Compounds of HQHG were scanned by LC-MS/MS, and the target profiles of compounds were identified based on SwissTarget Prediction. ITP target proteins were collected from various databases. Then, KEGG pathway and GO enrichment analyses were performed to explore the signaling pathways related to HQHG for ITP. The PPI and drug-herbs-compounds-targets-pathways network were constructed using Cytoscape 3.7.2. Finally, Discovery studio software was used to confirm the key targets and active compounds from HQHG. β-D-glucoside, rutin, scopoletin, and tianshic acid are the most important active compounds, which were validated by molecular docking simulation.  A total of 187 interacting targets of 19 potentially active compounds in HQHG and 3837 ITP-related targets were collected. Then, 187 intersection targets were obtained. A total of 20 key targets including EGFR, CASP3, TNF, STAT3, and ERBB2 were identified through PPI network analysis. These targets were mainly focused on the biological processes of positive regulation of protein phosphorylation, cellular response to organonitrogen compound, and cellular response to nitrogen compound. 20 possible pathways of HQHG in the treatment of ITP were identified through KEGG enrichment. EGFR, CASP3, TNF, and STAT3 are the four most important target proteins, while adenosine, caffeic acid, ferulic acid, quercetin-3 This study demonstrated that HQHG produced relief effects against ITP by regulating multitargets and multipathways with multicompounds. And the combined data provide novel insight of drug developing for ITP. Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. The annual incidence rate of ITP in adults reported is 2 ~ 10)/100000, while the annual incidence rate in children is about 4 ~ 5/100000 0/100000,.Huaiqihuang Granule (HQHG), a Chinese patent medicine, is composed of Lycium barbarum, Polygonatum, and extracts of Trametes robiniophila, which could supplement the vital energy. It is applicable for children's and old people's physical weakness, such as repeated cold, dizziness, weariness, parched mouth, palpitation, hidrosis, loss of appetite, and constipation. Lycium barbarum mainly contains Lycium barbarum polysaccharide, Lycium barbarum pigment, flavonoids, alkaloids, amino acids, trace elements, and other components . PolygonIn this study, network pharmacology was conducted to establish the components-targets-pathways network to investigate the potential mechanisms of HQHG in the treatment of ITP  and veriHuaiqihuang Granules (Lot: KH23) was provided by Gaitianli Pharmaceutical Co., Ltd.HPLC grade methanol was purchased from Fisher Scientific (USA). Water was purified using a ULUPURE water system . Other chemicals were of analytical grade.μm microporous membrane. The active compounds of HQHG extracts were identified by Q Exactive Orbitrap LC-MS/MS (Thermo Fisher Scientific). The XBridgeBEHC18  was applied for sample separation at 35°C. Methanol (A) and 0.1% formic acid-water (B) (95 : 5) were used as mobile phase, and the solvent gradient was set as follows: 5% B, 0 min; 95% B, 40.0 min. Detection wavelength was set at 280 nm. Nitrogen was applied as auxiliary gas and sheath gas at a flow rate of 12 l/min. The mass determination was carried out based on positive and negative scanning mode with the m/z of 100-1000.HQHG (5 g) was mixed with methanol (10 ml), ultrasonic for 15 min, and the supernatant was filtrated and collected, then centrifuged at 3000 rpm for 5 min. The supernatant was filtered through a 0.22 http://www.swisstargetprediction.ch/) to predict the targets. The probability of targets greater than 0.6 was collected. Targets of these compounds were collected through high-throughput screening and reverse docking. The corresponding targets of Trametes robiniophila were collected from literatures and merged with other targets.All the chemical structures of the identified active compounds were imported into SwissTarget Prediction (https://www.disgenet.org/) [http://tcmspw.com/index.Php) [https://go.drugbank.com/) [https://db.idrblab.net/ttd/) [https://www.genecards.org) [https://omim.org/) [Keywords such as “immune thrombocytopenia,” “thrombocytopenia immunologic,” or “idiopathic thrombocytopenic purpura” were used to search the known targets related to ITP from some databases, including DisGeNET (et.org/) , TCMSP (dex.Php) , DrugBannk.com/) , Therapeet/ttd/) , GeneCarrds.org) , and OMIim.org/) . The cheProtein-protein interaction (PPI) networkhttps://string-db.org/) to obtain PPI network, with the species of human and a highest confidence of 0.9 [The intersection targets were imported into the STRING database (e of 0.9 . The PPIe of 0.9 . The keyhttps://metascape.org) [P value 0.05, minimum count of 3, and enrichment factor of >1.5) were displayed. The bubble chart of GO and KEGG enrichment analyses was performed on the bioinformatics platform (http://www.bioinformatics.com.cn/).The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed on the Database for Metascape (ape.org) . GO funcA network of drug-components-disease-targets-pathways was constructed to characterize the therapeutic mechanisms of HQHG for ITP. The nodes with different colors and shapes in the network represent the drug, components, disease, targets, or disease-related pathways, respectively, and the “edge” between the nodes shows the association.http://www.rcsb.org/) [According to the KEGG path analysis results, the related targets were screened from the rcsbpdb database (sb.org/) . Compounsb.org/)  and prepAs shown in As shown in The intersection targets were input into the STRING database, and the PPI network diagram were drawn by the Cytoscape software, as shown in p < 0.01). The size of the dot in bubble chart indicates the number of target genes in the corresponding function pathway, and the enrichment expresses the ratio of the number of target genes belonging to all the annotated genes located in the pathway. For BPs, the top 3 significant enrichment with the key targets were GO:0001934 positive regulation of protein phosphorylation, GO:0071417 cellular response to organonitrogen compound, and GO:1901699 cellular response to nitrogen compound. For CCs, the top 3 significant enrichment were GO:0045121 membrane raft, GO:0098857 membrane microdomain, and GO:0048471 perinuclear region of cytoplasm, while GO:0140272 exogenous protein binding, GO:0019900 kinase binding, and GO:0004672 protein kinase activity were the top 3 significant enrichments in MFs.The top 10 significantly enriched BPs, CCs, and MFs' terms were shown in p < 0.01) were picked out for further analysis based on the 20 key target genes, including hsa05205 Proteoglycans in cancer, hsa05200 Pathways in cancer, and hsa05417 Lipid and atherosclerosis as the top ones.The KEGG enrichment showed how HQHG acts on the pathway, thereby playing a therapeutic role in ITP. As shown in The drug-components-targets-pathways network was shown in β-D-glucoside. LibDockScore of the testing compounds was acquired to indicate the binding affinities with each target (β-D-glucoside showed good binding affinities with EGFR, CASP3, TNF, and STAT3. These findings revealed that rutin, tianshic acid, and quercetin-3β-D-glucoside could be forecasted as the active compounds of HQHG for ITP, while EGFR, CASP3, TNF, and STAT3 were the major targets for reaching this effect. The representative molecular docking results of the major targets and active compounds of HQHG were exhibited in A total of 5 target genes in “pathways in cancer” which showed strong interactions with other targets, namely, EGFR, CASP3, TNF, STAT3, and ERBB2, were selected to binding with active compounds in HQHG, including caffeic acid, tianshic acid, scopoletin, ferulic acid, adenosine, rutin, and quercetin-3h target . All thePrimary immune thrombocytopenia is an autoimmune bleeding disorder, and the risk of fatal hemorrhage of ITP is around 1.6–3.9% per year . The firγ elicited by viruses [α signaling pathway was reported that decreased HIF-1α may contribute to impaired megakaryopoiesis in ITP and may provide a potential therapy for ITP patients [α blockade decreased antibody-mediated platelet destruction and may be a promising therapeutic strategy for the management of ITP [α therapy reduced the number of nonclassical monocytes and M1 macrophages, ameliorated the retention of platelets in spleen and liver, and increased the platelet count of ITP mice [A total of 20 key genes were screened in this study as the most important ones that contribute to ITP treatment of HQHG. These potential target genes were placed in the Metascape for KEGG pathway analysis, and 20 significant pathways that may be regulated by HQHG in the treatment of ITP were identified, including pathways in cancer, lipid and atherosclerosis, endocrine resistance, human cytomegalovirus infection, hepatitis B, microRNAs in cancer, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, and TNF signaling pathway. Interestingly, the relationships between some pathways and ITP were also confirmed in other studies –35. It h viruses . Human c viruses , 38. Pat viruses . HIF-1α patients . TNF-α bt of ITP . Anti-TNITP mice . To actiITP mice –42.β, and NF-κB in the inflammatory response [α action [α signaling pathway, resulting in remarkable attenuation of antibody-mediated platelet destruction [In this study, rutin showed the highest LibDockScores and forms many close interactions including conventional hydrogen bonds, Pi-Sulfur, Pi-alkyl, and carbon hydrogen bond to the residues of EGFR, CASP3, TNF, and STAT3, which indicated that rutin in HQHG plays an important role in the treatment of ITP. Rutin has been exploited for its potential role in inhibiting cancer cell growth and metastasis. It is proved that rutin could modulate STAT signaling via the repression of SRC phosphorylation and block the translocation of STAT to the nucleus  and furtresponse , drasticα action . A cliniα action . Other aα action , 50. It truction .This study investigated the intricate mechanism of HQHG treatment for ITP by utilizing an integrated network pharmacological and pharmacokinetics strategy. These novel findings provide a rational pharmacological basis and support for treating ITP and may help to expand the use of this formula in the treatment of ITP. However, several limitations should be noted in this study. Firstly, large molecular compounds such as polysaccharides and proteins in HQHG were not included in this study, and it still needs to focus on further research. Secondly, the underlying mechanism of HQHG against ITP involves multiple pharmacological actions, and more research is needed to reveal complex synergistic mechanisms by combining high-throughput detection technologies such as transcriptome, proteome, and metabolome. Moreover, the contribution of the active ingredients to efficacy and the synergistic effect among the active ingredients were not explained in our research, and we will conduct research in this area in the future to more fully interpret the modern theory of TCM efficacy.α signaling pathway were observed to play an important role in the mechanism of HQHG for ITP treatment. In addition, EGFR, CASP3, TNF, and STAT3 were speculated to be the most important target proteins. Network pharmacology results showed a close relationship between the TNF-α pathway and ITP, while HQHG is capable of blocking the TNF-α pathway. This study provided novel indications for further mechanism research of HQHG in the treatment of ITP. However, more research is necessary to make the results available.Collectively, 19 active compounds were identified from HQHG, and 187 targets of active compounds in addition to 3837 ITP-related targets were collected. A total of 20 key targets were screened out in PPI analysis, and signaling pathways including pathways in cancer, lipid and atherosclerosis, endocrine resistance, human cytomegalovirus infection, hepatitis B, microRNAs in cancer, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, and TNF-


C. elegans and found that GS-Rd prolongs lifespan, improves resistance to heat stress, delays physical paralysis and increases oxidative stress responses. Overall, these results suggest that GS-Rd protects against the toxicity of Aβ. The RNA-seq analysis revealed that GS-Rd achieves its effects by regulating gene expressions like daf-16 and skn-1, as well as by participating in many AD-related pathways like the MAPK signaling pathway. In addition, in CL4176 worms, GS-Rd decreased reactive oxygen species (ROS) levels and increased SOD activity. Additional research with transgenic worms showed that GS-Rd aided in the movement of DAF-16 from the cytoplasm to the nucleus. Taken together, the results indicate that GS-Rd significantly reduces Aβ aggregation by targeting the MAPK signal pathway, induces nuclear translocation of DAF-16 to activate downstream signaling pathways and increases resistance to oxidative stress in C. elegans to protect against Aβ-induced toxicity.Alzheimer’s disease (AD) is a serious public health issue but few drugs are currently available for the disease, and these only target the symptoms. It is well established that oxidative stress plays a crucial role in AD, and there is compelling evidence linking oxidative stress to β-amyloid (Aβ). An exciting source of potential new AD therapeutic medication possibilities is medicinal plants. Ginsenoside Rd (GS-Rd) is one of the main bioactive substances in ginseng extracts. In our study, we used a network pharmacology analysis to identify overlapping GS-Rd (therapeutic) and AD (disease)-relevant protein targets, gene ontology (GO) and bio-process annotation, and the KEGG pathway analysis data predicted that GS-Rd impacts multiple targets and pathways, such as the MAPK signal pathway and the JAT-STAT3 signaling pathway. We then assessed the role of GS-Rd in  Alzheimer’s disease (AD) is a common clinical neurodegenerative disease characterized by progressive cognitive impairment and memory loss . The mosvia the regulation of B cell lymphoma 2 (Bcl-2), BCL-2-like protein four and caspase-3 . GS-Rd (ginsenoside Rd) is one of its monomer components, the chemical structure of GS-Rd is shown in aspase-3 . The neuaspase-3 . Furtheraspase-3 . Furtheraspase-3 , Ling Liaspase-3  demonstrIn the past 10 years, the emerging discipline of network pharmacology has been widely used in the discovery of drugs and active compounds in traditional Chinese medicine. Network pharmacology has also provided new scientific and technological support for the research and development of new drugs . To explCaenorhabditis elegans (C.elegans) AD model to further clarify the pharmacological effects and mechanisms in the GS-Rd treatment of AD. Due to its short lifespan, 42% of its genes being related to human genes, and its possessing an intact nervous system, C. elegans is frequently used as a model for fundamental biology research .Based on these analyses, we utilized the research . The traaralyzed . This searalyzed  demonstraralyzed . To our Caenorhabditis Genetic Center (CGC). All worms were propagated at 16 °C except TJ356, which was propagated at 20 °C on nematode growth medium (NGM) and seeded with the standard food resource of E. coli OP50.Ginsenoside Rd (with a purity >98%) was purchased from Chengdu Must Biotechnology, A stock solution of GS-Rd (50 mM) was prepared with DMSO . The following strains were used in this work: CL4176 (dvIs27 [myo-3p:Aβ (1–42):let-851 30 UTR) + rol-6 (su1006)]); CL 2006 (dvIs2 [pCL12 (unc-54/human Aβ peptide 1–42 minigene) + pRF4]) and TJ356 (zls356 ; the heatmap of differential gene clusters is shown in Using RNA-Seq, it was possible to pinpoint the likely causes of the GS-Rd-treated CL4176 worms’ lower paralysis rate by identifying the altered gene expression and signaling networks. As shown in https://metacpan.org/pod/GO::TermFinder) to identify the gene networks that were regulated in GS-Rd-treated CL4176. The aggregate of highly differentially expressed genes was clustered into the functions of DNA binding and kinase activity in molecular functions, transcription regulation process in biological process, cytoplasm and the nucleus in cell components  . To asce results . TherefoSince Aβ aggregation causes oxidative stress , increasThis study was conducted to explore the potential mechanism of ginsenoside Rd for the treatment of Alzheimer’s disease with the help of network pharmacology. We found that GS-Rd might perform an anti-AD effect by down-regulating the expression level of Aβ mRNA level based on the CL4176 worm experiment. 311 AD-related proteins were identified as prospective GS-Rd targets from the relevant database and the top 10 core targets were identified using topological analysis and linked to 136 signaling pathways, many of which were connected to the development of cancer, for example, the JAK-STAT signaling pathway, the MAPK signal pathway and other pathways associated with cancer. 311 potential targets exert therapeutic effects on AD through 29 biological processes, 11 molecular functions and 15 cellular compositions, including immune system processes, cell proliferation, transcriptional regulation, antioxidant activity, cells and membranes. It is suggested that GS-Rd is a multi-pathway and multi-target treatment for AD.via stimulating estrogen-like activity. However, there are few reports of GS-Rd preventing Alzheimer’s disease by inhibiting Aβ deposition, and the molecular mechanism of treatment is still poorly understood, therefore, we empirically validated the hypothesis that was generated by network pharmacology regarding the potential mechanism of GS-Rd treatment of AD, providing a foundation for further studies. Using the CL4176 transgenic nematode model, we confirmed the results predicted by network pharmacology using the CL4176 transgenic AD model. The frequency of paralyzed worms in the GS-Rd-treated group was considerably lower than that in the control group, demonstrating that GS-Rd might delay the paralysis of CL4176 worms caused by Aβ deposition. The expression of Aβ mRNA was significantly down-regulated in worms that were treated with different concentrations of GS-Rd, indicating that GS-Rd was able to reduce Aβ-induced toxicity. In the meantime, we performed RNA-Seq assays on GS-Rd-treated CL4176 nematodes and showed that GS-Rd was able to regulate the expression of a variety of genes, including 94 up-regulated genes and 203 down-regulated genes. GO analysis of the genes with more than a 2-fold difference in expression revealed that these genes were mainly involved in the regulation of DNA transcription and enzymatic activity, and KEGG analysis showed that these significantly altered genes were concentrated in the MAPK signal pathway, which is closely related to AD and various cancer-related signal pathways. These results were highly consistent with the results predicted by network pharmacology.Alzheimer’s disease is the leading cause of dementia and is rapidly becoming one of the world’s most costly and fatal diseases. . Extracedaf-16 and skn-1 in CL4176 nematodes by suppressing the MAPK signaling pathway and reducing the ROS content and SOD-3 content in CL4176. According to data from fluorescence microscopy, GS-Rd encourages DAF-16 entrance into the nucleus to stimulate the expression of a number of genes.Mitogen-activated protein kinase (MAPK) is a group of highly conserved serine/threonine proteases in eukaryotes that regulate cell proliferation, differentiation, growth and apoptosis . PreviouUsing network pharmacology analysis, we identified 311 protein targets common to both GS-Rd and AD and certain targets of GS-Rd , which are known to be important in AD biology . Some hiIn conclusion, this study found that GS-Rd regulates APP transcription through the MAPK pathway to reduce Aβ mRNA levels, thereby reducing the production of Aβ and ROS in AD and providing a new target for the treatment of AD. According to the results discussed above, GS-Rd can operate as a neuroprotective agent by reducing the neurotoxic effects of Aβ through anti-oxidative stress. This suggests that GS-Rd may be a novel target for the treatment of AD. It should be acknowledged that further studies are needed to determine how the MAPK signaling pathway has been altered.


A 49-year-old man sustained an accidental injury when he was changing and inflating the tyre of a truck, and there was a sudden explosion of the truck tyre at the service station, which was by the roadside of the highway. With the pressure of air generated due to the exploding tyre, the victim was blown to around 6 feet away. He was declared dead on admission. The medico-legal examination was conducted, and death was determined to be multiple organ injuries  caused by the shock wave produced due to tyre explosion. Tyre blast injuries are not so common. A meticulous post-mortem examination is fundamental in formulating and recording the pattern of traumatic injuries. Preventive occupational measures should be put in place. Tyre burst injuries are relatively rare but are known for causing severe injury patterns. Tyres can be thought of as air tanks, but the same safety rules that apply to pressure tanks do not apply to tyres. Blast overpressure is described as an increase in pressure above atmospheric values, as witnessed in a blast of explosive weapons. Air compression before the blast wave accelerates air molecules and generates a pressure peak . This hiThe tyre explosion has a high potential for destruction, yet it has garnered little attention in the medical literature,  commonlyA 49-years-old man sustained an accidental injury on the 31st of December when he was changing and inflating the tyre of a truck and there was a sudden explosion of the truck tyre at the service station which was by the roadside of the highway. With the pressure of air generated due to the exploding tyre, the victim was blown to around 6 feet away. He was taken to the hospital in an unconscious state and was declared brought dead on admission at the All-India Institute of Medical Sciences in Delhi in the emergency department. The police were intimated and accordingly, the case was registered as a medicolegal case. Inquest papers were prepared by the Investigating Officer and the body was shifted to the Morgue associate with the Forensic Medicine and Toxicology Department at AIIMS, Delhi for medico-legal post-mortem examination.The Incident occur at about 4:00 PM on 31st Dec. Postmortem conducted at 12:30 PM and concluded at 1:45 PM on 1st January. A detailed complete and meticulous post-mortem examination was performed, rigor mortis all over the body in the supine position, cadaveric lividity on the back, dependant parts of the body. The lividity was fixed. There was the presence of clotted blood and oozing on left ear ossicles  in exterOn internal examination, subarachnoid haemorrhage with underlying brain parenchyma, contusion in patches were present on both cerebral hemisphere , the cheThe situations surrounding the incident, as well as the location of injuries anatomically, suggested that a middle-aged mechanic was injured accidentally as a result of a tyre explosion while changing and inflating at a service station along the highway. Because such work-related unintentional injuries are underreported, the forensic literature may be deficient in this regard. Multiple organ injuries  caused by the shock wave produced due to tyre explosion was concluded as the cause of death. An inflated tyre contains a high amount of potential energy.A 63000 ft-lb of energy is released when a truck tyre inflated to 90 psi explodes. The estimated force is around 2000 g, which is enough to raise a 3000 lb car 21 ft off the ground . The blaIn literature there have been instances in certain cases wherein a tyre burst did not result in a fatality, however, reports of cases with fatal incidents have been reported by certain authors. Fatal pneumatically induced injuries are rare in forensic literature; high-energy released due to blast of tyre results in fatal injuries .Deforming injuries and fatalities caused by explosions among mechanics servicing trucks, in the existing literature, it’s only been mentioned a few times. The size of the tyre’s air pressure and the distance between the tyre and the victim at the moment of the blast influence the severity of the injury. Explosions are common during tyre servicing, particularly when inflating the tyres; bystanders are also injured and may sustain fatal injuries , 22.A tyre burst can happen while servicing or inflating tyres. High inflating pressure and a short safety distance are the two most common causes of fatal injuries. The high energy produced by a large tyre burst can result in severe injuries with high morbidity and mortality. A meticulous post-mortem examination is fundamental in formulating and recording the pattern of traumatic injuries. Preventive occupational measures should be put in place.


Gynostemma pentaphyllum (G. pentaphyllum), a traditional medicinal material with a high medicinal and health value, has recently been reported for its anticancer activity. However, the pharmacological mechanism of G. pentaphyllum in non-small-cell lung cancer (NSCLC) remains to be elucidated.  Lung cancer represents the leading cause of cancer death on a global scale. G. pentaphyllum were obtained from the TCMSP database and known therapeutic targets of NSCLC from the GeneCards and OMIM databases. Disease-drug common targets are subjected to protein-protein interaction (PPI), GO enrichment analysis, and KEGG pathway enrichment analysis. A molecular docking strategy was performed to verify the interaction between molecules.  The active ingredients of G. pentaphyllum fulfilling OB ≥ 30% concomitant with DL ≥ 0.18 and corresponding 81 target genes in the TCMSP database, with 5062 NSCLC-related genes collected in the GeneCards and OMIM databases. The network consisting of the disease-target compound was obtained, including 8 active ingredients and 69 common targets. The PPI network with 65 nodes and 645 edges was visualized. After functional enrichment analysis, it was revealed that the therapeutic effects of G. pentaphyllum on NSCLC were achieved through response to ketone, gland development, and cellular response to xenobiotic stimulus. After molecular docking analysis, it was revealed that the two active ingredients of G. pentaphyllum, quercetin and rhamnazin, bound well and stably to their targets .  We found a total of 24 compounds of G. pentaphyllum when it is used to treat NSCLC. Our study, based on network pharmacology, identifies active ingredients, targets, and pathways model mechanism of  In spite of remarkable progress in understanding of pathogenesis, application of predictive biomarkers, immunologic control, and therapeutic strategies for lung cancer in the past two decades, lung cancer still contributes to a heavy global burden of cancer mortality and morbidity, with an estimated 2 million new diagnoses and 1.76 million deaths each year . When paGynostemma pentaphyllum (G. pentaphyllum) is a creeping perennial herb that is sourced from the family Cucurbitaceae, which is widely used as a herbal medicine and distributed in Asian regions, especially in China [G. pentaphyllum was first recorded as Traditional Chinese Medicine (TMC) in Jiuhuang Bencao, written by Zhu Su, who was a Chinese botanist in AD 1406 [G. pentaphyllum have been widely reported.Emerging studies with experimental models have proved therapeutic effects of herbal medicines on several common human cancers including NSCLC , 8. Gynoin China . G. pent AD 1406 . The ant AD 1406 , antiobe AD 1406 , antioxi AD 1406 , and antG. pentaphyllum for NSCLC based on network pharmacology followed by molecular docking analysis.TMC has several characteristics, such as multicomponent and multitarget synergistic effects, while it is difficult to systematically and comprehensively detect the exact mechanism of TMC through traditional methods. Network pharmacology is a novel approach that was proposed by Hopkins in 2008 to study the molecular mechanisms of drug action, for example, natural herbs and TCM, by establishing computer-aided networks on the basis of “multigene,” “multitarget,” and “multichannel” linking with multiple compounds . In addiG. pentaphyllum, we performed a computer-based retrieval in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform , which is a popular pharmacology database where there are almost 500 formulae of TCM concomitant with 30,069 compounds. Two pharmacokinetic parameters related to ADME , oral bioavailability (OB), and drug-likeness (DL), were evaluated to retain the active ingredients of G. pentaphyllum for further investigation. In this study, the active ingredients of G. pentaphyllum must fulfill OB ≥ 30% and DL ≥ 0.18. The protein targets of the active ingredients were also predicted in the TCMSP database, with the gene names obtained from the UniProt Knowledgebase (UniProtKB), which collects functional information on proteins covering accurate, consistent, and rich annotation.To obtain the active ingredients of https://omim.org/). The proteins (only “Homo sapiens”) associated with NSCLC were chosen. Using the Venn functional intersection in the R software, overlapping targets of G. pentaphyllum and NSCLC were obtained.The known therapeutic targets of NSCLC were dug up in the GeneCards combined database with the Online Mendelian Inheritance in Man database  to estimate their PPIs. The species should be “Homo sapiens” and the confidence score should be no less than 0.4. The PPI network was visualized by importing the TSV-based file to Cytoscape software (3.8.1). According to the degree value obtained using the cytoHubba plug-in of Cytoscape, the key genes were extracted.The common targets of G. pentaphyllum and NSCLC were subject to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the “clusterProfiler” package in R/Bioconductor. We mapped them into the DAVID Bioinformatics Resources 6.7 (https://david-d.ncifcrf.gov/), with the species set as “Homo sapiens” and P value < 0.05 set as a cutoff value. Results of GO analysis were visualized using the OmicShare platform (https://www.omicshare.com/), focusing on three levels: biological process (BP) analysis, cellular component (CC) analysis, and molecular function (MF) analysis.The disease-drug overlapping targets of https://www.rcsb.org/). The molecular docking verification was performed on the Systems Dock Website (https://systemsdock.unit.oist.jp/iddp/home/index) to evaluate the binding strength and activity between those protein ligand complexes and the active ingredients of G. pentaphyllum, with core chemical compounds sorted out.The protein ligand complexes of targets with high-ranking degree values in the PPI network were obtained using the RCSB database ,24(28)-trien-3β-ol, cucurbita-5,24-dienol, cyclobuxine, and 10 from gypenoside.When searching the active ingredients of aphyllum , includiG. pentaphyllum in the TCMSP database. Subsequently, we searched for known therapeutic targets of NSCLC in authoritative open databases and obtained 4861 and 201 targets in the GeneCards and OMIM databases, respectively. After the removal of duplicate values, 4970 therapeutic targets of NSCLC were obtained. Venn intersection analysis by R software showed there were 69 overlapping target genes of NSCLC and G. pentaphyllum (3′-methyleriodictyol (1 node), rhamnazin (9 nodes), sitosterol (2 nodes), spinasterol (16 nodes), campesterol (1 node), isofucosterol (16 nodes), CLR (2 nodes), and quercetin (107 nodes).After removal of duplicate values and conversion of protein names into gene symbols, we obtained 81 target genes of the active ingredients of aphyllum . We thenaphyllum . The 8 bG. pentaphyllum into the STRING database. As shown by We imported 69 common targets between NSCLC and the active ingredients of p < 0.05). At the level of BP, the active ingredients of G. pentaphyllum were associated with 1106 terms. At the level of CC, the active ingredients of G. pentaphyllum were associated with 11 terms. At the level of MF, the active ingredients of G. pentaphyllum were associated with 94 terms. After KEGG pathway analysis, we found 11 KEGG pathways were significantly enriched by these disease-drug common targets (p < 0.05).Next, we conducted GO annotation and KEGG pathway analyses of 69 disease-drug common targets. After GO analysis, we found 1211 GO terms were significantly enriched by these disease-drug common targets  within the human body and that “invigorating Qi for consolidation of the exterior” is an effective way to treat lung cancer . With thg cancer –20. NeveG. pentaphyllum stood out, which may responsible for the leading therapeutic effects of G. pentaphyllum on NSCLC, including 3′-methyleriodictyol, rhamnazin, sitosterol, spinasterol, campesterol, isofucosterol, CLR, and quercetin. Quercetin ranked the highest with 107 targets of NSCLC, followed by the other three bioactive compounds, isofucosterol, spinasterol, and rhamnazin. Quercetin has a variety of biological properties, such as antioxidant, anti-inflammatory, and antiapoptosis [quercetin by biotinylated mixed micelles and demonstrated a high accumulation of quercetin-loaded mixed micelles at the tumor site and showed good anticancer activity in the mouse model of NSCLC [rhamnazin may exert a therapeutic effect on pulmonary fibrosis by alleviating inflammation, oxidation, and collagen deposition via the TGF-β/Smad axis [rhamnazin were also proved in hepatocellular carcinoma [spinasterol might have activity against cancer cells in triple-negative breast cancer [spinasterol [isofucosterol is still rarely reported for its anti-obesity effect rather than its antitumor effect [In the system of TCM, compounds fail to be delivered to the target organs to produce biological activities due to a lack of proper pharmacokinetic properties . The netpoptosis –24, whicpoptosis . More prof NSCLC . The antmad axis . The antarcinoma . Wahyunit cancer . Ravikumnasterol . Howeverr effect .G. pentaphyllum, referring to quercetin and MYC, rhamnazin and ESR1, and quercetin and HIF1A. c-Myc regulates multiple genes, which are associated with cell proliferation in many cancers, including NSCLC [quercetin notably repressed cancer cell proliferation by downregulating c-Myc expression in pancreatic ductal adenocarcinoma [quercetin could block the Akt/mTOR/c-Myc axis to inhibit the epithelial-mesenchymal transition of cancer cells [Quercetin induces mesenchymal-to-epithelial transition by changing the nuclear localization of β-catenin and regulating β-catenin target genes, including c-Myc in triple-negative breast cancer [quercetin and HIF1A. The overexpression of HIF1A was found to predict poor survival in lung cancer [quercetin could enhance the cytotoxic activity of gemcitabine or doxorubicin on cancer cells by inhibiting HIF1A expression [quercetin to modulate glucose uptake/metabolism by affecting the stability of HIF1α [rhamnazin and ESR1 interaction in experimental models of lung cancer cells is not investigated in reported studies, which may be required for further profound functional validation. The importance of network pharmacology has been emphasized in medical research [Three candidate targets with higher degree values in the core PPI network, MYC, ESR1, and HIF1A, were subjected to further molecular docking and analysis, and it was revealed that the three core targets had good affinity with the active compounds of ng NSCLC . Guo et arcinoma . Chen eter cells . Quercett cancer . In addig cancer . Hassan pression . Tumova of HIF1α . Fulvestof HIF1α , suggestresearch .G. pentaphyllum treats NSCLC is not completely characterized as the public databases we used in the study have been updated continuously. Second, only three key genes received molecular docking and a lack of functional studies may weaken the reliability of the clinical translation. Third, experimental validation in vivo and in vitro focusing on the suppressive effects of two potential active compounds of G. pentaphyllum, quercetin and rhamnazin, on NSCLC cells as well as the expressions of MYC, ESR1, and HIF1A is warranted to improve the preliminary nature of the study.When interpreting our results, several limitations should be noted. First, the molecular mechanism by which G. pentaphyllum, especially its main active compounds, quercetin and rhamnazin, may exert therapeutic effects on NSCLC through the modulation of multiple targets, such as MYC, ESR1, and HIF1A. The present work also supports that the network pharmacology prediction method with molecular docking verification may provide a preliminary but systemic exploration focusing on the pharmacokinetic properties and mechanism of TCM in human diseases, offering opportunities to develop micelles for targeted delivery of TCM to tumor sites.In conclusion, our study demonstrates that


Prunella vulgaris L. is effective in the treatment of breast cancer (BRCA); however, the underlying mechanism is still unclear. The aim of this study was to elucidate the mechanism of treatment of BRCA by P. vulgaris using network pharmacology and molecular docking technology, and to verify the experimental results using human BRCA MDA-MB-231 cells. P. vulgaris were determined using the TCMSP™, SwissTarget Prediction™, and TargetNet™ databases. GeneCards™ and OMIM™ provided BRCA targets. After obtaining common targets, a protein-protein interaction (PPI) network was constructed using the STRING™ database, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the Xiantao™ academic database. Cytoscape™ was used to construct “single drug-disease-component-target” and “single drug-disease-component-target-pathway” networks. The Human Protein Atlas™ was used to determine protein expression levels in BRCA cell lines. AutoDock tools™ were used to carry out molecular docking for the first 10 targets of quercetin and the PPI network. Finally, the abovementioned results were verified using cell experiments.  Active components and action targets of P. vulgaris. The results of molecular docking showed that the first 10 targets of quercetin interacted well with the protein network. Cell experiments showed that quercetin effectively inhibited the proliferation of MDA-MB-231 cells by regulating apoptosis and cell cycle, which may be partly related to the MAPK signaling pathway.  We obtained 11 active components, 198 targets, and 179 common targets, including DUOX2, MET, TOP2A, and ERBB3. The results of KEGG pathway analysis screened 188 related signaling pathways and indicated the potential key role of PI3K-Akt and MAPK signaling pathways in the antibreast cancer process of P. vulgaris could provide a theoretical basis for further study on its complex anti-BRCA mechanism. Synergistic effects of multiple components, targets, and pathways on the anti-BRCA activity of  In recent years, the incidence rate of breast cancer (BRCA) has been increasing worldwide, at a rate of 3% per year . TherefoPrunella vulgaris L, a perennial herb in the family Lamiaceae, has a medicinal and edible homology. It has a medicinal history of thousands of years and was first mentioned in the Shennong herbal classic. Its Chinese name means “grass will wither after the summer solstice.” Prunella vulgaris is bitter, spicy, and cold, and it acts along the liver and gallbladder meridians. It can clear the liver and dissipate fire, clear eyesight, dissipate nodules, and disperse swelling [P. vulgaris have various pharmacological effects, including antitumor, antibacterial, anti-inflammatory, immunosuppressive, free radical scavenging, antioxidative, and antiviral properties [P. vulgaris and its extract, its related pharmacological effects are being gradually clarified, and clinical research on it is becoming increasingly extensive.The dried ear of swelling , pain, pswelling –11. Modeoperties –14. WithP. vulgaris has been shown to be clinically effective in the treatment of BRCA in a vast number of studies, its underlying mechanism is unclear. This study aimed to predict the potential components, targets, and molecular pathways of P. vulgaris against BRCA based on network pharmacology. Furthermore, we used molecular docking and cell experiments to verify and provide a theoretical basis for the potential mechanism  were used to get self-healing active ingredients and potential targets [Prunella vulgaris L” as the retrieval word, oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 are assumed as the screening conditions, and the eligible active ingredients are collected [Prunella subtilis. Using the UniProt database (https://www.uniprot.org/) [Homo sapiens,” and the gene names corresponding to the targets of the active ingredients of Prunella subtilis were collected. From the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) [https://www.swisstargetprediction.ch/) [Homo sapiens, and potential acquisition targets. Then, using the UniProt specification TargetNet of targets, the chemical composition of the canonical SMILES number was entered into TargetNet (https://targetnet.scbdd.com/), prob >0 genes were selected, and used the UniProt specification TargetNet of targets. The targets of the three databases were merged and the duplicate targets were deleted.The TCMSP database and analysis platform ( targets . Taking ollected . The corih.gov/) , in chemion.ch/) , the chohttps://www.genecards.org/) (correlation score ≥1.0) [https://www.omim.org/) [Using the GeneCards database (re ≥1.0)  and the im.org/) , using “P. vulgaris and BRCA were obtained from the Xiantao academic database (https://www.xiantao.love), and the Wayne map was drawn using the Venny plate. The “single-substance drug-disease-component-target” network was constructed using Cytoscape 3.7.1 software.The common targets of https://string-db.org/), the PPI network was constructed. The target points of P. vulgaris and BRCA obtained in previous step were introduced into the STRING database, the species “Homo sapiens” was defined, and the confidence level was >0.4. The light blue line represents the interprotein interaction from the database and the purple line represents the protein intercropping verified by experiments. GraphPad prism 7.0 was used to display the adjacent number of the top 35 proteins in the PPI network.Using STRING database . The top 10 biological processes, molecular functions, cellular components, and KEGG pathways were selected (P < 0.05), and the results were visualized by GraphPad prism 7.0. Then, the experimental data such as active ingredients, targets, and the top 10 KEGG pathways were imported into Cytoscape 3.7.1 software to construct a “single-substance drug-disease-component-target-pathway” network.To further study the molecular mechanism of P. vulgaris, was selected for molecular docking with the top 10 targets in the PPI network. The 2D structure of quercetin was downloaded using the PubChem database (https://www.ncbi.nlm.nih.gov/Pccompound/) and saved in an SDF format file. The SDF format file of quercetin was modified by ChemBio3D software to minimize its structural energy and save it in a mol format. From the PDB database (https://www.pdb.org/), the crystal structure of the target was downloaded and PyMOL software was used for pretreatment, including water molecule removal and hydrogenation. Then, the 3D structure and target crystal structure of quercetin were converted into PDBQT format files using Autodock Tools, and the molecular docking was carried out by AutoDock Vina software. The binding activity between quercetin and the target was evaluated by binding energy. Finally, PyMOL software was used to visualize the docking file.For further verification, quercetin, a key active component of P. vulgaris in the treatment of BRCA, the selected important pathways were input into the KEGG database (https://www.kegg.jp/). The role of the screened target in this pathway is queried and displayed in the human protein map database (https://www.proteinatlas.org/). The final verification of the screened target genes is carried out to verify whether they are enriched in BRCA tissues and cells, so as to provide a sufficient theoretical basis for future studies.To further verify the important role of the screened pathways and targets in Human BRCA MDA-MB-231 cells were purchased from Shanghai Fu Heng Biotechnology Co., Ltd., and quercetin (purity >97%) was purchased from Shanghai McLean Biological Co., Ltd.  Fetal bovine serum©   L15 Medium©   3--2,5-diphenyltetrazolium bromide (MTT)©   ERBB3 polyclonal antibody©   MET polyclonal antibody©   Cleaved poly ADP-ribose polymerase© (Cleaved PARP)   Cleaved caspase-3©   p38 polyclonal antibody©   Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody©   Secondary antibody sheep antirabbit IgG©   Bicinchoninic acid (BCA) protein detection kit©   Emitter coupled logic (ECL) chemiluminescence kit©   Pennexin V apoptosis detection kit I cell apoptosis kit©   Cell cycle detection kit©   ELX800 chemiluminescence enzyme labeling instrument®   DYCZ-24DN double vertical electrophoresis instrument®   DYCZ-40D transfer electrophoresis instrument®   Clinxchemiscope 6000 chemiluminescence instrument®   Facscantoii flow cytometry® 4 well−1), 5 multiple wells in each group. After 12 h of cell adhesion, 0, 10, 20, 30, 40, and 50 μg/mL were added, respectively. Quercetin was treated for 24 or 48 h, respectively, and the cell morphology was observed. After treatment with quercetin for 24 or 48 h, a 5 mg/mL MTT solution was added to each well and incubated in the cell incubator for 4 h. Carefully suck and discard the culture supernatant in the well, add 150 μL of dimethyl sulfoxide to each well, and oscillate at low speed in the dark at room temperature for 10 min to fully dissolve the crystal. The optical density (OD) of each well was measured by using a microplate reader at 570 nm. The relative cell viability and cell inhibition rate were calculated with reference to the OD of the blank control group.The effect of quercetin on the proliferation of human BRCA MDA-MB-231 cells was detected using the MTT method. Logarithmically grown MDA-MB-231 cells were inoculated into 96-well plates (5 × 104/well). MDA-MB-231 cells were treated with quercetin at different concentrations for 24 h, digested with trypsin, and collected. Cell cycle analysis was carried out. After the abovementioned treatment, the cells were centrifuged at 1500 rpm for 5 min. After that, 1 mL of phosphate buffered saline (PBS) was added for cleaning and centrifugation, and the supernatant was sucked out. Then, 2 mL of 75% ethanol was added and fixed overnight at 4°C. The supernatant was then centrifuged and removed, and the cells were resuspended twice with PBS. Finally, a 500 mL mixture of propidium iodide (PI) and ribonuclease (RNaseA) was prepared in each tube. After being kept away from light at 37°C for 30 min, the cell cycle was detected by flow cytometry. After the abovementioned treatment, the cell density was adjusted to 1 × 105 L−1. Then, 100 μL of binding buffer was added to each tube to resuspended the cells. Furthermore, 5 μL of annexin v-phycoerythrin (annexin V-PE) and 5 μL of 7-amino-actinomycin D (7-AAD) were added and mixed well. Then, it was incubated at room temperature in the dark for 20 min. Finally, the cells were washed with PBS, and 500 μL of PBS was added to resuspend the cells to detect apoptosis by flow cytometry.The effect of quercetin on the cell cycle and apoptosis of human BRCA MDA-MB-231 cells was detected using flow cytometry. Logarithmically grown MDA-MB-231 cells were inoculated into 96-well plates  membrane. The PVDF membrane was transferred to skimmed milk powder containing 5%, and the membrane was shaken on a decolorization shaker for 1 h at room temperature. The membrane was removed from the blocking solution, placed in a hybridization bag, and the following specific primary antibodies were added: cleaved caspase-3 (1 : 1000), or cleaved PARP (1 : 1000), or ERBB3 (1 : 1000), or MET (1 : 1000), or p38 (1 : 1000), and GAPDH (1 : 5000). After incubation at 4°C overnight, wash with Tris buffered saline tween (TBST) on a decolorizing shaker at room temperature 3 times for 8 minutes each time. The membrane was incubated in secondary antigoat antirabbit IgG (1 : 2000) at room temperature for 1 h and washed on a decolorizing shaking table with TBST 3 times at room temperature for 8 min each time. ECL chemiluminescence developing drops were added onto the PVDF film, the film was put into a chemical imaging analyzer for exposure, and photos were taken. ImageJ 1.4.1 software was used to collect the gray value of the protein and to conduct semiquantitative analysis.After the abovementioned treatment, human BRCA MDA-MB-231 cells were added to the protein lysate and lysed for 30 min on ice. The total protein concentration was measured using the bicinchoninic acid (BCA) assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 20 P. vulgaris were obtained in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Based on the screening criteria of OB ≥ 30% and DL ≥ 0.18, 11 potential active components were obtained, all of which were terpenoids and sterols. To facilitate the follow-up study, these components were numbered PL1–PL11  were excluded. The active ingredients of P. vulgaris and the common target of P. vulgaris and BRCA were introduced into Cytoscape 3.7.1 software to build the single-substance drug-disease ingredient-target network (P. vulgaris (light red round), 8 active ingredients screened from P. vulgaris (purple triangle), and 179 P. vulgaris and common target nodes of BRCA (light green and green ellipse). Quercetin is the most active component of P. vulgaris against BRCA. It is directly related to 59 targets, such as DUOX2, MET, TOP2A, ERBB3, and the interleukin family .The 198 target sites of ne chart . Only ei network . The netP. vulgaris and BRCA were introduced into the STRING database, and the species “Homo sapiens” was defined and screened with a confidence level >0.4. The resulting PPI network had 175 nodes, representing all prediction targets  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 179 common targets of P. vulgaris and BRCA were carried out using the Xiantao academic database. A total of 2905 GO enrichment entries was obtained, including 2573 biological process entries, 224 molecular functions, and 108 cellular components. In the biological process, P. vulgaris was mainly involved in (GO: 0007188 n = 42), lipopolysaccharide , reaction to the bacterial source , reaction to the metal ions , and cell response to drugs . P. vulgaris was mainly related to membrane raft , membrane microdomain , membrane region , transcription factor complex , and plasma membrane raft . In the process of molecular function enrichment, P. vulgaris was linked with ubiquitin-like protein ligase , endopeptidase activity , DNA binding transcriptional activator activity and RNA polymerase II specificity , receptor ligand activity , and cytokine receptor binding , which have a great impact. According to P < 0.05, the top 10 GO enrichment entries were screened and visualized by GraphPad prism , MAPK signaling pathway , hepatitis B , human cytomegalovirus infection , and the signaling pathway of the diabetes in the complication of diabetes mellitus. According to P < 0.05, the top 10 pathways were screened out and visualized by GraphPad Prism , BRCA (light purple octagon), 8 active ingredients of P. vulgaris (light pink hexagon), 179 P. vulgaris and BRCA common targets (light yellow diamond), and the top 10 signal pathways (light green V font). From this picture we can see quercetin (PL11). Multipurpose targets are the core components of P. vulgaris against BRCA. This network map directly reflects the mechanism of P. vulgaris against BRCA through multicomponent, multitarget, and multichannel.In order to further study the mechanism of ad prism . Using tad Prism . Finally network . The netP. vulgaris for the treatment of BRCA. Using AutoDock Vina software, quercetin was docked with the top 10 targets in the PPI network, and the binding activity of components and targets was evaluated by the binding energy. It is generally believed that binding energy < −4.52 kcal/mol indicates that there is a certain binding activity between ligand small molecules and receptor proteins; binding energy < −5.0 kcal/mol showed that they had a good binding activity; binding energy < −7.0 kcal/mol indicates that the ligand has a strong binding activity with the receptor [−1, indicating that quercetin was well docked with these targets  were selected for molecular docking with receptor . Molecul targets . PyMOL s targets . As showP. vulgaris in the treatment of BRCA. Therefore, the abovementioned two pathways were further validated. The names of the two pathways were entered into the KEGG database, and the target selection points  were entered into the target plate. The results of further analysis are shown in Previous KEGG pathway analysis revealed that the PI3K-Akt and MAPK pathways were crucial in the process of 50 at 24 or 48 h is 31.99 ± 0.98 and 37.26 ± 0.97 μg/mL, respectively.The effect of quercetin with different concentrations on the proliferation of the human BRCA cell line MDA-MB-231 was detected using the MTT method at 24 or 48 h . Quercetμg/mL) on the cell cycle of MDA-MB-231 cells for 24 h  on the apoptosis rate of MDA-MB-231 cells for 24 h (P < 0.05), indicating that quercetin induced apoptosis in human BRCA MDA-MB-231 cells. The effect of quercetin on MDA-MB-231 apoptosis was further detected by western blotting. Caspase-3 and PARP are the core executive proteins in the apoptosis pathway, ERBB3 and MET are the screened core proteins, so the expression of these proteins was detected. As shown in P < 0.05), indicating that quercetin may induce apoptosis of human MDA-MB-231 cells through caspase-3 and PARP. At the same time, ERBB3 and MET proteins decreased with the increase in quercetin concentration (P < 0.05). It is predicted that ERBB3 and MET could become biomarkers of quercetin, promoting MDA-MB-231 cell apoptosis. According to the results of KEGG pathway, the p38MAPK pathway played an important role in the action of P. vulgaris against BRCA. Based on the abovementioned reasons, this study speculated that quercetin induced apoptosis in human BRCA MDA-MB-231 cells may be related to the p38 channel, so the expression of p38 protein was detected. Quercetin increased the expression of p38 in a dose-dependent manner (P < 0.05), suggesting that the p38MAPK pathway may be involved in the process of apoptosis.In this study, flow cytometry was used to detect the effect of quercetin at different concentrations  have nine subtypes, namely Nav1.1–Nav1.9, with different functional characteristics. Nav1.5 belongs to TTX-R VGSC and SCN5A is the genotype encoding Nav1.5. At present, studies have reported that the functional expression of SCN5A is found in many malignant tumor cells, and its increased expression and activity are closely related to the malignant biological characteristics of some tumors, which may be involved in the occurrence and development of tumors [In vivo hydrolysis of MAP2 protein can increase the antitumor effect of docetaxel in pancreatic ductal adenocarcinoma [P. vulgaris against BRCA, which reflect the synergistic effect of multiple targets.In this study, 179 common targets of f tumors . An abnof tumors  showed tf tumors . Studiesarcinoma . DNA toparcinoma . TOP2A iarcinoma . In addiarcinoma , 48. TheP. vulgaris exerts anti-BRCA effects through multiple signals, including the PI3K-Akt signaling pathway, the MAPK signaling pathway, and the AGE-RAGE signaling pathway. The PI3K/Akt signaling pathway promotes BRCA formation through a variety of cell processes, including promoting cell growth, proliferation, viability, transforming glycolysis metabolism, increasing cell migration, and reducing cell apoptosis [β1. The relationship between the MCP-1 gene and other genes needs to be confirmed by further research.In this study, KEGG pathway analysis showed that poptosis . PI3K sipoptosis , 51. Thepoptosis . In recepoptosis , but PIKpoptosis . BecauseERBB3 plays an important role in the development of normal breast tissue. The expression level of ERBB3 is very low in normal breast tissue in the embryonic stage. Through the process of postnatal maturation, the expression of ERBB3 increases. In the second and third trimesters of pregnancy, it can be observed that the phosphorylation level increases after ERBB3 is activated, and the expression of ERBB3 also increases in the mammary duct epithelium and stroma of pregnant rats. In functionally differentiated or nontransformed breast epithelial cells, the expression and activation of ERBB3 are downregulated . ERBB3 wFurther studies have found the importance of ERBB3 in ERBB2 transformed cells. When ERBB3 expression is inhibited, ERBB2 cannot effectively transform cells . Althougα stimulated by serum, the level of ERK/MAPK phosphorylation and activity duration in cells were higher than those in ER-positive BRCA cell MCF-7, indicating that ERK/MAPK activation can promote the invasive growth of tumor cells. Song et al. found that in MCF-7, estrogen can produce rapid induction by binding with mer, activate SHC to form a SHC-GRB2-sos complex, activate the MAPK signal pathway, and finally affect the normal cycle of cell growth and abnormal proliferation of breast cells [The MAPK signaling pathway plays an irreplaceable role in the proliferation, growth, and differentiation of breast tumor cells. It is a serine/threonine protein kinase widely found in eukaryotic cells. It can phosphorylate some target proteins and activate a series of cascade reactions to produce some biological effects in cells. The MAPK signaling pathway can transduce extracellular stimulation signals into cells and nuclei through a three-level kinase cascade: extracellular signal ⟶ MAPK kinase ⟶ MAPK kinase ⟶ MAPK. This pathway is involved in the processes of cell proliferation, growth, and apoptosis, and plays an important role in the occurrence and development of a variety of tumors. Filardo and Gper have shown that in NER-negative SKBR3 BRCA cells, estrogen can accelerate the phosphorylation of ERK1/2 and promote the proliferation of cancer cells . Moreovest cells . At the st cells . Jingjinst cells , 76.P. vulgaris against BRCA. Second, this study was only carried out in vitro and only used human BRCA MDA-MB-231 cells. In future, further multidimensional validation is needed. It is very meaningful to study the effect of P. vulgaris on the role of anti-BRCA through various signaling pathways. For future research, the MAPK signaling pathway can be selected, and other signaling pathways, such as the PI3K-AKT signaling pathway, can be further studied. In addition, although quercetin is the main active component of P. vulgaris, it still cannot fully represent P. vulgaris, which still needs further experimental verification in the future. In conclusion, the mechanism of P. vulgaris against BRCA should be further studied in vivo and in vitro.The study also has some limitations. First, the public database is updated in real time, so the results of this study only reveal to a certain extent the mechanism of P. vulgaris against BRCA was preliminarily investigated by using network pharmacology, molecular docking, and in vitro experiments. The results showed that P. vulgaris played an important role in anti-BRCA through multicomponent, multitarget, and multisignal channels. Quercetin played an important role in this process. In addition, quercetin can effectively inhibit the proliferation of human BRCA cell MDA-MB-231 by regulating cell apoptosis and the cell cycle, which may be related to the MAPK signaling pathway. This study provides new inspiration for future experimental and clinical treatment of BRCA.In this study, the potential mechanism of


We postulated that repetitive peripheral magnetic stimulation (rPMS) might treat idiopathic persistent hiccups. This study aimed to determine the clinical effect of rPMS on hiccup alleviation.Seven patients with idiopathic persistent hiccups experienced the cervical rPMS session  in this prospective clinical series from November 2018 to May 2021. The rPMS session was applied once daily until the hiccups were utterly relieved. During the treatment, the round coil was transversally positioned over the upper nape area, and the center of the coil was placed at the level of the C4 vertebrae. The subjective assessment scale (SAS) scores and the hiccup frequency were assessed before and after rPMS treatment.A total of 7 patients were enrolled. All were male post-stroke patients  with dysphasia, 3 patients (3/7) were fed with a nasogastric tube, and 4 patients (4/7) were with dysarthria. The mean duration of hiccups was 4.14 ± 3.63 days (range 2–12 days). The rPMS therapy eliminated hiccups in all 7 patients. The mean sessions which stopped hiccupping were 3.43 ± 2.57 (range 1–9). The mean value of the SAS scores before rPMS therapy was 7 ± 1 (range 6–8), and it was decreased to zero after the therapy (0). No recurrence of hiccups was observed within 2 weeks of the last rPMS session. rPMS therapies were not associated with severe adverse effects.The cervical rPMS therapy is beneficial in treating idiopathic persistent hiccups, particularly in post-stroke patients. Golomb described the hiccup as a respiratory arrhythmia rather than reflexes. Clinically, the classification of hiccups is based on their duration. Transient hiccups last less than 48 hours, persistent hiccups last more than 48 hours, and intractable hiccups last more than 1 month. Pathological factors may bring on long-lasting and challenging-to-cure hiccups. Idiopathic hiccups refer to hiccups for which the cause cannot be identified. Persistent or intractable hiccups can cause exhaustion and anxiety, even make the underlying condition worse. Thus, it should be addressed.Hiccups are caused by a series of involuntary spasmodic contractions of the diaphragm and intercostal muscles, followed by a sudden closure of the glottis, which results in a peculiar “hic” sound.Multiple different medications and non-pharmacological interventions  have been used to stop persistent or intractable hiccups. However, the “perfect method” (a specific treatment for hiccups) has not yet been discovered, and structured guidelines have not yet been established.–12 Cervical magnetic stimulation, which stimulates the phrenic nerves, has been shown to cause bilateral diaphragm contractions. Therefore, we hypothesized that magnetic stimulation might prevent hiccups when rPMS is applied to the neural structures that comprise the hiccup reflex arc. Furthermore, no reports of adverse reactions to rPMS have been found. Therefore, rPMS can be considered a safe and feasible method to treat hiccups. This study aimed to determine the clinical effect of rPMS on hiccup alleviation.Repetitive peripheral magnetic stimulation (rPMS) has been effectively used to treat low back pain by regulating the function of the peripheral nerve and muscles.Seven hospitalized patients with idiopathic persistent hiccups were enrolled in the study between November 2018 and May 2021. All patients underwent a systemic examination to rule out the contraindications to rPMS. Conditions that may cause hiccups have not been identified. The preconditions for acceptance in this study were that the hiccups were diagnosed as idiopathic hiccups lasting at least 48 hours. Before rPMS therapy, physical maneuvers or medication for hiccups were allowed. The periods between previous empiric therapy (baclofen or metoclopramide) and rPMS therapy were at least 12 hours to exclude the influence of these medications. Prior to and following rPMS therapy, the frequency and severity of hiccups were measured using a subjective assessment scale (SAS), which ranged from 0 (no hiccupping) to 10 (unbearable hiccupping).This study was approved by the Institutional Ethics Committee of the hospital (HK-LLWYH-2018013), which adheres to the tenets of the Declaration of Helsinki. Patients provided informed consent to participate in the study. Written informed consent was received from the patients for publication of the data and any accompanying images.In this study, a magnetic stimulator with a round coil, model CCY-1 , was employed. The parameter of rPMS sessions followed the security guidelines and protocols. The treatment was carried out in a comfortable and quiet room. The round coil was transversally positioned over the upper nape with the help of a metal arm. The center of the round coil was positioned over the C4 vertebra, for it is where the hiccup reflex arc center located, and the nerve roots of phrenic nerves come from. In the seated position, the patient’s head should be bent forward as much as feasible to eliminate the consequences of the cervical lordosis  of patients were determined in order to evaluate the excitability of each patient’s nervous system, as in repetitive transcranial magnetic stimulation therapy. The RMT was measured by placing the round coil over the primary motor cortex (M1) of the unaffected cerebral hemisphere. The RMT was defined as the 90% of the lowest stimulation intensity capable of eliciting the slightest observable movement of the accompanying hand or fingers. Parameters of the 15-minute stimulation protocol consisted of 82 trains of 8 pulses at a frequency of 1 Hz, followed by a 3-second inter-train interval  at a tolerable intensity (80%–120% of the RMT). Patients received an rPMS session once a day. The rPMS therapy was planned to continue until the hiccupping stop. If hiccups reoccur within the following 2 weeks, rPMS therapy will be applied again. The SAS was assessed, and the adverse effects were also observed. Total hiccup relief was defined as having no hiccups within 2 weeks and receiving a score of 0 for both SAS and hiccup frequency.The history of hiccups and treatment details are provided in Table During the hiccupping days preceding rPMS treatment, all 7 patients sought to apply pressure on the eye, take baclofen orally, or inject metoclopramide intramuscularly, but none of them were successful.The rPMS therapies were not associated with severe adverse effects. Following rPMS treatment, all 7 patients were completely free of hiccups. The mean SAS scores before and after therapy were (7 ± 1) and (0), respectively. The average number of rPMS sessions required for these patients to stop hiccupping was 3.43 and 2.57 (range 1–9). Each session of rPMS could give at least part of hiccup relief. The No. 5 patient experienced complete hiccup resolution following just 1 session of rPMS therapy. No hiccup recurrence was noticed within 2 weeks after the final rPMS session.In the present study, we assessed the effectiveness of rPMS in treating hiccups. All hiccups in the survey disappeared, with no severe adverse effects and no recurrence within 2 weeks.,7 Male patients are more likely to suffer from hiccups. The findings of this investigation revealed that the neural tissue dysfunction might be the idiopathic hiccups in post-stroke patients.All 7 of the patients with idiopathic persistent hiccups in this study were men who had recently experienced a stroke and had dysphasia; 4 patients (4/7) also had dysarthria, and 3 patients used a nasogastric tube. Thus, the male, post-stroke patients, especially those with dysphasia and dysarthria, may be more susceptible to idiopathic persistent hiccups. It is consistent with the findings of previous researchers who found that idiopathic hiccups are assumed to be caused by the digestive system or the central nervous system.The results showed that the rPMS therapy could relieve hiccups immediately and keep no hiccupping in the following 2 weeks. The effects of the rPMS therapy on hiccups are like its impact on low back pain. The evidence indicated that rPMS therapy could relieve hiccups or pain through a short-term or long-term mechanism. The long-term mechanism of hiccup relief is uncertain, despite some rPMS studies on the reduction of low back pain attributing pain reduction to proprioceptive-induced brain reorganization. and then trigger trains of diaphragm contraction. We adopted the low frequency rPMS at a frequency of 1 Hz, which was higher than the upper mean value of the hiccup rate . As a result, the rPMS-induced diaphragm contraction may suppress the rhythmic diaphragm contraction associated with hiccups, subsequently ceasing the hiccups. The overdrive suppression phenomenon occurred after hiccup relief, which may be analogous to what happened during cardiac arrhythmia treatment. The overdrive suppression mechanism may be the most plausible explanation for the results of this investigation. Whether the hiccups are controlled by a Glomb-described respiratory rhythm center or an unidentified hiccup rhythm center is uncertain. It is also uncertain what causes the overdrive suppression phenomena, whether it is the frequency or the intensity of the magnetic stimulation. If a mechanism of overdrive suppression inhibition exists, studies employing a higher frequency of rPMS treatment may be more fruitful.In this study, the rPMS could penetrate to the deep structures beneath the skin, form electrical fields, depolarize the phrenic nerves, such as holding one’s breath, applying pressure on the eyes, massaging the carotid massage, and self-induced vomiting. These noninvasive techniques were always used before the medication or invasion therapies. As for the persistent and intractable hiccups, these physical manipulations are undoubtedly a failure. Medication is the primary therapy for persistent or intractable hiccups. The medicines such as metoclopramide, baclofen, gabapentin, and pregabalin are for hiccups originating in the central nervous system. And the medicines such as cisapride and omeprazole are for hiccups originating in the digestive system. However, a Cochrane systemic review concluded inadequate evidence to support the notion that pharmacological medicines are the most effective treatment for hiccups. Georg applied a combination therapy with cisapride, omeprazole, and baclofen for the idiopathic chronic hiccup, 60% of patients gained substantial relief or even better (disappearance), but the poor compliance and the long course (12 months) of treatment attempts are the limiting factors. Phrenic nerve blockade or radiofrequency ablation was even regarded as a “final resort” to treat intractable hiccups.,18 However, since the phrenic nerve is not the only nerve innervating the diaphragm, sometimes this intervention can fail even if both sides of the phrenic nerve are blocked.,20 And cases of acute dyspnea respiratory compromise after phrenic nerve blockade have been reported.–23 Therefore, the phrenic nerve block cannot be regarded as an ideal technique for hiccup elimination. Moreover, the blockade can be merely operated by a well-experienced anesthetist, these negative factors limit clinical application. Consequently, cervical rPMS therapy for hiccups is more effective and practical than medication therapy and intervention therapy.According to the findings, rPMS therapy provided 100% (7/7) alleviation from hiccups with a short course of treatment . Therefore, rPMS therapy is an effective way to eliminate hiccups. Hundreds of basic physical manipulations aim to stop hiccups by interrupting or suppressing the reflex arc,Under the circumstances of this study, neither double-blind nor placebo controls were feasible. Therefore, this study was not masked or placebo controlled. Consequently, one of the limitations of our study is that we cannot rule out the coincidence and placebo effect as a possible explanation for eliminating hiccups. In addition, the limited sample size and homogeneous patients may restrict the generalizability of the study’s findings.Cervical rPMS therapy appears to be a safe, workable, and effective treatment for idiopathic persistent hiccups, particularly in post-stroke patients. Since the mechanism remains unclear, further research should be conducted.Conceptualization: Weisen Cai.Data curation: Zongguang Tian, Feng Xiong.Formal analysis: Jiajing Yang.Investigation: Weisen Cai, Guangqing Xu.Software: Zongguang Tian, Feng Xiong, Jiajing Yang.Writing – original draft: Weisen Cai, Guangqing Xu.Writing – review & editing: Tong Wang.Dr. Weisen Cai’s name has been corrected from Weiseni Cai.


Background: Abdominal aortic aneurysm (AAA) is a degenerative disease that causes health problems in humans. However, there are no effective drugs for the treatment of AAA. Artemisia annua L. (A. annua) is a traditional herbal that has been widely used in cardiovascular disease. Based on network pharmacology and molecular docking technology, this study predicted the practical components and potential mechanisms of A. annua inhibiting the occurrence and development of AAA.Methods: The main active ingredients and targets of A. annua were screened through the TCMSP database; the GeneCards, OMIM, PharmGkb, and TTD databases were used to search for the targeted genes of AAA and map them to the targets of the active ingredients to obtain the active ingredient therapy of A. annua. The targets of AAA were to construct a protein interaction network through the STRING platform. R software was used to carry out the enrichment analysis of GO and KEGG for relevant targets, and Cytoscape was used to construct the active ingredient-target network prediction model of A. annua. Finally, AutoDock Vina was used to verify the results of the active ingredients and critical targets.Results: The main active ingredients obtained from A. annua for the treatment of AAA include quercetin, luteolin, kaempferol, isorhamnetin, and artemetin, as well as 117 effective targets, including RELA, MAPK14, CCND1, MAPK1, AKT1, MYC, MAPK8, TP53, ESR1, FOS, and JUN. The 11 targeted genes might play a key role in disease treatment. Enriched in 2115 GO biological processes, 159 molecular functions, 56 cellular components, and 156 KEGG pathways, inferred that its mechanism of action might be related to PI3K-Akt signaling pathway, fluid shear stress, atherosclerosis, and AGE-RAGE signaling pathway. Molecular docking results showed that the top five active components of A. annua had a good affinity for core disease targets and played a central role in treating AAA. The low binding energy molecular docking results provided valuable information for the development of drugs to treat AAA.Conclusion: Therefore, A. annua may have multiple components, multiple targets, and multiple signaling pathways to play a role in treating AAA. A. annua may have the potential to treat AAA. Abdominal aortic aneurysm (AAA) is mainly characterized by local progressive dilation of the abdominal aorta, the most high-risk vascular degenerative disease in vascular surgery . Once AAArtemisia annua L. (A. annua) is a kind of traditional Chinese medicine. With the award of the 2015 Nobel Prize in Physiology or Medicine to a Chinese scientist, Artemisia has attracted global attention . ②Disease Target Database: GeneCards (https://www.genecards.org/); Online Mendelian Inheritance in Man , Pharmacogenomics Knowledgebase ; Therapeutic Target Database ; ③Protein database, UniProt (https://www.uniprot.org); Protein Data Bank ; ④Protein interaction analysis platform, String (https://String-db.org/); ⑤Network analysis and mapping software: Cytoscape 3.8.0;R (R4.0.3 for Windows); ⑥Biological information analysis packet: VennDiagram packet; Bioconductor(https://Bioconduct.org/biolite.r) and its: org.hs.eg.DB, ⑦Molecular Docking Software: AutoDock Vina 4.1, PyMOL 2.4.①Drug component target database: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform . The classification standards were defined based on drug-likeness (DL) greater than or equal to 0.18 and oral bioavailability (OB) greater than or equal to 30% (http://www.uniprot.org/) to obtain the standardized gene symbol.All active ingredients in l to 30% . Then, tA. annua intersected with targets related to AAA to screen out the targets related to the treatment of AAA in A. annua.In the Genecards, OMIM, PHARGKB, and TDD databases, “abdominal aortic aneurysm” was input as the keyword for retrieval to obtain related AAA targets. The Venn diagram packet was then run in R to obtain compositional targets of The ClusterProfiler software package in R software (version 4.0.3) was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the intersection genes . When thA. annua for treating AAA were input into Cytoscape software to construct a “component-target” network (The targets of  network . The acthttps://string-db.org/) to obtain the possible intersection points and establish the relationship between the targets  for each module were shown in A. annua was mainly associated with oxidative stress, cellular response to chemical stress, and response to metal ion. Among the cellular components, A. annua was primarily associated with membrane raft, membrane microdomain, membrane region, and other cellular components. A. annua was mainly related to DNA-binding transcription factor binding, ubiquitin-like protein ligase binding, signaling receptor activator activity, and other molecular functions among the molecular functions.GO enrichment analysis was performed to analyze 117 genes of drug-disease intersection by using the ClusterProfiler package in R software (version 4.0.3). They grouped the functions of the genes into three components: biological processes (BP), cellular component (CC), and molecular function (MF), and enriched 2115 GO BPs, 159 MFs, and 56 CCs . The topA. annua might act together through multiple pathways, such as the PI3K-Akt signaling pathway, fluid shear stress, atherosclerosis, and AGE-RAGE signaling pathway in diabetic complications. The size and color of the nodes in the bubble map were determined by the number and p-value of the associated genes. The node size indicated how many target genes were associated, and the color from purple to yellow reflected the p-value from high to low.KEGG enrichment analysis was performed using the ClusterProfiler package (version 4.0.3) in R software for 117 genes targeted at drug-disease crossover. The results of KEGG analysis showed that these genes mainly enriched in 156 KEGG pathways , and theA. annua for treating AAA were input into Cytoscape software to construct a “component-target” network. The active ingredients and targets were represented as “nodes”, and the interaction between nodes was defined as “edges”. The details were presented in A. annua in the treatment of AAA, and green represented the potential targets.A total of 117 targets of A. annua for the treatment of AAA were entered into the STRING platform; the parameter was set to Homo sapiens, with the highest confidence (0.900), and the rest of the parameters were set to default to build the target protein interaction network, as shown in A. annua in the treatment of AAA.The 117 targets of A. annua in the treatment of AAA, 11 centers for topological analysis of genetic screening for potential A. annua center for gene therapy AAA. Therefore, we docked the active ingredients to the hub target genes. We downloaded the 3D structures of the five active ingredients from PubChem and the protein structures of eleven hub genes from the PDB database. All active ingredients and hub genes were docked, with binding free energies calculated by running Vina, and the results were presented as a thermal diagram in A. annua in the treatment of AAA. We showed the top 10 molecular docking maps with low binding energies in According to the “component-target” network, quercetin, luteolin, kaempferol, isorhamnetin, and artemetin were the top five active ingredients of Artemisia has gained global attention  had the best binding ability and could be used as a potential drug therapeutic target in the future. More literature had reported that these selected genes were closely related to AAA (A. annua in multigene targeting, it is more promising to bring good news to AAA patients.After our filtering, there are 22 active ingredients in Artemisia annua. The top compound is quercetin with a degree-value of 89, and the last compound with a degree-value of 1. The difference between the two values is too large. We predicted the compounds and targets that play a major role in s studies, we seles studies. The resd to AAA . These gd to AAA , we do nA. annua.In addition, KEGG enrichment analysis showed that several pathways, such as the PI3K-Akt signaling pathway, fluid shear stress, atherosclerosis, and the AGE-RAGE signaling pathway, were closely associated with the treatment of AAA by via the PI3K/Akt signaling pathway (A. annua might inhibit AAA by acting on related signaling pathways or targets.The relationship between the above pathways and AAA had been extensively studied. Inhibition of Notch1-mediated inflammation prevented AAA  pathway , and AGE pathway . Daidzei pathway . The MAP pathway . Lithium pathway . TherefoA. annua in the treatment of AAA and speculated the potential mechanisms from multiple approaches and perspectives. Therefore, A. annua might have multiple components, multiple targets, and multiple signaling pathways to play a role in treating AAA. Among them, quercetin corresponds to the most targets and has the strongest activity, and it will be one of the possible potential drugs in the future drug treatment of abdominal aortic aneurysm.In summary, the study used network pharmacology and molecular docking technology strategies to predict the significant active compounds and critical targets of A. annua in the treatment of AAA, aiming to promote more comprehensive development and research of A. annua. The potential molecular mechanism of the active ingredients of A. annua in the treatment of AAA could support its subsequent clinical research and be vital for exploring the pharmacological treatment of AAA. At the same time, the current work also has some shortcomings. This study lacks corresponding experimental verification, which will be further verified in future research. In addition, the loss and incompleteness of some database information will also have a particular impact on the prediction results.Based on network pharmacology combined with molecular docking technology, this study systematically summarized the molecular targets of


Recent studies have shown that the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteogenic lineages can promotes bone formation and maintains bone homeostasis, which has become a promising therapeutic strategy for skeletal diseases such as osteoporosis. Fructus Ligustri Lucidi (FLL) has been widely used for the treatment of osteoporosis and other orthopedic diseases for thousands of years. However, whether FLL plays an anti-osteoporosis role in promoting the osteogenic differentiation of BMSCs, as well as its active components, targets, and specific molecular mechanisms, has not been fully elucidated. First, we obtained 13 active ingredients of FLL from the Traditional Chinese Medicine Systems Pharmacology (TCSMP) database, and four active ingredients without any target were excluded. Subsequently, 102 common drug-disease targets were subjected to protein-protein interaction (PPI) analysis, Gene Oncology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The results of the three analyses were highly consistent, indicating that FLL promoted the osteogenic differentiation of BMSCs by activating the PI3K/AKT signaling pathway. Finally, we validated previous predictions using in vitro experiments, such as alkaline phosphatase (ALP) staining, alizarin red staining (ARS), and western blot analysis of osteogenic-related proteins. The organic combination of network pharmacological predictions with in vitro experimental validation comprehensively confirmed the reliability of FLL in promoting osteogenic differentiation of BMSCs. This study provides a strong theoretical support for the specific molecular mechanism and clinical application of FLL in the treatment of bone formation deficiency.  Bone protective effects of FLL might result from achieved by promotingosteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Beta-sitosterol, kaempferol, taxifolin, Lucidumoside D_qt, (20S)-24-ene-3β,20- diol-3-acetate, eriodictyol, syringaresinol diglucoside_qt, luteolin, quercetin might be the major active compounds of Fructus Ligustri Lucidi (FLL) promoting osteogenic differentiation of BMSCs.FLL might be target AKT1 and then activate the PI3K/AKT signaling pathway to promote the osteogenic differentiation of BMSCs.1.Bone marrow mesenchymal stem cells (BMSCs) play an important role in regenerative medicine, cell therapy, and tissue engineering which based on growth factors, scaffolds, and seed cells . BMSCs hFructus Ligustri Lucidi (FLL), a traditional Chinese medicine, is obtained from the fruit of Ligustrum lucidum Ait, a member of the Oleaceae family, which is distributed in the Yangtze River Valley and south of China, including Zhejiang, Jiangsu, and Yunnan . AccordiMoreover, as the osteogenic differentiation of BMSCs plays a core role in maintaining bone homeostasis, the promotion of osteogenic differentiation of bone marrow mesenchymal stem cells has become a concern for an increasing number of researchers. The positive therapeutic effect of FLL on osteoporosis and other diseases with an imbalance of bone homeostasis has been confirmed. However, whether FLL exerts its therapeutic effect by promoting the osteogenic differentiation of BMSCs, as well as its active components, targets, and exact molecular mechanisms underlying the osteogenic differentiation of BMSCs, has not been fully elucidated.As a result, our study is the first to identify potential bioactive compounds in FLL and elucidate its mechanisms in promoting the osteogenic differentiation of bone marrow mesenchymal stem cells by using the network pharmacology approach. The main goals of this study were to 1) screen potential FLL targets for promoting osteogenic differentiation in BMSCs; 2) use network pharmacology to analyze the underlying mechanisms of FLL promoting osteogenic differentiation in BMSCs; and 3) confirm the underlying pathway of FLL promoting osteogenic differentiation in BMSCs.The findings of this research might lead to a new therapeutic strategy for skeletal diseases. The graphical abstract depicts the present study’s technical plan.2.2.1.http://lsp.nwu.edu.cn/tcmsp.php) is a unique database covering the chemical aspects of natural compounds, oral bioavailability, drug-likeness, intestinal epithelial permeability, and blood-brain barrier [https://pubchem.ncbi.nlm.nih.gov/) [http://www.swisstargetprediction.ch/) to confirm the predicted target proteins of the active ingredients [https://www.uniprot.org/), duplicate items were deleted, and the data were combined and converted into gene symbols [The Traditional Chinese Medicine System Pharmacological analysis platform  . Moreoveredients ,17. Fina symbols .2.2.http://www.genecards.org/), and the Online Mendelian Inheritance in Man (OMIM) database (http://www.omim.org/) [The target proteins related to osteogenic differentiation were respectively obtained from the GeneCards database (im.org/) . Uniprot2.3.www.cytoscape.org/) [First, based on the above targets, we screened out the common targets of drugs and diseases and showed them using a Venn diagram. Second, we constructed a complex network of interactions among FLL, active ingredients, targets, and osteogenic differentiation. Finally, the network was analyzed, edited, and visualized using Cytoscape v3.8.2 (pe.org/) .2.4.https://string-db.org/, updated on 17 October 2020) and searched using the “multiple proteins’ option to construct the corresponding PPI network [After converting the previously obtained common targets of drug and disease into gene symbols, these were inputted into String v11.0b . Three types of GO enrichment analysis were performed: biological process (BP), molecular function (MF), and cellular components (CC). This method has been used to predict the specific molecular mechanism by which FLL promotes osteogenic differentiation [http://www.bioinformatics.com.cn  [KEGG pathway enrichment and GO enrichment analyses of the common targets were performed using the Metascape database (ntiation . The resization) .2.6.http://www.pdb.org/) [The interaction between the candidate active ingredients and key targets was further verified by molecular docking simulation, which provided a sufficient basis for FLL to promote osteogenic differentiation. Briefly, the SDF files of the three-dimensional chemical structures of candidate active ingredients were downloaded from the PubChem database, and the three-dimensional chemical structures were optimized using ChemBio3D Ultra 14.0 software. The structure of the target protein was obtained from the RCSB Protein Data Bank  . We thendb.org/) . Finally2.7.2 inhalation and soaked in 75% alcohol for about 10 min. The hind limbs were transected along the groin, and soaked in 75% alcohol again for about 5 min. Soft tissues on the bone surface were carefully dissected under aseptic conditions and washed with sterile PBS. The epiphysis at both ends was excised, and the bone marrow cavity was exposed. The bone marrow cavity was repeatedly flushed with complete culture medium until its appearance changed from red to white. The liquid was collected in a 15 mL centrifuge tube and centrifuged centrifugation at 1000 r/min for 5 min. The supernatant was removed after centrifugation. Cells were resuspended in complete culture medium, then transferred into a T25 culture flask, and placed into incubator with volume fraction of 5% CO2, 37°C for culture. After two days, the solution was changed by half. Thereafter, the complete medium was changed every 2–3 days. Cell passage was performed when the cells reached 80%-90%. The third to fifth passages of the cells were used for subsequent experiments. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University .The BMSCs were extracted as previously described . Briefly2.8.4 cells/well. After 24 h of culture, the cells were divided into six groups: control group, FLL  was added to each well. The absorbance (A450 nm) value of BMSCs was detected using a microplate [FLL was acquired from a Chinese medicine store in Harbin, China. Raw FLL (250 g) was cooked twice in 4 L distilled water under reflux for 2 h. The filtered aqueous extracts were collected. The filtrate was lyophilized into a powder and concentrated under decreased pressure at 50°C. The yield of extraction was 20% (w/w). Chemical indicators oleanolic acid and ursolic acid had concentrations of 0.01% (w/w) and 0.015% (w/w), respectively. Prior to use, the extract powder was stored in a desiccator at room temperature . Third passage BMSCs were harvested and cultured in 96-well culture plates at 2 × 10croplate .2.9.4 cells/well or 6-well culture plates at 1.5 × 105 cells/well. The cells were treated with different concentrations of FLL at 80% confluence. The specific concentrations were as follows: FLL ; FLL , and FLL . Those not treated with FLL were used as control groups [The third passage of BMSCs was seeded in 24-well culture plates at 5 × 10l groups . Next, 1l groups . BMSCs wl groups .2.10.According to a previously established method , RIPA  software was used for statistical analysis. All experiments were independently repeated at least three times. The results are expressed as mean ± standard deviation (SD). One Way ANOVA was used for comparison between groups. P < 0.05 was considered statistically significant.3.Based on pharmacological and experimental validation, the goal of this study was to investigate the pharmacological mechanisms of FLL in boosting the osteogenic differentiation of BMSCs. There are three aspects to this approach. First, we examined FLL’s active components and their probable targets, and the OMIM and Genecards databases were used to identify osteogenic differentiation targets. Second, the target genes of FLL that promote BMSCs osteogenic development were assessed, followed by GO and KEGG enrichment analyses. Third, for molecular docking, AutoDock Tools 1.1.2 was utilized. Finally, experimental evidence shows that FLL activated the PI3K/AKT signaling pathway, promoting the osteogenic differentiation of BMSCs. Finally, we used in vitro tests, such as alkaline phosphatase staining, alizarin red staining, western blot analysis of osteogenic-related proteins, and PCR to confirm earlier predictions.3.1.Strictly following the screening criteria of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, we acquired 13 active ingredients of FLL -24-ene-3β,20-diol-3-acetate, eriodictyol, syringaresinol diglucoside_qt,lucidusculine,olitoriside, olitoriside-qt, luteolin, and quercetin) from the TCMSP database . Because3.2.We combined the GeneCards and OMIM databases to screen osteogenic differentiation-related targets as comprehensively as possible, analyzed the data, removed duplicate target proteins, and finally obtained 1739 target proteins related to osteogenic differentiation.3.3.By analyzing the related targets of FLL and osteogenic differentiation, it is reasonable to believe that the intersection items are the potential targets of FLL promoting osteogenic differentiation, which are displayed through a Venn diagram. There were 102 common targets that can be considered key targets of FLL in promoting osteogenic differentiation . We pres3.4.PPI analysis of 102 common targets of FLL and osteogenic differentiation using the STRING database resulted in a complex network consisting of 102 nodes and 1757 edges . Nodes r3.5.To further illustrate the biological functions of common targets, we performed GO and KEGG enrichment analyses of these targets using the Metascape database. GO enrichment analysis histograms were plotted with GO terms as the X-axis and −log10(P) as the Y-axis, target-related biological processes (BP), cellular components (CC), and molecular functions (MF) represented by green, purple, and blue columns, respectively . We foun3.6.Based on the prediction that AKT1 is the core target of FLL in promoting osteogenic differentiation, we selected three active ingredients of FLL , which contain AKT1 as the target, for molecular docking analysis, to investigate the potential way in which FLL exerts its therapeutic effect through AKT1. Our results suggest that the interaction between FLL and AKT1 may be determined by hydrogen bonds formed between kaempferol and residues GLU-17 and ARG-67 in AKT1 ; luteoli3.7.Compared with the control group, FLL at concentrations of 25, 50, and 100 µg/mL stimulated the proliferation of BMSCs. Among them, the 50 µg/mL and 100 μg/mL groups had the most significant effects of promoting proliferation, but there was no statistical difference between the two groups. However, FLL had a significant inhibitory effect on BMSCs at concentrations of ≥ 200 µg/mL . Therefore, we chose the FLL group, which had no inhibitory effect on BMSCs, for subsequent experiments.3.8.The results of alkaline phosphatase staining on the 7th day show that the expression of alkaline phosphatase in each group was increased to different extents after FLL treatment compared with the control group. In particular, the stained areas and depth of color in the FLL  group were significantly higher than those in the other groups . Calcium3.9.We tested the inhibitory effect of the PI3K/AKT signaling pathway on osteogenic differentiation using a PI3K/AKT inhibitor (LY294002) to verify whether FLL promoted osteogenic differentiation of BMSCs through this signaling pathway . We foun3.10.The mRNA levels of AKT1 and the osteogenic genes RUNX2 and OPN were higher in the FLL group than in the control group (P < 0.05). The mRNA levels of AKT1 and the osteogenic genes RUNX2 and OPN in the FLL group were also higher than those in the FLL+ LY294002 group (P < 0.05) .4.BMSCs are driven by different signaling molecules and can differentiate into bone, cartilage, and fat which fully reflects the multidirectional differentiation of these cells. The impaired ability of BMSCs to differentiate into different lineages can lead to a variety of diseases including aging and osteoporosis . The weaThe effectiveness of FLL in the treatment of skeletal diseases such as osteoporosis has been confirmed by numerous studies . HoweverIt has been reported that tyrosine and serine/threonine kinases are indispensable in the process of cell differentiation resulting from extracellular factors, such as drugs and growth factors ,43. As aIn summary, we comprehensively verified the molecular mechanism by which FLL promotes the osteogenic differentiation of BMSCs through activation of the PI3K/AKT signaling pathway through a combination of network pharmacological prediction as well as in vitro experiments. However, more in-depth research is required. Nevertheless, our study provides new insights into the treatment of bone formation deficiency diseases by FLL, based on the new perspective of promoting the osteogenic differentiation of BMSCs.5.In this study, we combined network pharmacological prediction and in vitro experimental validation to confirm that FLL acts on AKT1 through a variety of effective active ingredients and promote BMSCs osteogenic differentiation by activating the PI3K/AKT signaling pathway. This provides a valuable theoretical basis for the specific molecular mechanism and clinical application of FLL in the treatment of bone formation deficiency diseases, aid in FLL drug screening, and serve as a helpful reference for the investigation of other TCM therapeutic mechanisms.Click here for additional data file.