yudephritis . OverallChebulae Fructus (Hezi), Carthami Flos (Honghua), Radixet Rhizoma Rubiae (Zangqiancao), Mountain alum leaf (Shanfanye), Swertia petiolata (Zhangyacai), Lithospermum erythrorhizon (Zicaorong), Canavaliae Semen (Daodou), Alpinia Katsumadai Hayat (Doukou), Slag breaking cream (Zhaxungao), and Sabina chinensis (Yuanbai) (Shiwei Hezi pill (SHP) is a classic Tibetan medicine prescription commonly used to treat nephritis, which consists of ten species of medicinal herbs, including Yuanbai) . The effYuanbai) . HoweverPanax Notoginseng Saponins  and TCMID (http://tcm.cmu.edu.tw) databases  database was retrieved to annotate the target protein sequence, after that, the corresponding targets of the bioactive ingredients in SHP were obtained. We then established the ingredient-target data set by converting the target sites into gene names with the species limit to “Homo sapiens” by the DAVID database (https://david.ncifcrf.gov)  .https://pubmed.ncbi.nlm.nih.gov/) (http://omim.org/) (https://www.genecards.org/) (The nephritis-related targets were filtered by searching the PubMed (ih.gov/) , OMIM (him.org/) , and Gends.org/)  databasehttps://bioinfogp.cnb.csic.es/tools/venny/) to construct a Venn diagram of common targets of SHP and nephritis.The SHP and nephritis-related targets were imported into the Venny 2.1.0 online platform (https://cn.string-db.org/) with the species limit to “H. sapiens”. The PPI was obtained and saved as a “tsv” format file. Afterwards, the file was input into Cytoscape 3.6.1 to build a PPI network.The data set of common targets of SHP and nephritis was imported into the STRING database Version 10.5 (http://bioinformatics.com.cn/). By entering a list of target gene names and restricting the species to human, all target gene names were corrected to their official gene symbols. The top 15 and top 20 entries, including biological processes (BP), cellular components (CC) and molecular functions (MF), were selected for analysis with p-value as a screening criterion. The histograms and bubble charts were plotted to further explore the biological significance of common targets of SHP and nephritis.GO functional annotation and KEGG pathway analysis were performed via the Bioinformatics website   database (sb.org/) . Using AChebulae Fructus, Carthami Flos, Canavaliae Semen, Alpinia Katsumadai Hayat, and S. petiolata) were obtained from the TCMSP and TCMID databases, while the active ingredients of the remaining 5 herbs were identified through the literature search. Notably, despite madder diester and n-butanol with OB < 30% and DL < 0.18, they still showed a high content and potential anti-nephritis value, making them as active ingredients. Cytoscape software was then used to construct a compositional network diagram, and 75 active ingredients were gained after removing duplicate ingredients of this herb  were provided. The top 10 significant enrichment results of each category are shown in The binding interactions between main active ingredients  and key targets  was verified by molecular docking. The molecular docking binding performance is presented in Nephritis is characterized by various pathological forms and clinically presents with albuminuria, hematuria, hypertension and edema. If left untreated, it can lead to renal shrinkage and decreased function. Therefore, it is urgent to develop safer and more effective anti-nephritis drugs . In thisin vitro and in vivo , a prostaglandin endoperoxidase, exerts a key effect in prostaglandin biosynthesis. It has been reported that there is a certain relationship between COX-2 inhibitors and acute interstitial nephritis  of SHP in nephritis treatment, we further conducted a KEGG analysis to reveal the signal pathways of these main targets. The results showed that the AGE-RAGE, IL-17 and TNF signal pathways were the important pathways of SHP acting on nephritis. Studies have shown that the accumulation of diseases . Additiodiseases . IL-17 hdiseases . Similardiseases . The TNFdiseases . Moreovediseases . In concTo further verify the relationship between active ingredients  and key targets , we carried out molecular docking. The results showed that the binding energies of quercetin and luteolin with TNF, AKT1 and PTGS2 were lower than −5.0 kJ/mol, indicating the potential for forming an effective and stable complex between the ligand and receptor. In short, the core active compounds in SHP appeared to regulate SHP-related pathways by acting on the important genes linked to nephritis, thus offering therapeutic value for nephritis treatment.However, our research has some limitations. Bioactive ingredients of SHP were screened only from existing public databases and literatures, rather than using mass spectrometry and other methods. Additionally, there is a lack of animal experiments and clinical trials to verify our findings. To further improve our research, more animal experiments and clinical trials will be conducted in the future.In this study, we combined network pharmacology and molecular docking to explore the mechanism by which SHP exerts its anti-nephritis effects. We found that quercetin, kaempferol and luteolin are likely the main active compounds of SHP responsible for its therapeutic effects against nephritis. Moreover, SHP can target the expression of TNF, AKT1 and PTGS2 via the AGE-RAGE, IL-17 and TNF signaling pathways. Overall, although more researches are needed to clarify the exact mechanism, this study provides a valuable insight into the application of SHP for nephritis treatment and the potential for future anti-nephritis drug development.


Ginkgo biloba L. leaves (GBLs) play a substantial role in the treatment of vascular dementia (VD); however, the underlying mechanisms of action are unclear.This study was conducted to investigate the mechanisms of action of GBLs in the treatment of VD through network pharmacology, molecular docking, and molecular dynamics simulations.The active ingredients and related targets of GBLs were screened using the traditional Chinese medicine systems pharmacology, Swiss Target Prediction and GeneCards databases, and the VD-related targets were screened using the OMIM, DrugBank, GeneCards, and DisGeNET databases, and the potential targets were identified using a Venn diagram. We used Cytoscape 3.8.0 software and the STRING platform to construct traditional Chinese medicine–active ingredient–potential target and protein–protein interaction networks, respectively. After gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of potential targets using the DAVID platform, the binding affinity between key active ingredients and targets was analyzed by molecular docking, and finally, the top 3 proteins–ligand pairs with the best binding were simulated by molecular dynamics to verify the molecular docking results.A total of 27 active ingredients of GBLs were screened and 274 potential targets involved in the treatment of VD were identified. Quercetin, luteolin, kaempferol, and ginkgolide B were the core ingredients for treatment, and AKT1, TNF, IL6, VEGFA, IL1B, TP53, CASP3, SRC, EGFR, JUN, and EGFR were the main targets of action. The main biological processes involved apoptosis, inflammatory response, cell migration, lipopolysaccharide response, hypoxia response, and aging. PI3K/Akt appeared to be a key signaling pathway for GBLs in the treatment of VD. Molecular docking displayed strong binding affinity between the active ingredients and the targets. Molecular dynamics simulation results further verified the stability of their interactions.This study revealed the potential molecular mechanisms involved in the treatment of VD by GBLs using multi-ingredient, multi-target, and multi-pathway interactions, providing a theoretical basis for the clinical treatment and lead drug development of VD. It is often considered one of the most common causes of dementia after Alzheimer’s disease, accounting for approximately 15% to 20% of dementia cases in Western developed countries and possibly up to 30% in Asia and developing countries,,3 with a median survival time of approximately 4 to 9.4 years.,5 Cerebrovascular factors play a key role in the pathogenesis of VD and cerebrovascular diseases such as chronic cerebral hypoperfusion (CCH), cerebral small vessel lesions, cerebral amyloid angiopathy, and mixed vascular lesions can trigger the molecular mechanisms leading to VD.,6 Among these conditions, CCH is considered a key etiological factor in the progression of VD. However, the specific pathogenesis of VD remains unclear; therefore, its management is highly limited and there are currently no approved standard VD treatments to date. Cholinesterase inhibitors and memantine have been found to show some cognitive improvement, but the effect is very limited and expensive, and the price of weak efficacy increases the risk of adverse events in patients.,9 The ideal drug for VD should not only improve cognitive impairment but also have multiple other effects, including angiogenesis and neuroprotection. The unique advantages of multi-component, multi-target, and multi-pathway interactions of traditional Chinese medicine (TCM) provide new ideas for the development of ideal drugs for VD. Numerous studies have shown that TCM have clinical effects on VD, and compared to singular drugs, they have much more complicated targets and mechanisms. Compared to current prescription drugs, TCM exhibits fewer adverse effects, lower costs, and higher applicability for long-term use and has considerable potential in the treatment of VD.Vascular dementia (VD) is a neurocognitive disorder caused by the global or local impact of vascular diseases that leads to stroke injury or other changes in tissue perfusion and is characterized by behavioral abnormalities, motor and autonomic dysfunction, and neurocognitive impairment.Ginkgo biloba L. leaves (GBLs) are a commonly used TCM for treating cardiovascular and cerebrovascular diseases. They are rich in various natural active ingredients with a wide range of pharmacological activities, playing an important role in food, health care and medicine and have a high medicinal value. The Chinese Pharmacopoeia (2020 edition) records that GBLs have the effects of promoting blood circulation for removing blood stasis, regulating collaterals for relieving pain, removing turbidity and lowering lipids. Therefore, it is mainly used clinically to treat patients with dementia, memory decline, ischemic stroke, coronary angina and other brain dysfunction, degenerative diseases, and peripheral and coronary vascular diseases. Western medical research has found that GBLs have antioxidant, anti-inflammatory, anti-apoptosis, enhanced neuroplasticity, reduced blood viscosity, and enhanced microperfusion activity; show good safety and tolerance; and are increasingly used for the treatment of cognitive impairment with or without cerebrovascular disease because of their known neuroprotective effects and cerebrovascular benefits.Ginkgo biloba extract (GBE) is derived from GBLs and is used as a nutritional supplement and drug to improve cardiovascular and cerebrovascular diseases in many countries. The Anatomical Therapeutic Chemical classification system classifies GBE as an antidementia drug, along with acetylcholinesterase inhibitors (AChEIs) and memantine. The expert consensus in recent years states that the use of GBE alone or as an additional therapy plays an important role in the treatment of dementia, especially when patients have poor efficacy with AChEIs or memantine. Although GBLs are widely used to treat dementia, systematic studies on the direct effects and mechanisms of GBLs on VD are still lacking. The current study, therefore, used network pharmacology combined with molecular docking and molecular dynamics simulations to investigate and predict the underlying mechanisms of GBLs for the treatment of VD and provides ideas for clinical applications and experimental studies of GBLs and VD  . All ingredients were screened, and their targets were recorded with oral bioavailability ≥ 30% and drug-likeness ≥ 0.18 as the qualifying conditions. The normalized SMILES structures of each active ingredient were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and entered into the Swiss Target Prediction Database (http://www.swisstargetprediction.ch). Target prediction was performed using the species “Homo sapiens” and a probability > 0.1 as the screening criteria. The GeneCards human gene database (http://www.genecard.org/) was used to collect unpredicted active ingredient targets. Finally, the target proteins were converted and normalized using the UniProt database (http://www.uniprot.org) and the targets of GBLs were integrated and de-duplicated.All active ingredients of GBLs were searched through the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) database (http://omim.org/), DrugBank (http://www.drugbank.ca), DisGeNET (https://www.disgenet.org/), and GeneCards databases for disease target search and screening, integrated, and de-duplicated to obtain VD disease targets.Using “Vascular dementia” as the keyword, the disease targets were searched and screened in OMIM was used to construct the “TCM–active ingredients–potential targets” network. The nodes in the network diagram represent the drugs, active ingredients, and potential targets and the edges represent the interactions between the 3. The topological properties of the network were analyzed using the Network Analyzer tool in Cytoscape and the 3 parameters of degree, betweenness centrality, and closeness centrality were used to analyze the key active ingredients of GBLs in treating VD. The node size reflects the degree value.The Venn diagram was constructed to obtain the intersection of GBLs and VD disease targets, i.e., the potential targets of GBLs for VD treatment. Cytoscape 3.8.0 software (https://string-db.org) to construct a PPI network for the species Homo sapiens, with a confidence level of 0.4. Default settings were used for the other parameters and the PPI network of potential targets was obtained. The network files were imported into Cytoscape to visualize and analyze the topological properties of the PPI network; the target color and size reflected the node degree value. The first screening was qualified with greater than or equal to twice the median degree value; the second screening was subsequently performed to identify targets with greater than the median degree, median betweenness, and median closeness, i.e., key targets.To identify the interaction targets of GBLs that may be relevant to VD pathology and treatment, the potential targets were uploaded to the STRING databaseP < .05) were performed using the DAVID database (https://david.ncifcrf.gov) for potential targets with the species limited to Homo sapiens. The top 10 GO entries and top 20 KEGG signaling pathways were screened based on their P values and a bubble diagram and histograms were generated using Microscopic Letters (http://www.bioinformatics.com.cn) and GraphPad Prism.To further investigate the underlying mechanisms of GBLs for VD treatment, GO functional enrichment analysis  and KEGG pathway enrichment analysis (https://www.rcsb.org/). The target proteins and small molecules were processed using AutoDockTool 1.5.6 software to remove water, add hydrogen, add charge, and set rotatable bonds. Then, all processed small molecules, as well as proteins, were converted to PDBQT format. Finally, semi-flexible molecular docking was performed using AutoDock Vina 1.1.2 and the docking results were visualized using Discovery Studio 4.5 and PyMol 2.5.2 software to obtain 2D and 3D images.The top 11 key targets in the degree ranking were subjected to molecular docking analysis with the 4 key active ingredients and the docking binding energy was calculated. The three-dimensional (3D) structures of the key active ingredients were downloaded from the PubChem database and the 3D stereo structures of the core target proteins were obtained from the PDB database , OMIM, DisGeNET, and DrugBank databases, respectively. A total of 1665 VD targets were obtained by collation and deduplication.–active ingredients–potential targets network, which included 302 nodes and 1203 pairs of interactions , positive regulation of gene expression (GO:0010628), aging (GO:0007568), inflammatory response (GO:0006954), negative regulation of apoptotic process (GO:0043066), and response to hypoxia (GO:0001666). Cellular composition mainly involved the plasma membrane, extracellular space, cell surface, membrane raft, and synapse. Molecular functions mainly involved enzyme binding (GO:0019899), identical protein binding (GO:0042802), protein kinase binding (GO:0019901), neurotransmitter receptor activity (GO:0030594), and RNA polymerase II transcription factor activity (GO:0004879).To investigate the potential synergistic mechanism of GBLs for VD treatment, GO and KEGG analyses of potential targets were performed using the DAVID database, which contained 938 biological processes, 110 cellular compositions, 192 molecular functions, and 181 signaling pathways. Figure P value, and Figure  playing an important role in mechanisms related to learning and memory consolidation.,30 The repair of various organs in the human body usually proceeds through the PI3K/Akt pathway, especially in the central nervous system. After being phosphorylated by upstream signals, the PI3K/Akt pathway can stimulate downstream proteins to induce a cascade response that is involved in neuroprotection by inhibiting apoptosis, alleviating inflammation and oxidative stress, stimulating angiogenesis, improving cerebral circulation, and many other biological processes , and neurotransmitter system dysfunction have also been suggested to be associated with VD. These pathological mechanisms are not independent and may interact with each other to produce a pathogenic cascade response. In this study, GO analysis revealed that potential targets were associated with biological processes, such as apoptosis, inflammatory response, cell migration, lipopolysaccharide response, hypoxic response, and aging, which are consistent with the pathological process of VD. In addition, this study identified quercetin, luteolin, kaempferol, and ginkgolide B as the main active ingredients of GBLs for the treatment of VD. The first 3 belong to the class flavonoids and the latter to diterpene lactones. They are all closely related to VD and play essential roles in neuroprotection in terms of anti-apoptosis, anti-inflammation, anti-oxidation, and promotion of blood circulation. Therefore, we believe that GBLs are promising anti-VD lead drugs for further development.VD is a cerebrovascular disease characterized by severe cognitive and memory impairment. Apoptosis, inflammatory response, and oxidative stress due to cerebrovascular causes  are thought to be the molecular mechanisms closely associated with VD. It was found that quercetin enhanced neuronal resistance to oxidative stress and excitotoxicity by modulating cell death mechanisms, increasing the expression of longevity factors, and preventing neuroinflammation in the hippocampus by decreasing astrocyte markers and pro-inflammatory factor protein levels. In addition, quercetin plays a protective role in the apoptosis and degeneration of hippocampal neurons. Luteolin reduces brain reactive oxygen species (ROS) levels and modulates inflammatory factors through anti-oxidative stress, anti-inflammatory, and anti-apoptotic effects to achieve protective effects for neurons and inhibit or prevent neuroinflammation and cognitive decline caused by neuronal death. Kaempferol can bind to vascular endothelial growth factor (VEGF), enhance the angiogenic effects of VEGF in different models, and prevent vascular damage by alleviating oxidative stress and reducing inflammatory marker levels.,39 Ginkgolide is a key constituent of GBE and the most studied one is ginkgolide B, which is recognized as a natural neuroprotective agent through its beneficial effects of reducing neuroinflammation, inhibiting neuronal apoptosis, increasing cerebral blood flow, and promoting learning and memory capacity in various neurological disease models.,41Quercetin has been widely used in cardio–cerebrovascular and neurodegenerative diseases because of its anti-inflammatory, antioxidant, anti-apoptotic, and neurogenesis-enhancing properties, which exert neuroprotective effects in various research models. Neuronal apoptosis, especially in hippocampal neurons, is the main cause of symptoms of dementia. It has been shown that increased AKT1 phosphorylation can downregulate Caspase-3 (CASP3) activation, thereby increasing the survival of hippocampal neurons and reducing learning and memory impairment. AKT1 can be activated by extracellular signaling through a phosphatidylinositide 3-kinases (PI3K)-dependent mechanism, and activated AKT1 is involved in the cell survival pathway by inhibiting the apoptotic process, which is the core of the PI3K/Akt signaling pathway. Based on the results of KEGG analysis, the PI3K/Akt signaling pathway appears to be a critical pathway for the GBLs involved in VD. PI3K-activated AKT can mediate the inhibition of neuronal apoptosis and promote neuroprotection by phosphorylating multiple downstream target molecules, including GSK-3β, mTOR, CREB, Bcl-2 protein family, and CASP3. CASP3 is the most important protease in the apoptotic signaling pathway and is considered a key mediator of neuronal apoptosis. Considerable studies have shown that Casp3 expression is increased in the hippocampus of VD rats and that reducing apoptotic proteins in the hippocampus of VD rats can inhibit neuronal apoptosis, thereby alleviating the symptoms of VD and improving cognitive impairment.,47 The Bax/Bcl-2 ratio is an important indicator of apoptosis, and a higher Bax/Bcl-2 ratio indicates a stronger pro-apoptotic capacity. According to immunohistochemical findings, VD rats had substantially higher levels of Bax expression and substantially lower levels of Bcl-2 expression, which raised the Bax/Bcl-2 ratio.,47 It was found that GBE inhibited apoptosis of hippocampal neurons in mice after stroke by regulating the expression of Bax/Bcl-2 and Casp3. The PI3K/Akt signaling pathway could also downregulate the Bax/Bcl-2 ratio, alleviate apoptosis, and exert neuroprotective effects.,51 Therefore, we hypothesized that GBLs might be involved in regulating neuronal apoptosis primarily through the PI3K/Akt pathway to mitigate the developmental process of VD.Although apoptosis, inflammation, and oxidative stress usually exhibit distinct mechanisms, they are usually involved in the molecular regulatory pathways of neuronal survival and death induced by vascular dysfunction. These molecular regulatory pathways are also activated in a sequential and interdependent manner. Similarly, the key targets obtained in this study were closely related to these 3 processes. Molecular docking and molecular dynamics simulations showed that the top 11 key targets, according to the degree value, had good binding affinity for the key active ingredients and the top 3 protein–small molecules had good binding stability to each other. Among these, AKT serine/threonine protein kinase 1 (AKT1), which has the strongest binding capacity to luteolin and quercetin, is the main target of GBLs and plays a key role in the inhibition of apoptosis. GO analysis revealed that molecular functions, such as enzyme binding, identical protein binding, general protein binding, and protein kinase binding, are involved in the overall neuroinflammatory response. Among the key targets, increased levels of the inflammatory cytokines interleukin-6 (IL6), tumor necrosis factor (TNF), and interleukin-1β (IL1B) all independently contribute to cognitive impairment. A recent meta-analysis showed that moderate to high elevations in blood levels of IL6 and TNF-α were associated with a diagnosis of VD and that for each unit increase in IL-6 levels, the risk of VD increased by 28%. TNF-α is a key mediator of neuroinflammation and can induce enhanced pro-apoptotic responses in combination with other pro-inflammatory cytokines in an inflammatory environment. It has also been found that TNF-α expression in the brains of patients with VD is highly correlated with p53 expression, and both of these proteins act together to initiate the neuronal apoptotic program, leading to neuronal death. IL1β enhances the activity of matrix metalloproteinase-9 (MMP9), which in turn causes BBB impairment, leading to significant vascular endothelial dysfunction, inflammation, and a more rapid decline in cognitive function. These pro-inflammatory cytokines can also contribute to the development of VD by activating microglia, damaging neurons through oxidative stress, directly damaging white matter, and inducing dysfunction of brain microvascular endothelial cells. Microglia are the major inflammatory cells in the central nervous system, and their mediated neuroinflammation plays an important role in VD. SRC kinase (SRC) plays a key role in triggering microglia activation and is a key factor involved in the development of neuroinflammation. In addition, both pro-inflammatory cytokines and vascular endothelial dysfunction can induce overexpression of vascular cell adhesion molecule 1 (VCAM1). Previous studies have confirmed that VCAM1 in brain endothelial cells can activate microglia, inhibit hippocampal neural progenitor cell activity, and impair cognitive function. Quercetin, a key ingredients of GBLs, can improve the function and energy metabolism of cerebrovascular endothelial cells, reduce neurovascular injury, and participate in the prevention and delay of neurodegenerative diseases by targeting VCAM1. Oxidative stress is considered one of the contributing factors in the pathogenesis of VD, where excessive production of ROS in the brain and imbalance with cellular antioxidant systems eventually leads to cellular damage, including mitochondrial damage and neuronal apoptosis.,60 GO analysis involves biological processes such as the inflammatory response, response to lipopolysaccharides, response to hypoxia, and aging, all of which are closely related to oxidative stress and inflammation. Sustained oxidative stress can activate chronic inflammatory mechanisms and inflammation increases ROS production, which in turn induces oxidative stress and further exacerbates the inflammatory response, resulting in a vicious oxidative stress–inflammation cycle that can lead to cell death in severe cases.,62 The PI3K/Akt pathway regulates downstream targets that play a role in neuroinflammation and oxidative stress, and the potential target NFE2L2 encoding the nuclear factor erythroid two-related factor (Nrf2) is a major regulator against inflammation and oxidative stress. It was found that activation of the Nrf2/heme oxygenase 1 (HO-1) pathway downregulated the production and release of pro-inflammatory mediators in microglia and subsequently protected neurons from inflammatory damage, and that upregulation of Nrf2 and HO-1 was reliant on activation of the PI3K/Akt signaling pathway. In addition, quercetin was found to reduce lipopolysaccharide-induced ROS production and inhibit inflammatory responses through the ROS-regulated PI3K/Akt/NF-κB signaling pathway, exerting a protective effect in atherosclerosis models.Neuroinflammation is increasingly recognized as a risk factor for dementia. Numerous studies have demonstrated that inflammatory factors play a key role in the pathogenesis of VD and are important biomarkers for VD diagnosis. After the onset of VD, cerebral microangiogenesis can promote neurological recovery by improving cerebral microcirculation and the blood oxygen content of surrounding tissues, thus improving learning and memory capacity.,67 Vascular endothelial growth factor (VEGF) is by far the most significant and specific pro-vascular growth regulator. In addition to its classical effects, such as angiogenesis and increased vascular permeability, VEGF can also exert neurotrophic, neuroprotective, and neurogenic activity by binding to its receptor VEGFR2. It was found that VEGF can activate the downstream PI3K/Akt pathway, which is involved in regulating endothelial cell differentiation and angiogenesis to repair brain tissue and neurological damage.,71 In addition, the epidermal growth factor receptor (EGFR) can activate the PI3K/Akt/mTOR pathway and increase VEGF secretion. Endothelial nitric oxide synthase (eNOS) deficiency leads to endothelial dysfunction. This results in severe brain damage and cognitive deficits.,74 Akt1 is the major Akt isoform that regulates normal physiological functions of endothelial cells and preferentially regulates the phosphorylation of eNOS.,76 VEGF stimulates Akt-mediated eNOS phosphorylation and promotes nitric oxide (NO) release thereby participating in the regulation of vascular tension, angiogenesis, vascular remodeling, and maintenance of vascular homeostasis.,78 SRC regulates vascular endothelial cell proliferation and differentiation and is involved in growth factor-induced (including VEGF) vascular permeability, BBB destruction, and cerebral angiogenesis after ischemic stroke.,80 The PI3K/Akt pathway is involved in regulating the expression of angiogenic factors such as NO and angiopoietin. In addition, SRC mediates the PI3K/Akt signaling pathway to stimulate eNOS phosphorylation,,83 and eNOS catalyzes the synthesis of low levels of NO, thereby protecting the brain tissue.Neurorepair of VD is closely related to angiogenesis, which has been shown to mitigate the reduction in cerebral blood flow caused by different types of brain injury and to prevent and delay the occurrence of VD./in vitro experiments are still needed to further validate their mechanism of action. This study comprehensively elucidates the underlying pharmacological mechanism of GBLs in the treatment of VD and lays the foundation for further optimization of experimental design to obtain more reliable results.In this study, the molecular mechanisms of GBLs in VD treatment were investigated using network pharmacology, molecular docking, and molecular dynamics simulations. Quercetin, luteolin, kaempferol, and ginkgolide B are the key active ingredients of GBLs against VD and act on multiple signaling pathways, such as PI3K/Akt, lipid and atherosclerosis, fluid shear stress, and atherosclerosis through the regulation of AKT1, TNF, IL6, VEGFA, IL1B, and other targets, among which the PI3K/Akt signaling pathway seems to play a key role. These findings suggest that GBLs play a neuroprotective role by reducing apoptosis, alleviating inflammation, limiting oxidative stress, and promoting angiogenesis through multi-ingredient, multi-target, and multi-pathway interactions involved in the treatment of VD, which is generally consistent with the therapeutic pathways of VD reported in previous studies. Molecular docking and molecular dynamics simulations suggested that the interactions between AKT1–luteolin, AKT1–quercetin, and SRC–quercetin may be an important link in this mechanism. GBLs may serve as promising complementary and alternative drugs against VD; however, in vivoConceptualization: Jienuo Pan, Jiqin Tang.Data curation: Jienuo Pan, Jiqin Tang, Jialin Gai.Formal analysis: Jienuo Pan, Yilan Jin, Bingshun Tang.Funding acquisition: Jiqin Tang.Methodology: Jienuo Pan, Yilan Jin, Xiaohua Fan.Software: Jienuo Pan, Jiqin Tang.Supervision: Jiqin Tang, Xiaohua Fan.Visualization: Jienuo Pan, Jialin Gai, Bingshun Tang.Writing – original draft: Jienuo Pan, Jiqin Tang, Yilan Jin.Writing – review & editing: Jienuo Pan, Jiqin Tang, Jialin Gai, Yilan Jin, Bingshun Tang, Xiaohua Fan.


Polygonum capitatum Buch.-Ham. ex D. Don (THL) is a traditional vaccine that has antibacterial and anti-inflammatory effects. In this research, to investigate the mechanism of action of THL in the intervention of pulmonary fibrosis by network pharmacology and molecular docking related research methods, in order to provide a theoretical basis for expanding the scope of THL medication. A total of 49 active ingredients were analyzed and screened in Cephalus cephalusis, including 35 pulmonary fibrosis targets, and 10 key targets such as ALB, EGFR were screened after software analysis. The molecular docking results showed that there were 44 binding energies less than –3 kcal·mol−1 in the 60 docking results, indicating that most proteins had strong binding energies with compounds. The key targets of KEGG enrichment analysis were mainly enriched in 20 core action pathways, such as hemostasis-related pathway, regulation of kinase activity. This study shows that based on network pharmacology, the multicomponent–multitarget–multipathway effect of THL intervention in pulmonary fibrosis is discussed.Pulmonary fibrosis (PF) is a serious interstitial disease that includes diffuse collagen deposition of lung tissue.  At present, it is in the global pandemic of Corona Virus Disease 2019 (COVID-19). Epidemiological surveys show that severe patients are mostly accompanied by PF, so controlling the development process of PF is of great significance for treating the development of COVID-19.Pulmonary fibrosis (PF) is a serious interstitial disease. Its pathological characteristics include diffuse collagen deposition of lung tissue, poor elasticity, weakening of the regeneration ability of pulmonary epithelial cells, and the destruction and fusion of alveolar structure, leading to airway remodeling, reduced lung ventilation, and ventilation. It seriously affects the quality of life of patients, and shortens the survival time of patients. Patients generally have a mortality rate of 50% for 3 to 4 years after diagnosis, and the treatment options are limited. Nidanib, pyrimidine, and acid inhibitors are recommended in the diagnosis and treatment guidelines for lung fibrosis, but the above drugs have adverse reactions and are expensive.Polygonum capitatum Buch.-Ham. ex D. Don (THL) is a perennial herb of THL and the main ingredient of Relinqing Granules . Relinqing granules have the effects of clearing heat and diasipation and diuresis. Relevant clinical reports show that Relinqing granules have a significant effect on urinary tract infections and other diseases. Some basic studies also show that Relinqing granules have the effects of reducing the expression of inflammatory factors, inhibiting bacterial growth, and antioxidant damage. Modern pharmacological studies have found that THL has a spectrum of anti-inflammatory effects, which is mainly dependent on Toll-like receptor 4, Phosphoinositide-3-kinase,mitogen-activated protein kinases, and other signaling pathways, and is related to inhibiting the expression of inflammatory factors and reducing the phosphorylation of related proteins It is worth noting that THL contains a variety of active ingredients such as flavonoids, lignin, and volatile oil, which have a certain anti-inflammatory effect, but at present, there is a lack of research on the pharmacological mechanism of THL interfering lung fibrosis.,4,5 Since there is no mechanism and material basis for the treatment of lung fibrosis, this paper uses network pharmacological research methods to screen the chemically active substances, targets, and mechanisms of THL interfering with PF, to provide a scientific theoretical basis for the clinical application of the drug in the later stage.Network pharmacology research is a scientific theoretical method to analyze and predict the mechanism of drug-acting diseases by using network big data screening, bioinformatics analysis, and network visualization technology to analyze and predict drug-target-disease interaction networks.Through a large number of references, the chemical composition of THL is summarized, and the structure of each compound is drawn with ChemDraw software. Enter the structure of each compound drawn into the Swiss ADME database and specify that the criteria for selecting potential compounds are: GIabsortion is “High,” indicating that this ingredient has good oral bioavailability and is easy to absorb; the results of the 5 types of drug tests: Lipinski, Ghose, Veber, Egan, and Muegge have no less than 2 “yes.”Subsequently, according to the results of the literature, if the pharmacological effect of the compound is highly correlated with PF, it is still retained and the final potential compound is obtained. Then use the PharmMapper database based on the drug-effect group matching method to obtain the core action targets of different potential compounds, select the target protein of NF  ≥ 0.9, and combine with relevant literature reports to synthesize the core action target of active compounds.To ensure the correctness and comprehensiveness of the target data, using “Pulmonary Fibrosis,” “lung fibrosis,” “Idiopathic pulmonary fibrosis,” “IPF” as the terms related to PF were the key search terms, Search GeneCards database, DRUG BANK database, OMIM database, and Therapeutic Target Database to obtain the core targets of PF diseases. Among them, the DRUG BANK database is a collection of clinical test drug data, GeneCards, OM, and Therapeutic Target Database based on literature data construction, Therefore, we use different databases for disease target point collection and obtain more comprehensive target point information. The collected target information is re-merged, and the potential targets of disease targets with pharmaceutical chemicals are uniformly standardized as Gene Symbol using the UniProt protein database and then map the two to obtain the target of potential intervention of THL in lung fibrosis.Upload the obtained intersection target to the STRING 11.5 database, select the Organisms as “Homo sapiens,” set the confidence to 0.40, conduct protein–protein interaction (PPI) network relationship analysis, and import the results into CytoScape3.7. 2 Software. The software is used to analyze the interaction relationship between proteins and obtain the potential functions of the relevant proteins.P value of the statistical analysis results is set to P ≤ .01, and the top 20 GO enrichment analysis results and KEGG pathways were selected according to the LogP value.The Metascape database is used for bioinformatics enrichment analysis of core targets, including gene ontology (GO) analysis of biological processes, molecular function and cell composition, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The Synthesize the results of bioinformatics enrichment analysis obtained by “1.4” and drug diseases obtained by “1.2” intersect the target and introduce them into Cytoscape 3.7.2 software to build: drug composition-target-channel network diagram. CytoScape3.7.2 software was used to obtain the topological analysis results of the potential chemical composition and targets of THL, including betweenness and degree, etc. According to the network topological analysis results, the core target and the main activities of intervention in lung fibrosis were obtained ingredients.Selective analysis to obtain the medium value of THL composition-PF target network diagram ranked in the top 6. Download its pdb format through the RCSB database and convert the core compound to pdb format through Open Babel software. Proteins are dehydrated, liganded, hydrogenated, and stored in pdbqt format using pymol and Autodock Tools software. Treat and store potential compounds in pdbqt format by reducing energy and assigning ligand atomic types. Autodock software is used for molecular docking, and the binding energy Affinity is used as a reference for judging the binding activity.The current analysis does not require ethical approval, because our analysis only collects uploaded data information from the public database search.A total of 100 chemical structures related to THL were obtained from the relevant literature, and their main components were flavonoids, lignans and volatile oils, including terpenes, tannins and other components. Forty-nine active ingredients were screened by SwissADME, and the structure of the 10 most active ingredients is shown in Table A total of 5807 targets related to PF diseases were obtained in the Genecards database, and the result of Score ≥ 4.50 was selected as the potential disease targets of PF, and 1449 related disease targets were collected. Combined with the Therapeutic Target Database, DRUGBANK, and OMIM databases, and deduplication and merger, 1638 PF-related disease targets were collected. 114 targets of THL active ingredient were intersected with 1638 PF-related targets to obtain 35 intersection targets, and the results were plotted as VENNA  degree value is 39, the intermediate degree is 0.139, and the tightness is 0.4 20, and THL27 is predicted to be the main component of THL intervention in PF, followed by THL41 (hexadecanoic acid) connection degree of 33, intermediate degree of 0.225, tightness of 0.433, THL29  connection degree of 25, intermediate degree of 0.051, and tightness of 0.395 , ESR1, and Signal transducer and activator of transcription 3 (STAT3) obtained 60 groups of compound-protein docking results. Among them, there were 44 docking results of Affinity < -3 kcal·mol–8 Quercetin is one of the representative substances of THL flavonoids, and it has been found that quercetin can reduce the levels of malondialdehyde and hydroxyproline in mice with PF model, improve the total antioxidant capacity, regulate the expression of metalloproteinase-1 and then downregulate the expression of type III collagen, suggesting that quercetin has anti-fibrotic effects to a certain extent, and its mechanism may be related to the regulation of oxidative balance.,10 Inflammation can participate in the occurrence and development of various stages of fibrosis, and excessive inflammation will promote the proliferation of a large amount of fibrous tissue and collagen deposition, thus becoming an important factor in the formation of fibrosis. Other studies have found that quercetin can achieve therapeutic effects on PF by regulating the release of various inflammatory mediators in the NF-κB signaling pathway. Transforming growth factor β1 (TGF-β1) is an important factor in promoting collagen deposition in lung tissue and plays an important role in the process of PF, and quercetin can inhibit the TGF-β pathway lung to alleviate fibrosis, in addition, quercetin can also affect the SphK1/S1P pathway and the Smad/β-catenin pathway, and these pathways have an important regulatory role in the course of PF.The main active ingredient of THL exerting anti-inflammatory and antibacterial effects is its flavonoid components, and the flavonoids in traditional Chinese medicine have a certain anti-fibrotic effect. Rhodiolaside regulates oxidative stress responses in a variety of cells,,16 which not only destroys lung tissue structure and affects alveolar ventilation, but also induces apoptosis and activates related signaling pathways to stimulate PF. JAK2/STAT3 and PI3K/AKT/MTOR signaling pathways are mainly involved in cell proliferation and differentiation and metabolic regulation and play an important regulatory role in the occurrence and development of PF.,19 Salidroside can affect the expression of tyrosine-protein kinase JAK2 and STAT3 proteins, and regulate the PI3K/AKT/mTOR signaling pathway to achieve an intervention effect on fibrosis.,21 NACHT, LRR, and PYD domains-containing protein 3 inflammasome can induce the release of a variety of downstream inflammatory factors, promote inflammation, promote oxidative stress, and promote the occurrence of fibrosis. Luteolin is widely found in a variety of traditional Chinese medicines, with anti-inflammatory, antioxidant stress response and other effects,,23 luteolin can inhibit NACHT, LRR and PYD domains-containing protein 3 inflammasome activation, while reducing the level of interleukin-1 beta, Interleukin-18 thereby reducing the effect of PF. In addition, this study found that there are chemical components in THL that have not been reported to intervene in PF, such as cetanic acid, rosaceamine, etc., which can be further designed for experimental verification, and predict that this component may become a potential therapeutic direction for future research on THL intervention in PF.In addition, the main active components of salidroside and luteolin in the intervention of PF were preliminarily screened out by network pharmacological research methods. Rhodiola rosea is one of the active substances of the traditional Chinese medicine Rhodiola rosea, and in recent years it has been reported to have antioxidant, anti-inflammatory, anti-aging, anti-cancer, and other effects.−1, suggesting that this component may be the key component of THL in the treatment of PF.The molecular docking results showed that the top 10 THL compounds all had a strong binding activity with the main protein targets, and the binding energy of THL37 (catechin) compounds to 6 proteins was <−9 kcal·mol The EGFR family is mainly involved in gene transcription and transduction, protein translation expression, apoptosis, and other processes, and it has been found that EGFR can transmit fibrotic signals downstream after activation, and has an important regulatory role in the physiological process of PF. SRC kinase can promote the activation of TGF, and at the same time it can regulate the proliferation of fibroblasts, induce pneumonia and PF, and related studies have shown that reducing SRC levels can reduce the proliferation and differentiation of myofibroblasts, control the apoptosis of epithelial cells, and play an important role in the formation of PF. HSP is a chaperone protein, which can bind to specific proteins to regulate cell proliferation, differentiation, and apoptosis, and studies have shown that HSP90 inhibitors have the function of inducing apoptosis in HSC cells, which may be by inhibiting TGF-β signaling pathways and oxidative stress response to play an intervention role in PF.The results of network topology analysis showed that there were 49 active components of THL, corresponding to 114 targets, including 35 common targets related to PF. Through the construction of the PPI network, it was found that ALB, EGFR, SRC, HSP90AA1, STAT3, ESR1, etc. have large degrees, which may be the most critical targets for the treatment of PF of THL. ALB is a binding protein synthesized by the liver and involved in various biological regulatory pathways such as inflammation and oxidative stress, which is often used as one of the markers for the evaluation of pneumonia, and when ALB is at a normal level, it is conducive to inhibiting the occurrence and development of fibrosis.Through GO function enrichment analysis, it can be seen that the biological process of THL intervention in PF includes regulating lipid binding, regulating kinase activity, regulating phosphotransferase activity, etc., and through KEGG enrichment analysis, it can be seen that the signaling pathway of THL intervention in PF is mainly related to the regulation of nociception response and kinase activity, such as wound healing related signaling pathway, kinase activity regulation pathway, injury response related pathway, etc. Kinase activity plays an important role in the treatment of PF. The extracellular regulated protein kinases (ERK) signaling pathway is mainly regulated by the expression of multiple protein kinases such as ERK1 and ERK2, and studies have shown that the high expression of ERK1 and ERK2 will promote the level of collagen synthase and thus promote the fibrotic deposition of extracellular matrix, resulting in the occurrence of fibrosis. The serine/threonine kinase (AKT) and [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme are both protein kinases, and the combination of the two can induce AKT phosphorylation into the nucleus to regulate the expression level of related cell growth factors and promote the formation of fibrosis. Inhibition of ERK/AKT-related protein phosphorylation plays an important regulatory role in intervening in the occurrence and progression of PF. It was found that the abnormal expression of STAT kinase was closely related to lung injury and the formation of pulmonary inflammatory response, and its inhibition of inflammatory factors and Janus kinase (JAK) and STAT3 protein content was of great significance for improving the physiological state of lung interstitial, among which the JAK/STAT signaling pathway was mainly composed of tyrosine kinase JAK and STAT protein kinase, which combined to lead to PF.,34Hemostasis is an important pathophysiological element of PF, and in KEGG enrichment analysis, the Hemostasis signaling pathway is the signaling pathway with the most enriched genes, so it is speculated that it may be the key pathway for THL to intervene in PF. Patients with PF are often accompanied by pulmonary tissue bleeding, and endothelial and epithelial tissue damage will stimulate the activation of the coagulation response and promote the occurrence of PF when the patient’s wound heals.In summary, according to relevant experimental reports, the biological information pathways related to PF diseases are mainly related to nociception response and kinase regulation, suggesting that THL may intervene in PF by regulating JAK/STAT, Hemostasis, ERK, and other signaling pathways.,36 However, there are few studies on its treatment of PF disease. In this study, network pharmacology and molecular docking related techniques were used to preliminarily elucidate the mechanism of THL multi-component and multi-target joint regulation of PF through target prediction, protein interaction network, GO enrichment analysis, and KEGG pathway enrichment analysis. THL may act on biological targets such as ALB, EGFR, SRC, HSP90AA1, STAT3, EGF, and ESR1 through active ingredients such as quercetin, salidroside, luteolin, etc., and participate in the regulation of JAK/STAT, Hemostasis, ERK, and other signaling pathways to play a role in the treatment of PF. Based on scientific theories, this study speculates on the mechanism of THL intervention in PF, providing a theoretical reference for the discovery of new drug effects of THL. But there are still certain limitations in network pharmacology research, and the follow-up research team will continue to pay attention to the progress of THL pharmacological research. Mitochondria-targeted nano drug delivery systems are currently the focus of research, which can accurately deliver drugs and control drug release rate, and facilitate group modification and efficient loading of compounds. Our team will also conduct research based on green nanomaterials to target cells and mitochondria to explore the application value of targeted therapy of THL. In the future, our research team will continue to pay attention to the pharmacological research progress of THL and further explore the mechanism of THL in the intervention of PF combined with animal and cell experiments, to provide more effective treatment guidelines for the prevention and treatment of PF with traditional Chinese medicine and the diagnosis and treatment of COVID-19.Modern pharmacological studies have found that Polygonum viviparum has a spectrum of anti-inflammatory effects, which is mainly dependent on TLR4, Phosphatidylinositol 4,5-bisphosphate 3-kinase, mitogen-activated protein kinase and other signaling pathways, and is related to inhibiting the expression of inflammatory factors and reducing the phosphorylation of related proteins.Conceptualization: Xiang Pu, Te Jiang, Yihui Chai.Data curation: Lailai Li.Formal analysis: Xiang Pu.Funding acquisition: Jingwen Tang, Xiang Pu.Investigation: Liping Lu.Methodology: Lailai Li.Resources: Mei Pan.Software: Te Jiang.Validation: Liyan Zhang.Visualization: Zhiliang Fan, Yihui Chai.Writing – original draft: Zhiliang Fan.Writing – review & editing: Zhiliang Fan, Qian Li, Yihui Chai.