The results revealed that Xiaoji recipe suppressed the viability and migration and promoted the apoptosis of pancreatic cancer cells via the inactivation of PI3K/AKT, MAPK, and STAT3 pathways. The findings of our study suggested the potential of Xiaoji recipe in the targeting therapy of pancreatic cancer.Traditional Chinese medicine (TCM) is applied in the anticancer adjuvant therapy of various malignancies and pancreatic cancer included. Xiaoji recipe consists several TCM materials with anticancer activities. In our work, we intended to analyze the molecular targets as well as the underlying mechanisms of Xiaoji recipe against pancreatic cancer. A total of 32 active components and 522 potential targets of Xiaoji recipe were selected using the TCMSP and SwissTargetPrediction databases. The potential target gene prediction in pancreatic cancer was performed using OMIM, Disgenet, and Genecards databases, and totally, 998 target genes were obtained. The component-disease network was constructed using the Cytoscape software, and 116 shared targets of pancreatic cancer and Xiaoji recipe were screened out. As shown in the protein–protein interaction (PPI) network, the top 20 hub genes such as TP53, HRAS, AKT1, VEGFA, STAT3, EGFR, and SRC were further selected by degree. GO and KEGG functional enrichment analysis revealed that Xiaoji recipe may affect pancreatic cancer progression by targeting the PI3K/AKT and MAPK signaling pathways. Moreover, we performed  Pancreatic cancer is a fatal malignancy and ranks the seventh leading cause of cancer-related death in both sexes, with approximately 5 million new cases and 466000 death cases in 2020 . Many riGanoderma lucidum can induce cancer cell apoptosis [Curcuma zedoaria , Polygonum cuspidatum , clematis root , Rhizoma Paridis , and Eupolyphaga Steleophaga . The main functions of Xiaoji recipe is to eliminate the heat and dampness and promote the blood circulation and can be used in the antitumor therapy for various cancers. Curcuma zedoaria (Zingiberaceae) is reported to inhibit the development of gastric carcinoma, breast cancer as well as liver cancer [Polygonum cuspidatum is used for the therapy of multiple diseases including hypertension, diabetes, and atherosclerosis [Rhizoma Paridis is revealed to suppress the cancer development in non-small-cell lung carcinoma, colon cancer, and hepatocarcinoma [Eupolyphaga Steleophaga is reported to be used in the treatment of fractures, falls, uterine fibroids, or menstrual problems [Traditional Chinese medicine (TCM), especially Chinese herbal medicines and acupuncture, is used to treat advanced cancers with low-toxic effects and is reported to increase physical function, reduce symptoms, and improve the life quality of patients , 7. Incrpoptosis . The prer cancer –14. Polyclerosis –17, and clerosis –20. The arcinoma –23. Eupoproblems , while iNetwork-based pharmacology is widely used in drug discovery by predicting potential mechanisms via exploring the targets of drugs, diseases, and their biomolecular networks , 26. In Signaling pathways such as the phosphoinositide 3 kinase/AKT (PI3K/AKT) signaling pathway, signal transducer and activator of transcription 3 (STAT3) signaling pathway, and mitogen-activated protein kinases (MAPK) signaling pathway are important in the pathological process of pancreatic cancer and are frequently activated in pancreatic cancer. They are associated with poor prognosis of pancreatic cancer. Aberrant activation of these pathways are involved in cell survival, cell cycle progression, and cell apoptosis , 30. TarIn our study, we intended to explore the potential core targets and pathways of Xiaoji recipe against pancreatic cancer based on the TCM network pharmacology approach. The findings of our study may provide clues for the targeting therapy of Xiaoji recipe in pancreatic cancer.2. To evaluate the effects of Xiaoji recipe on cell malignant behaviors in vitro, the CFPAC and PANC1 cells were treated with 150 μg/ml or 300 μg/ml Xiaoji recipe.Pancreatic cell lines ) were provided by the ATCC , CFPAC cell line was cultured in Iscove's modified Dulbecco's medium. PANC1 cell line was cultured in Dulbecco's modified Eagle's medium . Both culture mediums were maintained in an incubator supplemented with g 10% FBS  at 37°C and 5% COμl CCK-8 solution, followed with incubation for another 2 h at 37°C. A microplate reader  was used to determine the absorbance at 450 nm.The viability of pancreatic cancer cells was measured using a Cell Counting Kit-8 . The treated CFPAC and PANC1 cells were grown into 96-well plates at 2000 cells/well and incubated for 24, 48, and 72 h, followed with addition of 10 μl pipette tip. A microscope  was used to photograph the wound healing distance at 0 and 48 h.The treated CFPAC and PANC1 cells were plated into 6-well plates supplemented with medium and 1.5% fetal bovine serum and cultured to reach 90% confluence. Then, the plates were scratched using a 10 μl mixture with 5 μl Annexin and 10 μl propidium iodide . Finally, cell apoptosis rate was detected using a flow cytometer and analyzed with the FlowJo software. The apoptosis rate in each group was calculated by Q2 (late apoptosis) + Q3 (early apoptosis).The apoptosis of CFPAC and PANC1 cells after indicated treatments was assessed by flow cytometry analysis. CFPAC and PANC1 cells were harvested, washed with PBS, and resuspended in a 500 RIPA lysis buffer  was used to collect the protein in treated CFPAC and PANC1 cells. The concentration of collected proteins was evaluated using a BCA Protein Assay kit . Then, the proteins were separated with 10% SDS-PAGE gels and electrotransferred on the PVDF membranes. The Protein-Free Rapid Blocking Buffer  was used to block the membranes for 1 h, which were then cultured with the primary antibodies against AKT1 , STAT3 , EGFR , MAPK3  at 4°C overnight, and GAPDH  served as an internal reference. Next, the membranes were washed with TBST and cultured with corresponding secondary antibodies  for 2 h at room temperature. Finally, the protein signal was detected using a Biosharp ECL detection kit  and analyzed with ImageJ software.Curcuma zedoaria (E Zhu), Polygonum cuspidatum (Huzhang), clematis root (Weilingxian), Rhizoma Paridis (Zhonglou), and Eupolyphaga Steleophaga (Tubiechong). The active components of these TCM materials were searched on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  [http://119.3.41.228:8000/tcmid/) [https://pubchem.ncbi.nlm.nih.gov/) [http://www.swisstargetprediction.ch/) [Xiaoji recipe is a traditional Chinese prescription composed of msp.php)  under th/tcmid/) . The comih.gov/)  and TCMSion.ch/)  for the https://www.disgenet.org/) [https://omim.org/) [https://www.genecards.org/) [The potential target genes of pancreatic cancer were searched in the Disgenet (et.org/) , OMIM (him.org/) , and Gends.org/)  databaseThe 522 targets of Xiaoji recipe and 998 targets of pancreatic cancer were imported into the Venny2.1 software, and the obtained Venn diagram showed 116 shared targets of the pancreatic cancer and the components of Xiaoji recipe. The drug-component-target-disease network was constructed using the Cytoscape 3.8.2 software and analyzed with the Network Analyzer function. The hub genes were analyzed and selected based on the topological analysis, and the degree value indicates the relation between the components and targets .https://cn.string-db.org/) [Homo sapiens.” The protein was presented as nodes, and the association between proteins was shown as edges in the PPI network, and the size and shade of color represented the value of degree. Furthermore, based on the topological analysis, the results from the STRING database were analyzed with the Cytoscape 3.8.2 software using the Network Analyzer function, and the core targets were selected under the condition of degree value over the average. The top 30 targets were selected and exported the bar graph using the R 4.0.5 software. Based on the clustering analysis, the results from the STRING database were imported into the Cytoscape 3.8.2 software and analyzed using the Molecular Complex Detection (MCODE) plugin. Three gene clusters were obtained, and 2 core genes  were selected.The interaction of the 116 component-disease targets was explored on the STRING platform (db.org/)  under “Hp value < 0.05. The results were exported with the bar graphs and pathway maps.Gene Ontology (GO) analysis including the biological process (BP), molecular function (MF), and cellular components (CC) and the biological pathway (KEGG) enrichment analysis of 116 disease-component targets were performed using the R software with Bioconductor package under t-test was used to compare the difference between two groups, and one-way ANOVA was used for multiple group comparisons. p < 0.05 indicates statistical significance.All experiments were completed three times independently. The results were analyzed using GraphPad 8 software and presented as the mean ± SD. Student's Curcuma zedoaria (E Zhu), Polygonum cuspidatum (Huzhang), clematis root (Weilingxian), Rhizoma Paridis (Zhonglou) and Eupolyphaga Steleophaga (Tubiechong) based on the TCMSP database, under OB ≥ 30% and DL ≥ 0.18 and TCMID database , Polygonum cuspidatum (Huzhang), clematis root (Weilingxian), Rhizoma Paridis (Zhonglou), and Eupolyphaga Steleophaga. Totally, 522 target genes of these active ingredients of Xiaoji recipe were screened out, and there were 159 targets for Curcuma zedoaria, 313 targets for Polygonum cuspidatum, 107 targets for clematis root, 271 targets for Rhizoma Paridis, and 44 targets for Eupolyphaga Steleophaga  is indicated as a promising treatment option for multiple malignant diseases with unique clinical effects , 41. As The component-disease network showed the target genes regulated by these active components in pancreatic cancer. The genes include EGFR, CDK1, MMP7, BCL2, and PARP1. Moreover, the PPI networks revealed that TP53, HRAS, VEGFA, AKT1, STAT3, EGFR, and SRC were the core target genes in the PPI network. The target genes were not mutually independent but were interacted with each other. The Xiaoji recipe may inhibit the progression of pancreatic cancer by regulating the multiple proteins. In line with our finding, all mentioned core target genes have been reported as major driver genes for pancreatic cancer –60.GO and KEGG enrichment analysis revealed the biological process, molecular functions, cellular component as well as the related signaling pathways in the occurrence and progression of pancreatic cancer. There were 2144 BP, 50 CC expression process, and 132 MF-related process of these 116 component-disease targets. We also found that the enrichment of the 116 component-disease targets in the PI3K/AKT and MAPK pathways according to the KEGG analysis. PI3K/AKT pathway is critically involved in the regulation of malignant behaviors in pancreatic cancer, indicating that PI3K/AKT is a valuable target for pancreatic cancer therapy , 61. TheIn this study, the active ingredients and related targets of Xiaoji recipe in pancreatic cancer were explored, and the biological process, molecular function, cellular process, and related signaling pathways were under investigation based on the network pharmacology approach. Moreover, we also verified the effect of Xiaoji recipe on pancreatic cancer cell proliferation, migration, apoptosis, and relevant signaling pathways using in vitro assays. Xiaoji recipe significantly inhibited the proliferation, migration, AKT, MAPK, and STAT3 signaling pathways and induced the apoptosis of pancreatic cancer cells, which indicated its potential in clinical therapy of pancreatic cancer patients.However, our study also exists some limitations. First, the in vivo experiments of Xiaoji recipe on tumor growth were lack. Second, the specific mechanism of Xiaoji recipe on regulating the PI3K/AKT, MAPK, and STAT3 signaling pathways were not explored. More, the potential mechanism of Xiaoji recipe on regulating the core target genes of TP53, HRAS, VEGFA, AKT1, STAT3, EGFR, and SRC in pancreatic cancer were not shown. All these limitations will be perfected in the near future.


QiLing decoction (QLD) is a traditional Chinese medicine compound. This study aims to explore the therapeutic effect of QLD in castration-resistant prostate cancer (CRPC) and its potential bio-targets. A total of 51 active components and QLD 149 targets were identified using bioinformatics analysis. Additionally, five optimal hub target genes were screened including tumor protein P53 (TP53), interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), caspase-3 (CASP-3), and estrogen receptor-1 (ESR-1). The interrelated network between active components of QLD and their potential targets was constructed. The molecular function, biological processes, and signaling pathways of QLD-against CRPC were identified. Moreover, QLD was found to efficiently exert a repressive effect on CRPC tumor growth mainly by suppressing the activation of HIF-α/VEGFA and TNF-α/IL6 signaling pathways, and increasing the P53 expression level. These results successfully indicated the potential anti-CRPC mechanism of the active components of QLD. Prostate cancer (PCa) is a common malignancy with a poor prognosis and is the second primary cause of malignancy-related mortality worldwide . PCa is Recently, several studies have reported on the use of chemotherapy for CRPC. For example, the structurally unique androgen-receptor antagonist, darolutamide has been used to delay metastasis and death in CRPC patients . Abiratein vitro and in vivo experiments to verify the underlying mechanism regulating the therapeutic effects of QLD on CRPC.QiLing decoction (QLD) is a traditional Chinese medicine compound, consisting of 10 components, including raw astragalus (huangqi), rubescens (donglingcao), codonopsis (dangsheng), turmeric (jianghuang), rehmannia glutinosa (dihuang), shuyangquan, motherwort (yimucao), psoralen (buguzhi), shegan, and licorice (zhigancao). Extensive studies have reported that several components of QLD may be involved in mediating cancer progression. For example, rubescens may inhibit the growth and angiogenesis of breast cancer , and ast2 atmosphere. Formononetin (cat. no. 20100311) was purchased from Jubang Biotechnology Co., Ltd. , and Calycosin (20575-57-9) was purchased from Sigma-Aldrich , both of which were dissolved with dimethyl sulfoxide (DMSO). The cells in the experimental groups were treated with formononetin (10 µM) or calycosin (20 µM), and the control cells were treated with DMSO.PC3 cells were purchased from the American Type Culture Collection , and cultured in a Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified 5% COhttp://tcmspw.com/tcmsp.php) and the Traditional Chinese Medicine Integrated Database (TCMID). Oral bioavailability (OB) and drug likeness (DL) were used for the screening of active components, as previously reported. DL ≥ 0.18 and OB ≥ 30% were considered to be the threshold for candidate compounds.The active components of QLD in each herb were identified by the Traditional Chinese Medicine Systems Pharmacology (TCMSP) (https://omim.org/), DisGeNET (http://www.disgenet.org/), and GeneCards (https://www.genecards.org/) to screen the pathological targets of CRPC. The active components of QLD and potential pathological targets evaluated were predicted using Venn diagrams. Cytoscape 3.8.2 (https://cytoscape.org/) was used to reveal the active components and potential targets in the form of a network diagram.To further identify the target proteins on the active components, we applied the Online Mendelian Inheritance in Man  was used to visualize the results by analyzing the topological parameters . The maxhttp://www.interactivenn.net) was used for the GO and KEGG pathway enrichment analyses to determine the role of target proteins that interact with the active components of QLD in gene function and signaling pathway. The data were acquired using a cut-off value adjusted to P < 0.05.RStudio 3.6.1 software (n = 6) on day 7 after the injection. The groups included a control group , QLD group (9 g/kg QLD (body weight) was given intragastrically), Doc group (5 mg/kg QLD (body weight) was given intragastrically), and QLD + Doc group (QLD (9 g/kg) and Doc (5 mg/kg) were given intragastrically). The mice with QLD were fed once daily and while the Doc group was fed once a week. The treatment was maintained for 28 days and then all mice were euthanized. The tumor weight and volume were monitored. The tumor volume (mm3) was calculated as follows: tumor volume = (L × W2)/2 . IHC was conducted to detect the expression level of PCNA in cancer cells using PCNA antibody , in accordance with previous reports. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The protocol was approved by the Ethics Committee of LONGHUA Hospital Shanghai University of Traditional Chinese Medicine.To establish CRPC xenograft models, BALB/c nude mice  were provided by LONGHUA Hospital, which is affiliated with the Shanghai University of Traditional Chinese Medicine . The mice were injected with 100 µL phosphate-buffered saline (PBS) containing PC-3 cells into the left axilla of the mice, as described previously . The mic3 cells/well, 10% CCK-8 (Sigma-Aldrich) was added into 100 ul fresh mixture, followed by incubation at 37 °C for 1 h. Finally, the optical density (OD) value was measured at a wavelength of 450 nm using a spectrophotometer.The PC3 cells were cultured in 96-well plates and when the cells reached a density of 2  × 103 cells/well. Two weeks later, the colonies were fixed with 4% paraformaldehyde for 15 min at room temperature and visualized by a camera.The PC3 cells treated with the indicated reagents were seeded into 6-well culture plates at a density of 1  × 10Total proteins were extracted from cells using RIPA lysis buffer . The proteins were quantified using a BioRad Bradford protein assay kit and electrophoresed into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, followed by incubation with anti-HIF-α , anti-vascular endothelial growth factor A (VEGFA) , anti-tumor necrosis factor alpha (TNF-α) , anti-interleukin 6 (IL6) , anti-P53 , anti-cleaved-caspase-3 , anti-Bax , and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  at 4 °C overnight, and then washed with Tris Buffered Saline with Tween (TBST) three times. The horseradish peroxidase (HRP)-labeled secondary antibodies  were used for the secondary incubation for 1 h. Finally, imaging was performed by the ChemiScope 6200 Touch Chemiluminescence imaging system , and the band intensity was quantified using ImageJ software .t-test or one-way analysis of variance (ANOVA) were used to compare differences between groups. P < 0.05 was considered to be statistically significant.All of the data were analyzed by GraphPad Prism  and SPSS 22.0  software, and were presented as mean ± standard deviation (SD). The QLD consisted of 10 herbs, of which we identified six herbs with 1,157 active components using the TCMSP database. Specifically, there was one component in Baimaoteng, 515 components in Huangqi, 41 components in Jianghuang, 236 components in Shegan, 34 components in Shudihuang, and 330 components in Yimucao . A totalDetermining the effectiveness of QLD in preventing and treating CRPC depends on the synergic interaction between active components and their targets; thus, we evaluated the relationships among the active components and their targets using the compound-target network. The active components of six herbs of QLD and their target proteins related to CRPC were detected using TCMSP and TCMID. The interaction between the active components and target proteins was analyzed using the Cytoscape 3.2.1 software. The results are shown in via the UniProtKB. Next, the OMIM, DisGeNET, and GeneCards databases were used to collect the CRPC-related target genes. Venn diagrams were drawn to display the intersected genes of these two target sets, and 66 intersected genes were found. The PPI network was then established to display the intersection of the target genes of QLD against CRPC and CRPC-related biotargets based on the STRING database , cellular components (CC), and molecular functions (MF). A histogram and bubble chart were plotted using the ggplot2 package (version 1.0.2) in the R software . The topetc , microRNAs in cancer, the PI3K–Akt signaling pathway, AGE–RAGE signaling pathway in diabetic complications, HIF–1 signaling pathway, endometrial cancer, central carbon metabolism in cancer, IL–17 signaling pathway, rheumatoid arthritis, P53 signaling pathway, platinum drug resistance, etc . The remCytoscape software was used to determine the targets of QLD against CRPC and the hub target-associated pathways, and a network was constructed. All of the drug protein targets and disease-related proteins were screened. Cytoscape was used to detect the targets of QLD against CRPC, the hub target-associated pathways, and a network visualization of QLD against CRPC targets. An interaction diagram for core target-related pathways was plotted .in vivo. Considering the therapeutic significance of Docetaxel (Doc) on CRPC  and their potential targets were listed.Click here for additional data file.10.7717/peerj.13481/supp-2Supplemental Information 2GO enrichment analyses were used to explore the gene function of potential targets of active components of QLD, including the potential biological processes (BP), cellular components (CC), and molecular functions (MF).Click here for additional data file.10.7717/peerj.13481/supp-3Supplemental Information 3KEGG pathway enrichment were used to explore the signaling pathways of potential targets of active components of QLD.Click here for additional data file.10.7717/peerj.13481/supp-4Supplemental Information 4Click here for additional data file.


Results from epidemiological studies suggest that vitamin D (VD) deficiency (VDD) may be a cause of hypertension (HTN). However, the results of randomized clinical trials (RCTs) designed to address the impact of VD supplementation on reducing blood pressure (BP) remain equivocal. To determine whether VD might serve as a beneficial treatment option for a specific subset of hypertensive patients, we performed a stratified analysis of RCT data and addressed problems associated with some methodological issues.HTN is caused by multiple factors. VDD may be one of the factors contributing to the development of this disorder. There are more than 70 RCTs that examined the impact of VD supplementation on BP. These RCTs can be classified into four groups based on their respective study populations, including participants who are (1) VD-sufficient and normotensive, (2) VD-deficient and normotensive, (3) VD-sufficient and hypertensive, and (4) VD-deficient and hypertensive.Our evaluation of these studies demonstrates that VD supplementation is ineffective when used to reduce BP in VD-sufficient normotensive subjects. VD supplementation for five years or more may reduce the risk of developing HTN specifically among those with VDD. Interestingly, findings from 12 RCTs indicate that daily or weekly supplementation, as opposed to large bolus dosing, results in the reduction of BP in VD-deficient hypertensive patients. Our ongoing research focused on elucidating the mechanisms of VDD-induced HTN will ultimately provide evidence to support the development of etiology-specific prevention and treatment strategies focused on HTN in the VD-deficient population. Journal of the American Medical Association D) levels <20 ng/ml , 7. The At this time, findings are available from more than 70 RCTs that were designed to evaluate the role of VD in reducing BP. These can be classified into four groups based on baseline levels of serum 25[OH]D and BP in selected study populations; of note, several of the earliest RCTs are without serum 25[OH]D data. As a group, these studies include participants that are (1) VD-sufficient and normotensive, (2) VD-deficient and normotensive, (3) VD-sufficient and hypertensive, and (4) VD- deficient and hypertensive. We have also re-evaluated these findings based on the recent changes in the guidelines used to diagnose HTN. While the new guidelines, which have reduced the threshold for diagnosing HTN , may benIn 1998, Krause et al.  used ultIn contrast to exogenous BP-reducing drugs , VD is synthesized in skin exposed to sunlight and can also be obtained from dietary sources . VD is a well-known threshold nutrient, meaning that physiological responses  are dose-dependent at low concentrations; once a threshold value is reached, higher levels of the given nutrient promote limited beneficial effects ••, 16••.The ongoing vitamin D and Omega-3 HTN trial  was designed to examine long-term VD supplementation (2000 UI daily for five years) as a means to prevent HTN in participants aged ≥ 50 years who presented with normal BP. VITAL researchers have already published one report D with optimal benefits. By contrast, intermittent large bolus doses of VD do not achieve steady or effective levels of VD and 25[OH]D and should not be used in these treatment regimens ••.Meta-analyses aim to provide a more precise estimate of the effects of a specific situation or intervention as they can increase sample size and power by a combined review of primary studies with similar populations, controls, interventions, and outcomes. However, it has become clear that administration of VD to subjects in each of the four different aforementioned groups  results in dramatically different outcomes. Furthermore, large intermittent bolus doses of VD are ineffective in reducing BP, even in hypertensive VD-deficient patient cohorts. Thus, pooling data from the four different groups will introduce a considerable amount of heterogeneity. Likewise, meta-analyses that do not consider the specific dosing regimens will also provide mistaken support for the null hypothesis. While none of the published meta-analyses designed to evaluate the role of VD supplementation in reducing BP have provided uniformly positive results, several recent reviews do conclude that VD supplementation can reduce BP in VD-deficient hypertensive patients , 77. TheOther meta-analyses , 81 haveOf note, at the end of the Results section of this meta-analysis and review, Beveridge et al.  reportedUnlike pharmaceutical agents , VD is an endogenous nutrient with a specific threshold that limits its therapeutic efficacy in lowering BP. It is critical to recognize that VD is not a panacea as it is not effective in reducing BP in the VD-sufficient normotensive population. However, an appropriate VD regimen  can reduce BP in patients diagnosed with some types of HTN, most notably HTN secondary to VDD. Long-term VD supplementation (five years) may also reduce the risk of developing HTN in VD-deficient susceptible populations. Our ongoing studies focused on the molecular and cellular links between VDD and HTN will support the development of etiology-specific therapeutics for and prevention of HTN in this critical patient subgroup.


To explore the molecular targets and mechanism of YuPingFeng (YPF) for the treatment of asthma by using network pharmacology and molecular docking.  The potential active ingredients and relevant targets of YPF were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Asthma-related gene targets were retrieved from GeneCards, OMIM, DrugBank, PharmGKB, and TTD databases. The protein-protein (PPI) network between YPF and asthma common targets was constructed by SRING online database and Cytoscape software. GO and KEGG analyses were performed to explore the complicated molecular biological processes and potential pathways. Finally, a molecular docking approach was carried out to verify the results.  We obtained 100 potential targets of the 35 active ingredients in YPF and 1610 asthma-related targets. 60 YPF-asthma common targets were selected to perform PPI analysis. Seven core genes were screened based on two topological calculation methods. GO and KEGG results showed that the main pathways of YPF in treating asthma include TNF signaling pathway and PI3K-Akt signaling pathway. Finally, the molecular docking results indicated that the key ingredients of YPF had a good affinity with the relevant core genes.  This study reflects the multicomponent, multitarget, and multipathway characteristics of YPF in treating asthma, providing a theoretical and scientific basis for the intervention of asthma by traditional Chinese medicine YPF. The blockers , 5. Howeblockers , 7. Biolblockers –10. TherTraditional Chinese medicine (TCM) has a long history. In recent years, great attention has been focused on TCM because of its relative safety and unique superiority. TCM such as Xin Guan-1 Formula and LianHuaQingWen Capsule had significant beneficial effects in treating patients infected with COVID-19 , 12. YuPNetwork pharmacology is an emerging discipline that combines bioinformatics, molecular biology, and traditional pharmacology . It condhttps://tcmsp-e.com/) [https://www.uniprot.org/). Finally, valid gene symbols were obtained after removing mismatches and redundant duplicates.We obtained the YPF active ingredients and the related targets from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (-e.com/) , which i-e.com/) . The prohttps://www.genecards.org/, GeneCards), Online Mendelian Inheritance in Man , DrugBank Online , Pharmacogenomics Knowledgebase , and Therapeutic Target Database . The union of all the search results was used to establish an asthma-related gene set, and the set was visualized by R 3.6.3 software.Targets relevant to asthma were collected from five databases with the keyword “asthma”: the Human Gene Database  to construct the PPI network. Parameters were set to “Homo sapiens” in the organism and the minimum required interaction score cutoff set at 0.400. Disconnected nodes were hidden in the network. Then, the PPI network was constructed, and the result was visualized by Cytoscape v3.8.0 software.Overlaps between YPF-related targets and asthma-related targets were obtained to clarify the interaction between drugs and disease. These overlaps were put into the STRING database ,” “Closeness Centrality (CC),” and “Degree Centralities (DC),” were selected to calculate the gene scores. Genes with score values higher than the median value were obtained. To identify crucial core genes, the filter process was performed twice. A second approach involves using the Cytohubba plugin of the Cytoscape software. We used the plugin to select the top 10 genes based on a Maximum Neighborhood Component (MNC) calculation method. The common targets set from both ways were regarded as the core genes.q value < 0.05.To analyze the molecular biological functions of YPF-asthma common targets, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The GO enrichment analysis involved three main categories: biological process (BP), cellular component (CC), and molecular function (MF). KEGG enrichment analysis was utilized for revealing complicated biological pathways. R 3.6.3 software was used to perform both GO and KEGG analysis, with the screening criterion as the following filters: https://pubchem.ncbi.nlm.nih.gov/). Conversion of 2D structures into 3D formats with minimum energy was performed by ChemBio 3D software. The forms of protein receptors with the screen criteria of “Homo sapiens,” “X-ray Diffraction,” and “Protein” were downloaded from the RCSB Protein Data Bank database . The ligands and the receptors were prepared in PyMOL v2.4.0 and AutoDock v1.5.6 by removing water molecules, adding hydrogen atoms and charges. Afterward, AutoDock Vina was used to performing molecular docking. The results were analyzed by the Protein-Ligand Interaction Profiler (PLIP) web tool, and finally, the output results were visualized using LigPlot v2.2.4 and PyMOL software.Molecular docking simulation technology was used to predict molecular targets of YPF for treating asthma. The 2D structures of small molecule ligands were downloaded from the PubChem database , response to steroid hormone (GO:0048545), reactive oxygen species metabolic process (GO:0072593), cellular response to oxidative stress (GO:0034599), and response to oxidative stress (GO:0006979) . The stability of the ligand-receptor complex was correlated with the binding energy. The lower the value of binding energy, the more stable the docking complex . As showAsthma is a complicated respiratory tract disorder characterized by airway hyperresponsiveness and inflammation , 23. TheIn the YPF's active ingredient-target network, a total of 60 target genes by 35 active components in the YPF were selected. The top two ingredients, quercetin and kaempferol, with the degree of 44 and 25, respectively, were chosen to perform molecular docking with six related core genes.Quercetin is the principal representative of natural flavonoids and widely distributed in diverse food and plants . A varieIn this study, we constructed the PPI network; IL6, CASP3, EGFR, MAPK8, ESR1, CCND1, and PPARG were found to be the core genes of YPF in treating asthma. These genes are connected with host immunity, cell apoptosis, signal transduction, and cell cycle regulation. IL6 is a multifunctional cytokine and is generally related to eosinophil and neutrophil recruitment . High blThe signal nucleotide polymorphism in CCND1 was linked with obesity ; meanwhiGO analysis indicated the diverse and complex synergistic effects of YPF and showed a few BP categories crucially involved with asthma. The top ten BP terms revealed that YPF could regulate the oxidative stress process and the immune response. There is an extraordinarily complex network between multiple cytokines and a number of signal-transduction pathways involved in the pathophysiological process of asthma . KEGG enFurther molecular docking results suggested that the docked small molecule ligands exhibited the lowest binding energy with a good affinity toward macromolecular protein receptors. All the binding energies were less than 6 kcal/mol. Quercetin was verified as the most potent binding activity with EGFR, while kaempferol had the most robust combination with PPARG.In summary, this sufficiently thorough bioinformatic analysis provided plentiful testable hypotheses about the potential molecular mechanisms of YPF in treating asthma. It is indicated that the detailed action mechanisms of YPF in the treatment of asthma involve multiple ingredients, targets, and signaling pathways. Therefore, traditional Chinese medicine such as YPF could be considered as a supplementary regimen for future asthma therapy.


Preeclampsia (PE) is a hypertensive disorder of pregnancy that threatens the lives of millions of pregnant women and their babies worldwide. Without effective medications, there are thousands of maternal and child mortalities every year. Resveratrol (RSV), a non-flavonoid polyphenol extracted from multiple plants, has shown positive effects in treating hypertension, cardiovascular disorders, and even PE. This study aimed to explore the pharmacological mechanism of RSV in treating PE by using network pharmacology and bioinformatics.With the use of multiple databases, 66 intersecting targets were obtained from the 347 putative targets of RSV and 526 PE-related genes. Then, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted to investigate the functions of the intersecting targets. The protein–protein interaction network and target–pathway network were drawn and analyzed to illustrate the correlation between targets and pathways. Finally, molecular docking was conducted to calculate the binding energy between RSV and core targets.The results showed that the core targets of RSV were IL6, TNF, IL1B, VEGFA, STAT3, and EGFR. There existed good binding between RSV and IL6, TNF, IL1B, VEGFA, and EGFR. In addition, we found that RSV mainly functioned in the AGE-RAGE and HIF-1 signaling pathways, which are associated with the occurrence and development of PE.In conclusion, our findings indicated that RSV has the effects of regulating angiogenesis and anti-inflammation and can be a candidate medicine for treating PE. Preeclampsia (PE) is a multisystem disease in pregnancy, which is characterized by the onset of hypertension after 20 weeks of pregnancy, proteinuria, and malfunctions in multi-organs . PE now Resveratrol (RSV), a natural plant polyphenol, has been reported to exert many advantageous effects on the human body including anti-oxidation , anti-inHere, we conducted our work to identify the molecular mechanism of RSV in the treatment of PE. By employing bioinformatics and network pharmacology, we selected the core targets and the main signaling pathways of RSV in treating PE. Our findings verified the therapeutic effects of RSV and provided new options for the treatment of PE.https://pubchem.ncbi.nlm.nih.gov/) (http://www.swissadme.ch/)   and Swisdme.ch/) . AccordiThe putative targets of RSV were collected from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) , SwissTahttp://www.genecards.org/) (https://omim.org/) (The PE-related genes were collected from the GeneCards (ds.org/)  and Onliim.org/)  databaseThen, all putative targets of RSV and PE-related genes were imported to the UniProt database for the transformation of gene symbols, and the overlapping part was called the intersecting targets of RSV and PE.http://metascape.org/)   was emplhttps://string-db.org/) (http://www.cytoscape.org/) (All intersecting targets of RSV and PE were sent to the STRING database (db.org/)  to invespe.org/)  was applThe relationship between the intersecting targets of RSV and PE and the signaling pathways from the KEGG pathway enrichment analysis was also illustrated by the target–pathway (TP) network and visualized by Cytoscape software. Finally, the core targets and the main signaling pathways were obtained.https://pubchem.ncbi.nlm.nih.gov/) (http://www.pdb.org/)  , and thedb.org/) .To become a potential drug, a compound needs to be analyzed in accordance with Lipinski’s rule of five (RO5). The fewer violations of Lipinski’s rule there are, the greater drug likeness and pharmacokinetics a compound has. According to Lipinski’s rule, a drug-like molecule must have an MW less than 500 g/mol, an octanol/water partition coefficient (XLogP3) less than 5, no more than 10 hydrogen bond acceptors, no more than 5 hydrogen bond donors, and rotatable bonds less than 10.The parameters of RSV from the PubChem and SwissADME databases are listed in To collect the putative targets of RSV, five databases were applied including TCMSP, TargetNet, SwissTargetPrediction, STITCH, and SEA. As a result, a total of 347 proteins were collected from the databases mentioned above after deleting the duplicate targets. These targets were listed in The PE-related genes were collected from the GeneCards database and OMIM database. After the redundant information was deleted, a total of 511 genes were identified and listed in Then, the 66 overlapping targets of these two lists formed the intersecting targets of RSV and PE Figure 1To further identify the functions of the intersecting targets of RSV and PE, GO analysis and KEGG analysis were carried out. A total of 1,707 GO terms and 125 KEGG pathways were identified (As shown in As shown in As shown in Leishmania infection (hsa05140), pertussis (hsa05133), non-alcoholic fatty liver disease (NAFLD) (hsa04932), hepatitis B (hsa05161), IL17 signaling pathway (hsa04657), tuberculosis (hsa05152), toxoplasmosis (hsa05145), microRNAs in cancer (hsa05206), inflammatory bowel disease (IBD) (hsa05321), bladder cancer (hsa05219), NF-kappa B signaling pathway (hsa04064), amebiasis (hsa05146), longevity regulating pathway (hsa04211), African trypanosomiasis (hsa05143), cytokine–cytokine receptor interaction (hsa04060), pancreatic cancer (hsa05212), insulin resistance (hsa04931), endocrine resistance (hsa01522), PI3K-Akt signaling pathway (hsa04151), and measles (hsa05162).As shown in In order to obtain the interaction relationship among the intersecting targets, the STRING database was employed. All 66 targets were imported into the STRING database to illustrate the PPI network. Then, the PPI network was modified by using the Cytoscape software  and the HIF-1 signaling pathway (hsa04066), were chosen to be the potential signaling pathways of RSV in the treatment of PE.Furthermore, in order to elaborate on the correlation between the six core targets, the STRING database was used to construct their PPI network. As shown in To further investigate the physical binding between the core targets and RSV, molecular docking was carried out and visualized. The values of binding energy are shown in As shown in PE has become the major cause of maternal and fetal death worldwide. Furthermore, during their lifetime, women with preeclampsia are also more likely to suffer from cardiovascular disorders than those without. Unfortunately, there is still no effective treatment except termination of pregnancy. There is an urgent need to seek new drug targets and develop new effective therapies. RSV is a native polyphenol extracted from many plants. It has been reported to exert anti-oxidation, anti-inflammation, and anti-cancer effects. The positive influences on complicated pregnancy and a decrease in cardiovascular events both have been verified , 27.As a good source of RSV, the Mediterranean diet is rich in many phenols. As the main components of the Mediterranean diet, red wine is rich in RSV, and olive oil can increase the absorption of RSV , 29. ThePE is a complicated disease that affects multiple cells, organs, or systems including the placenta, trophoblast cells, and endothelial cells. Previous studies have shown that RSV upregulates VEGF  and inhiIn this study, we investigated the potential of RSV as a candidate medicine for the treatment of PE. There were 66 intersecting targets of RSV and PE between the 347 potential targets of RSV and the 526 PE-related genes. The results of GO showed that more than 38 GO terms widely participated in the pathophysiology of PE. These GO terms included pregnancy, regulation of blood pressure, positive regulation of angiogenesis, inflammatory factors, endothelial cell proliferation and migration, and oxidative stress. Previous studies have shown that these processes are involved in the pathological processes of PE. Specifically, PE is a main complication of pregnancy, manifested as dysregulation of blood pressure . The decIn order to further obtain the core targets among the intersecting targets, the PPI network between the intersecting targets and the TP network between the intersecting targets and the signaling pathways were constructed and analyzed. Eventually, six core targets, namely, TNF, IL6, IL1B, VEGFA, STAT3, and EGFR, were required.via activation of NOD-like receptor protein 3 (NLRP3) inflammasome , HIF-1 signaling pathway, and TNF signaling pathway. Further, the interaction between these eight pathways and the signaling pathways related to the six core targets were obtained, and two pathways, the AGE-RAGE signaling pathway in diabetic complications and the HIF-1 signaling pathway, were finally acquired.via possible involvement of placental oxidative and nitrative stress has been reported  can be found in the article/Conceptualization: QS and NJ. Investigation: JS and JW. Methodology: JS and JW. Supervision: QS and NJ. Visualization: JS and JW. Writing—original draft: QS, JS, and JW. Writing—review and editing: QS and NJ. All authors contributed to the article and approved the submitted version.


Immune checkpoint blockades (ICBs) have emerged as a promising treatment for cancer. Recently, tumour mutational burden (TMB) and neoantigen load (NAL) have been proposed to be potential biomarkers to predict the efficacy of ICB; however, they were limited by difficulties in defining the cut-off values and inconsistent detection platforms. Therefore, it is critical to identify more effective predictive biomarkers for screening patients who will potentially benefit from immunotherapy. In this study, we aimed to identify comutated signaling pathways to predict the clinical outcomes of immunotherapy.Here, we comprehensively analysed the signaling pathway mutation status of 9763 samples across 33 different cancer types from The Cancer Genome Atlas (TCGA) by mapping the somatic mutations to the pathways. We then explored the comutated pathways that were associated with increased TMB and NAL by using receiver operating characteristic (ROC) curve analysis and multiple linear regressions.+) could be used as a predictor of increased TMB and NAL and was associated with increased levels of immune-related signatures. In seven independent immunotherapy cohorts, we validated that SpHe-comut+ patients exhibited a longer overall survival (OS) or progression-free survival (PFS) and a higher objective response rate (ORR) than SpHe-comut− patients. Moreover, a combination of SpHe-comut status with PD-L1 expression further improved the predictive value for ICB therapy.Our results revealed that comutation of the Spliceosome (Sp) pathway and Hedgehog (He) signaling pathway (defined as SpHe-comut+ was demonstrated to be an effective predictor of immunotherapeutic benefit in seven independent immunotherapy cohorts and may serve as a potential and convenient biomarker for the clinical application of ICB therapy.Overall, SpHe-comutThe online version contains supplementary material available at 10.1186/s12967-022-03836-3. In recent years, immunotherapy with immune checkpoint blockades (ICBs) has emerged as a promising treatment for advanced-stage cancer. There are three main types of ICB therapies, including inhibition of programmed cell death 1 (PD-1) or ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4), which have been approved by the FDA for a range of solid tumours . HoweverTumour mutational burden (TMB), defined as the total number of somatic mutations per coding area of the genome in a tumour , has gaiPrevious studies have demonstrated that mutations in specific genes may be associated with ICB efficacy , 13. ForThus, several pathway-based biomarker identification methods have been developed. Li et al. revealed that substantial alterations of genes in the Notch pathway may predict the prognosis of NSCLC patients . AnotherIn this study, we comprehensively analysed the signaling pathway mutation status of 9763 samples across 33 different cancer types from The Cancer Genome Atlas (TCGA) to explore the association between the comutated pathway and immunotherapy response. ROC curve and multiple linear regression analyses were performed to determine the optimal combinations of comutated signaling pathways associated with increased TMB and NAL. Moreover, we further explored the associations between comutation in signaling pathways and the efficacy of immunotherapy. Collectively, we aimed to identify comutated signaling pathways to predict the clinical outcomes of immunotherapy, which may provide a potential and convenient biomarker for the future clinical application of immunotherapy.https://portal.gdc.cancer.gov/), and their somatic mutations (whole-exome sequencing), gene expression (RNA-seq), and clinical data  were downloaded accordingly. Then, the corresponding neoantigen data of 5446 TCGA cancer patients across 20 cancer types were downloaded from the TCIA (https://tcia.at/neoantigens) [We first collected 9763 cancer patients across 33 cancer types from the GDC TCGA data portal (ntigens) , and thentigens) . To furtntigens) ; 2) thentigens) ; (3) thentigens) , 32; (4)ntigens) ; (6) thentigens) . Patient thentigeWe then downloaded the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the MsigDB database  and manuUsing TCGA somatic mutation data, we constructed a non-silent gene binary mutation matrix, in which each column represents a sample and each row represents a mutated gene. If a gene mutation occurred in one sample, the element was 1; otherwise, it was 0. In our study, we extracted only non-silent somatic mutations in the genomic coding regions, including missense mutations, nonsense mutations, insertions, deletions, and splice mutations. Genes with mutation frequencies greater than 1% were considered. Then, we mapped the mutated genes into signaling pathways, and if a pathway involved at least one mutated gene, the pathway was defined as mutated. Pathways with a mutation frequency of at least 1% were retained. For each pathway, we used the multivariate Cox proportional hazard model to estimate the association between the pathway mutation status and overall survival (OS) by accounting for known covariates such as patient age, sex, and cancer type. The statistical significance levels  of pathways were then corrected with the false discovery rate (FDR) method proposed by Benjamini and Hochberg . Then, thttps://tcia.at/neoantigens). We intended to identify the comutated pathways associated with high TMB and NAL. To do this, we first divided the TCGA cancer patients into high TMB/NAL and low TMB/NAL groups according to the top quartile of TMB/NAL levels. Receiver operating characteristic (ROC) curve analysis was then used to determine the cut-off points for pathway mutation counts based on the highest Youden index. According to the cut-off of pathway mutation counts, we further screened which pathways were comutated. Multiple linear regression analysis was used to test the correlation between the survival-associated pathways and TMB/NAL. Considering the consistency of TMB and NAL data, the pathways with a suitable β coefficient were retained as comutated (comut+) pathways.High TMB and NAL were proposed to be associated with an improved response to ICB treatment . In thisSurvival analysis (OS or PFS) for the pathway comutation status was performed by Kaplan–Meier curves, and their significance was assessed by the log-rank method. Fisher’s exact test was used to identify the association between the pathway comutation status and ORR.+ and comut− patients. The resulting p values were corrected using the FDR method.We first collected immune response-related genes, including immune checkpoints, T-cell receptor genes, tumour microenvironment (TME)-related genes, interferon-gamma (IFNγ)-associated genes, etc., from the published articles –41, whic+ and comut− patients. The immune score was estimated by the “ESTIMATE” R package [Then, we compared the differences in immune-related signature features, including immune score, cytolytic activity (CYT) score, major histocompatibility complex (MHC)-I score, and T-cell-inflamed immune gene expression profile (GEP) score, between comut package . The CYT package  and MHC- package  were cal package  was esti+ and comut− patients, and then the functional gene sets or pathways were mapped to the ranked list. We performed these analyses using the “clusterProfiler” package [Furthermore, the GSEA method was applied to identify the pathways associated with pathway comutation status. Specifically, a ranked gene list was produced based on the t-score of expression values between comut package .+ status. We used I2 statistics to assess the heterogeneity across different immunotherapy cohorts. Pheterogeneity > 0.1 and I2 < 50% indicated that there was no significant inter-cohort heterogeneity, and the results were consistent in these cohorts.To pool outcomes across multiple immunotherapy cohorts, the random-effect inverse-variance weighted approach was employed, which is computed by hazard ratio (HR) and its 95% confidence interval (CI) to assess the magnitude of influence on therapeutic benefits such as ORR, PFS, and OS based on pathway comut+”, those with mutations in one or more signaling pathways; “Pathway−”, those without mutations in any of the signaling pathways. Comparing the TMB and NAL levels between the two subgroups, we found that both TMB and NAL levels were significantly higher in the Pathway+ patient subgroup than in the Pathway− patient subgroup by analysing the sequencing data from TCGA  by accounting for known covariates, including patient age, sex, and cancer type. With FDR < 0.05, six significant pathways, such as the ECM receptor interaction (ECMRI) pathway, Spliceosome (Sp) pathway, Hedgehog (He) signaling pathway, and RNA degradation pathway, etc., were used for the following analysis  pathway, Hedgehog (He) signaling pathway, and the ECMRI pathway, which have suitable β coefficients and contribute more to both TMB and NAL, were identified Fig. C, D. To + (7.19%) among the 9763 cases in the TCGA database. The number of SpHe-comut+ patients varied among different cancer types, ranging from 0% in KICH to 25.89% in SKCM , except for the NAL levels of KIRC and LIHC. This may be due to the relatively low NAL levels in KIRC and LIHC  status. The results showed that SpHe-comut+ contributed significantly to MSI-H  and predicted MSI-H with a sensitivity of 63.22% and a specificity of 94.15% /SpHe-comut+ to MSS/SpHe-comut−, a significant difference in TMB was also observed -I score, and T-cell-inflamed immune GEP score –45. To esis Fig. A. The CYoup Fig. B–D. It w+, GSEA was performed. A ranked gene list was constructed based on the gene differential expression levels (T-score) between the SpHe-comut+ and SpHe-comut− groups across all cancer patients, and the gene sets of hallmark pathways from the MsigDB database were mapped to the ranked list. The results revealed that several immune-related hallmark pathways were significantly enriched in the SpHe-comut+ patient group, such as the interferon-gamma (IFNγ) response, DNA repair, and inflammatory response pathways . We then compared the mean expression levels of these 39 genes between the SpHe-comut+ and SpHe-comut− groups in each cancer type. Interestingly, the heatmap of mean difference expression levels of these genes showed that the cancer types with an approved indication of immune therapy  clustered together, and these genes presented higher mean expression levels in SpHe-comut+ patients in these cancer types  family, which has been reported to promote the innate immune response to shape and improve the patient’s adaptive immune response [+ patients may tend to respond to immunotherapy.Additionally, we investigated the differential expression levels of immune response-related genes between the SpHe-comutresponse . These f+ status can be used to predict the efficacy of immunotherapy, three immunotherapy cohorts were obtained from published studies for this analysis, including the Inova cohort (melanoma), the Rizvi cohort (NSCLC), and the Miao cohort (pan-cancer) 1 plus anti-CTLA4, 25 patients were defined as SpHe-comut+, and those patients had a significantly longer PFS  vs. SpHe-comut− 7.5 months; HR, 0.27; 95% CI, 0.11–0.64; log-rank test p = 0.002; Fig. + patient group  vs. SpHe-comut− 5.2 months; HR, 0.31; 95% CI 0.091–1.1; log-rank p = 0.047; Fig. + 75% vs. SpHe-comut− 30.7%; Fisher’s exact test, p = 0.042; Fig. + 26.68 vs. SpHe-comut− 20.39 months; HR, 0.69; 95% CI 0.47–1; log-rank p = 0.050; Fig. + 39.4% vs. SpHe-comut− 21.5%; Fisher’s exact test, p = 0.002; Fig. + patient group. These results suggested that SpHe-comut+ had a good prognostic predictive ability in three immunotherapy cohorts and can be used as a predictor for the efficacy of immunotherapy.To explore whether the SpHe-comuter) see “”. The Sp+, the other four public immunotherapy cohorts with sufficient information on genomic alterations and survival were analysed. The four public immunotherapy cohorts included (1) the Hellmann cohort of 75 NSCLC patients treated with anti-PD-1 plus anti-CTLA4; (2) the Allen and Snyder cohort, a pooled analysis of two cohorts of 174 metastatic melanoma patients treated with anti-CTLA4; and (3) the MSKCC cohort of 1661 pan-cancer patients treated with anti-PD-(L)1 or anti-CTLA4. Our analysis revealed a significant positive association between SpHe-comut+ and TMB and NAL levels , 1.74; 95% CI 1.41–2.15; p < 0.001, Fig. heterogeneity > 0.10 and I2 < 50%), indicating the consistency of the association between SpHe-comut+ status and favourable benefits to ICBs across these cohorts.Next, a survival analysis was performed in the Hellmann, Allen and Snyder, and MSKCC immunotherapy cohorts to explore the impacts of SpHe-comut status on PFS and OS. The survival analysis showed that SpHe-comutrts Fig. A–C. The Furthermore, we performed multivariate Cox regression analyses of SpHe-comut status and survival by adjusting for clinical features  in the Inova cohort, Rizvi cohort, and Miao cohort. In each cohort, we found that SpHe-comut was an independent prognostic factor  patients had longer PFS than either single-positive (single comut+) or dual-negative (both comut−) patients  vs. not reached (NR) vs. 3.8 months, log-rank test, p = 0.012, Fig. + groups compared to the other two groups  and PD-L1 expression  were two independent predictor variables for anti-PD-(L)1 treatment  obtained more favourable PFS than either single positive (SpHe-comut+ or PD-L1high) or dual negative patients  vs. 14.5 vs. 2.1 months, log-rank test, p < 0.001, Fig. + and PD-L1high groups compared to the two other groups 1 blockade immunotherapy . In the In addition, we collected some mutated markers that have been regarded as stable immunotherapy markers in NSCLC, including those of the Li et al. method  and Bai Recently, immunotherapy with immune checkpoint blockades (ICBs) has emerged as a promising treatment for cancer. TMB is a biomarker that can be used to predict patient response to cancer immunotherapy, indicating that patients with high TMB have better responses to ICB therapy. An increased neoantigen load is associated with a better response to ICB treatment as well. However, these indicators have limited use due to the lack of a validated cut-off value. Cancer is a disease in which cells acquire the ability to divide and proliferate uncontrollably, usually through genetic changes in specific genes. Mutation profiles differ dramatically between individuals with cancer. They may be used as indicators for treatment response and disease progression. Applying a molecular pathway-based approach to cancer mutations can dramatically boost their biomarker potential. Therefore, investigation of genomic mutation pathways may lead to the discovery of effective biomarkers for immunotherapy.+ were validated in seven independent immunotherapy cohorts. In the melanoma cohort, the SpHe-comut+ group exhibited longer median PFS , indicating the consistency of the association between SpHe-comut+ status and clinical results of ICBs across these cohorts  had longer survival and higher response proportions to immunotherapy than either single-positive (single comut+) or dual-negative (both comut−) patients in the Rizvi, Inova, and Miao cohorts, respectively  or single-positive status (SpHe-comut+ or PD-L1high) obtained more favourable PFS and DCB than patients with dual-negative status (SpHe-comut− and PD-L1low status)  Fig. C, D. Bas+ pattern was handicapped by the limited information available regarding the functions of different mutations. This suggests that the development of immunotherapeutic biomarkers requires more advanced sequencing techniques and nanomaterials, such as carbon nanotubes [+ pattern may provide higher accuracy for efficacy prediction for ICB delivery.The advantage of our method is that it provides an optional and cost-effective approach by offering a small panel of genes in the signature pathways that can easily be translated into an easy-to-use clinical assay to identify potential immunotherapy responders. However, there are still limitations in our research. For example, we did not differentiate with respect to whether the gene mutations in the pathway were functional. Otto Warburg suggested that defects in mitochondrial respiration may be the underlying cause of cancer , and it anotubes , 56. If +) was associated with increased TMB and NAL levels, as well as increased levels of immune-related signatures. Furthermore, the patients with SpHe-comut+ exhibited a higher ORR and a longer OS or PFS than SpHe-comut− patients in seven independent immunotherapy cohorts. Our results suggest that SpHe-comut+ might provide an optional and cost-effective approach for identifying potential immunotherapy responders, which might complement recent immunotherapy biomarkers.In summary, the pan-cancer analysis revealed that comutation of Spliceosome (Sp) pathways and the Hedgehog (He) signaling pathway  for PFS or OS in the ICBs cohorts. Figure S5. Comparison of performance for predicting ORR among SpHe-comut, DDR-comut and TMB as a predictive biomarker. Figure S6. Combining SpHe-comut status with DDR-comut status for the prediction of ICB therapy. Figure S7. Comparison of SpHe-comut status with NOTCH and GMS for the prediction of ICB therapy in the Rizvi cohort. Table S1. Data source. Table S3. Gene list of immune-related gene signature. Table S4. The significant survival-associated signaling pathways identified by the multivariate Cox regression model adjusted by clinical factors. Table S5. Comparison of tumor mutational burden between SpHe-comut+ and SpHe-comut− groups across different cancer types. Table S6. Comparison of neoantigen load between SpHe-comut+ and SpHe-comut− groups across different cancer types.Additional file 2: Table S2. The Information for time period and dose selection of involved cohorts.


Epimedium (EPI) is a common Chinese herb with neuroprotective effects against a variety of central nervous system disorders, especially spinal cord injury (SCI). In this study, we performed network pharmacology and molecular docking analyses to reveal the mechanism underlying EPI treatment of SCI, then validated its efficacy using animal models.The active ingredients and targets of EPI were screened by Traditional Chinese Medicine Systems Pharmacology (TCMSP) and their targets annotated on the UniProt platform. SCI-related targets were searched from OMIM, TTD, and GeneCards databases. We employed the STRING platform to construct a protein–protein interaction (PPI) network then visualized the results using Cytoscape (3.8.2) software. We also subjected key EPI targets to ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, then docked the main active ingredients with the key targets. Finally, we established an SCI rat model to evaluate efficacy of EPI in treating SCI and validate the effects of different biofunctional modules predicted by network pharmacology.A total of 133 EPI targets were associated with SCI. GO terms and KEGG pathway enrichment results showed that EPI’s effect in treating SCI was significantly associated with inflammatory response, oxidative stress and the PI3K/AKT signaling pathway. Molecular docking results indicated that EPI’s active ingredients have a high affinity for the key targets. Results from animal experiments revealed that EPI not only markedly improved Basso, Beattie, and Bresnahan scores in SCI rats, but also significantly improved p-PI3K/PI3K and p-AKT/AKT ratio. Moreover, EPI treatment not only mediated a significant decrease in malondialdehyde (MDA) but also increased both superoxide dismutase (SOD), and glutathione (GSH). However, this phenomenon was successfully reversed by LY294002, a PI3K inhibitor.EPI improves behavioral performance in SCI rats through anti-oxidative stress, which may be mediated by activation of the PI3K/AKT signaling pathway. Spinal cord injury (SCI) is a serious traumatic disease of the central nervous system, characterized by a high disability rate, resulting in abnormalities or loss of motor, sensory, and autonomic functions . SCI hasEpimedium (EPI), a genus in family Berberidaceae with about 52 species, has been found to contain more than 260 compounds . In tradNetwork pharmacology, first proposed by Hopkins in the UK, is a technique used to systematically study the biological effects of complex drugs by integrating drug action with biological networks based on systems biology and multidirectional pharmacology . Numerouhttps://www.Uniprot.org/; Traditional Chinese Medicine Systems Pharmacology (TCMSP) is a comprehensive pharmacological database of traditional Chinese medicine, comprising 3,311 targets and 29,384 components of 499 Chinese herbal medicines included in The Chinese Pharmacopoeia . In the We searched GeneCards , OMIM H, and TTDWe employed the STRING database to build a PPI network, we uploaded the potential therapeutic targets of EPI for SCI to the STRING database and set the parameter to an interaction value >0.7 . We thenp ≤ 0.05.Next, we explored the biological processes and signaling pathways associated with EPI in treatment of SCI by performing GO and KEGG pathway enrichment analyses using the clusterProfiler package in R . StatistThe interrelationships between key targets and active ingredients of EPI were visualized using the Sankey diagram. We also generated 3D structures of the active compounds using Chem Office software. These 3D structures were then downloaded from the PDB database, then the proteins dehydrated and dephosphorylated using PyMOL software . AutodocAdult female Sprague–Dawley rats (10-week-old) were purchased from Beijing Huafukang Biotechnology Co (license no: SCXK (Jing) 2019-008). Prior to experimental treatments, all rats were acclimated for 7 days in a controlled environment under a 12-h light/dark cycle with free access to food and drink. All animal experiments were approved by the Experimental Animal Ethics Committee of Tianjin People’s Hospital. Rats were anesthetized by intraperitoneal injection (ip), using 1% sodium pentobarbital (50 mg/kg), with the appropriate anesthetic effect confirmed by the pain reflex in the tail. After anesthesia, the rats were fixed in prone position, and the dorsum routinely prepared and disinfected with skin. The rats were subjected to laminectomy, at the Tenth thoracic vertebra (T10) level by incising each subcutaneous layer sequentially to fully expose the T10 segment of the spinal cord, which was not subject to impact injury. Next, a 10-gram impactor device (diameter: 2 mm) was lowered into the spinal cord from a height of 5 cm, to generate moderate spinal cord contusion . After mEPI was decocted twice  in distilled water, then combined. The combined extracts were cooled to room temperature, and subsequently stored at −20°C in dry, sealed and contamination-free conditions until further use. LY294002 (154447-36-6), a PI3K inhibitor, was purchased from Aladdin, China and administered by intraperitoneal injection. After surgery, 40 rats were randomly divided into 4 groups and orally administered with EPI extract for 4 weeks. The treatments were as follows: sham group , SCI group , SCI + EPI group , and SCI + EPI + LY group . Since oral administration is the main route of herbal drug delivery, we administered EPI orally to rats. The EPI dosage was adopted from the Chinese pharmacopeia clinical dose converted to body surface area .Researchers, who were completely blinded to the study, assessed hind limb function of the rats by observing their hip, knee, and ankle joint walking movements for 5 min at1, 7, 14, 21, and 28 days post-operation. The function of the hind limbs was evaluated according to the Basso, Beattie, and Bresnahan (BBB) scores, where 0 and 21 considered complete paralysis and normal gait, respectively.th day after operation. Firstly, ventricular perfusion with 0.9% physiological saline was performed under anesthesia, and 4% paraformaldehyde (PFA) injected to fix the tissue. Next, tissue from the center of the injury (1 cm) was obtained and immediately stored at-80°C to await western blot analysis. For H&E staining, spinal cord samples were fixed with 4% PFA, dehydrated in ethanol, dipped in wax, embedded in paraffin and cut into 6 μm sections using a microtome. The sections were then dewaxed and rehydrated, then stained with hematoxylin followed by eosin. The sections were permeabilized using xylene then visualized under a light microscope.Spinal cord tissues were obtained from the rats on the 28Spinal cord tissues, 1.0 cm around the center of the lesion, were collected from 3 individual rats and lysed in RIPA buffer  with a protease inhibitor cocktail . The tissue lysates were centrifuged at 12,000 rpm for 10 min at 4°C, and protein concentration determined using the double Bicinchoninic Acid (BCA) method . Equal concentrations of protein samples were separated on a 12% SDS polyacrylamide gel electrophoresis, then transferred to a polyvinylidene fluoride membrane. Next, the PVDF papers were blocked using skim milk for 1.5 h, then incubated overnight with the primary antibodies against GAPDH , phosphor-Akt , Akt , phosphor-PI3K , and PI3K  at 4°C. The membranes were washed 3 times using Tris-buffered saline Tween, then incubated with the secondary antibodies for 1 h at room temperature. Using ImageJ software, chemiluminescence was identified and measured.p < 0.05.Data were first tested for normality using the Shapiro–Wilk normality test, then normally-distributed data subjected to one-way analysis of variance (ANOVA). Both non-normally distributed data and those with non-uniform variance were subjected to the Kruskal Wallis analysis. Statistical significance was set at Using OB ≥ 30% and DL ≥ 0.18 as filtering conditions resulted in a total of 24 active EPI ingredients from the TCMSP database . From thCombining the three databases, coupled with deleting of repeated targets resulted in 2456 SCI-related targets by GeneCards, OMIM, and TDD databases. Analysis of the intersection between 204 EPI-related and 2,456 and SCI-related targets, resulted in 133 potential targets of EPI for SCI .Based on 133 potential targets and 24 active ingredients, the compounds-targets network diagram of EPI to treat SCI was plotted . CollectNext, we used the STRING program to construct a PPI network of the 133 potential EPI targets for SCI . The resTo elucidate the potential mechanism underlying EPI’s action in SCI, we used the clusterProfiler package to perform GO and KEGG pathway enrichment analyses on the 133 potential EPI targets. The top 10 GO items and enriched KEGG pathways are shown in To validate results of the network pharmacology, we used molecular docking to evaluate the interaction between active ingredients and key targets. A resulting Sankey diagram revealed that the 10 key targets were related to active EPI ingredients. Particularly, quercetin was associated with 10 targets, luteolin corresponded to 9 targets, kaempferol corresponded to 5 targets, while other active ingredients corresponded to 1 target each . MoleculTo assess whether EPI promotes recovery of motor function in SCI rats, we first established an SCI rat model then analyzed the resulting BBB scores to understand behavioral performance. Rats in the sham group exhibited a BBB motor score of 21 at each postoperative time point , with no evidence of hindlimb motor dysfunction . ConversNetwork pharmacology analysis results revealed that EPI treats SCI by modulating the PI3K/Akt signaling pathway. To verify the importance of the PI3K/Akt signaling pathway in SCI treatment, we analyzed expression levels of p-PI3K, PI3K, p-Akt, and Akt proteins in the spinal cord tissue of rats after EPI treatment. We also used LY294002, a specific PI3K inhibitor, to inhibit PI3K/Akt signaling pathway to further validate the importance of EPI for SCI recovery. Western blots revealed that the p-PI3K/PI3K ratio was significantly higher in the spinal cord of SCI rats relative to those in the sham group, whereas the p-AKT/AKT ratio was not significantly different between the groups –C. p-PI3Next, we investigated whether EPI affects the ability to resist oxidative stress through PI3K/AKT pathway by analyzing the effect of EPI on spinal cord tissue in SCI rats. Experimental results revealed that GSH and SOD activities were significantly lower while MDA activity was significantly higher in spinal cord tissue of SCI rats, relative to those in the sham group, consistent with previous results by SCI, a major traumatic disorder of the central nervous system that can lead to permanent motor and sensory impairment, has been shown to affect approximately 930,000 people worldwide each year . In receIn the present study, GO terms indicated that EPI could treat SCI by modulating oxidative stress, inflammatory response, and neuron death, as well as other biological functional modules. The targets were significantly enriched in PI3K/Akt, MAPK, and IL-17 signaling pathway signaling pathways, which are closely related to the SCI signaling pathway. Among them, the PI3K-AKT signaling pathway appears to be the most enriched in SCI-related signaling pathways and seems to be the most important pathway for the treatment of SCI. Previous studies have shown that the PI3K/AKT signaling pathway plays important roles in secondary SCI, such as anti-inflammatory, anti-apoptotic, and regulating glial scar formation . In addiPhosphatidylinositol 3 kinases (PI3Ks), which belong to the lipid kinase family, are activated by various stimuli to produce phosphatidylinositol-3-phosphate (PIP3) which subsequently directly activates conversion of threonine proteins (AKT) into phosphorylated proteins (p-AKT) by stimulating serine-dependent activase PDK; . NotablyPrevious studies have shown that oxidative stress markedly exacerbates both SCI and diabetic neuropathy . MoreoveThis study had some limitations. Firstly, the data obtained from the online databases were based on researched and anticipated material, therefore unverified and undocumented active ingredients or genes might not be considered our study. Secondly, in addition to the biological modules validated in this experiment, EPI may also exert protective effects through other pathways. Further research explorations are needed to validate each biofunctional module. Thirdly, the basis of EPI’s treatment of SCI is that it contains a large number of compounds, and we will conduct more experiments to explore the role of key compounds in EPI.In summary, we employed a combination of network pharmacology and animal experiments to effectively reveal the biological-molecular mechanisms underlying EPI’s action in treatment of SCI. Particularly, our results indicated that EPI ameliorates SCI in rats through antioxidant effects, which may be related to the activation of the PI3K/AKT signaling pathway. These findings affirm EPI’s significance as a potential therapy for treatment of SCI.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Experimental Animal Ethics Committee of Tianjin People’s Hospital.XF, BM, MZ, and RZ designed the experiments and analyzed data. YC, LL, SK, CL, and ZJ performed the experiments. XZ, SF, HZ, and WH wrote the paper. All authors contributed to the article and approved the submitted version.This study was supported by Tianjin Key Medical Discipline  Construction Project (TJYXZDXK-064B), Foundation of Tianjin Union Medical Center (2019JZPY01), and Tianjin Health Commission Science and Technology Project (ZC20225 and KJ20061).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Shiliao Decoction (SLD) was developed for treatment and prevention of cancer-associated malnutrition (CAM) in China. In this study, we aim to discover SLD’s active compounds and demonstrate the mechanisms of SLD that combat CAM through network pharmacology and molecular docking techniques.All components of SLD were retrieved from the pharmacology database of Traditional Chinese Medicine Systems Pharmacology (TCMSP). The GeneCards database and the Online Mendelian Inheritance in Man database (OMIM) were used to identify gene encoding target compounds, and Cytoscape was used to construct the drug compound–target network. The network of target protein-protein interactions (PPI) was constructed using the STRING database, while gene ontology (GO) functional terms and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways associated with potential targets were analyzed using a program in R language (version 4.2.0). Core genes linked with survival and the tumor microenvironment were analyzed using the Kaplan–Meier plotter and TIMER 2.0 databases, respectively. Protein expression and transcriptome expression levels of core gene were viewed using the Human Protein Atlas (HPA) and the Cancer Genome Atlas (TCGA). A component-target-pathway (C-T-P) network was created using Cytoscape, and Autodock Vina software was used to verify the molecular docking of SLD components and key targets.The assembled compound–target network primarily contained 134 compounds and 147 targets of the SLD associated with JUN, TP53, MAPK3, MAPK1, MAPK14, STAT3, AKT1, HSP90AA1, FOS, and MYC, which were identified as core targets by the PPI network. KEGG pathway analysis revealed pathways involved in lipid and atherosclerosis, the PI3K/Akt signaling pathway, and immune-related pathways among others. JUN is expressed at different levels in normal and cancerous tissues, it is closely associated with the recruitment of different immune cells and has been shown to have a significant impact on prognosis. The C-T-P network suggests that the active component of SLD is capable of regulating target genes affecting these related pathways. Finally, the reliability of the core targets was evaluated using molecular docking technology.This study revealed insights into SLD’s active components, potential targets, and possible molecular mechanisms, thereby demonstrating a potential method for examining the scientific basis and therapeutic mechanisms of TCM formulae. Many malnourished cancer patients exhibit associated pathophysiological changes such as inflammation and immunosuppression, and a complex interplay exists between these two factors and cancer-associated malnutrition (CAM). Localized tumor cells and immune cells in the microenvironment interact to release various types of cytokines that promote systemic inflammation, contributing to malnutrition and poor outcomes by causing anorexia, altering metabolism, and increasing resting energy expenditure and muscle breakdown . AccordiNutrition and health have been incorporated into China’s national strategy in “Health China 2030” and the “National Nutrition Plan (2017-2030),” which place particular focus on rapidly improving the nutrition of cancer patients. Although there is a great need for effective therapeutic options to address CAM, there is a lack of pharmacological agents that can be safely administered as part of long-term treatment. Traditional Chinese medicine (TCM) is a major component of complementary alternative medicine and can play important roles in the entire cancer treatment process, up to and including palliative care in advanced stages. Our team has long been concerned about the metabolic and immune disturbances in tumor patients and have previously designed a therapy formula named Shiliao Decoction (SLD) aimed at improving nutrition levels, drawing on the ideas behind the Wu Zhi An Zhong and Da Jian Zhong decoctions . PreviouNetwork pharmacology can create a relationship prediction model between drugs and disease targets, integrate an interaction network to analyze drug interactions with specific nodes in each network module, and investigate the interaction relationship between drugs and potential targets from a systematic perspective , 6. NetwCodonopsis pilosula (Franch.) Nannf , Zanthoxylum bungeanum Maxim , Citrus reticulata Blanco , Coix lacryma-jobi L.var.ma-yuen (Roman.) Stapf , Dioscorea opposite Thunb , Typha angustifolia L., Amomum villosum Lour , Zingiber officinale Rosc , Ziziphus jujuba Mill. , Glycyrrhiza uralensis Fisch , all of which are plants known to have medicinal properties. Details on the composition of SLD can be found in 1 and drug names were confirmed using the Chinese Pharmacopeia 2020 edition.SLD is a decoction of 10 botanicals extracted by water, including 2  Database and Analysis Platform (TCMSP)2 . Oral bi2 . Target 2  to obtain a bubble map of the results of the GO and KEGG enrichment analyses. Biological process (BP), cellular component (CC), and molecular function (MF) are all included in the GO functional analysis.Target gene analysis was performed using the “cluster profile” and “pathview” function package in R, a well-known R package with dynamically updated data for KEGG and GO analysis. Results were screened using a threshold P-values with 95% confidence intervals (CIs) and hazard ratios (HRs) were calculated.The Kaplan-Meier Plotter  is a pow7  and the association with prognosis.The TIMER database7  contains8 and the expression of target genes in normal and cancer tissues was compared. Transcriptome data of cancer-related genes were obtained from The Cancer Genome Atlas (TCGA).9Immunohistochemistry (IHC) can reveal the relative distribution and abundance of proteins by utilizing the high specificity of antibody-antigen binding. Immunohistochemical data were obtained from the Human Protein Atlas (HPA),via Cytoscape (An appropriate component-target-pathway (C-T-P) network was constructed and visualized ytoscape  to analy10  Nannf., 88 compounds from Glycyrrhiza uralensis Fisch., 6 compounds from Coix lacryma-jobi L.var.ma-yuen (Roman.) Stapf., 19 compounds from Ziziphus jujuba Mill., 9 compounds from Amomum villosum Lour., 12 compounds from Dioscorea opposite Thunb.Target prediction of SLD ingredients using the TCMSP database and screening (for OB ≥ 30% and DL ≥ 0.18 as described in methods) identified 172 compounds with sufficiently high OB and DL. They consisted of 5 compounds from The UniProt database was used to search for information on potential targets of the active ingredients identified by the TCMSP database. Target gene names were standardized, and 261 target genes were obtained after removal of duplicates.2,581 CAM-related genes were identified by searching the GeneCards database, and 168 CAM-related genes were separately identified in the OMIM database. Following combination of these two sets and removal of duplicates, a total of 2,706 CAM-related genes were selected for further analysis.In order to identify potential gene targets of SLD with an effect on CAM, the set of 2,706 CAM-related genes selected earlier was compared with the set of 261 target genes of SLD components. A total of 147 potential target genes were found to be members of both these sets.Cytoscape software was used to visualize the compound-target relationships and rank the components of SLD in descending order of degree. The top 12 key compounds are listed in We created a PPI network by importing the 147 target genes selected earlier into the STRING database. Cytoscape software was used to visualize the PPI network. Screening was carried out using a threshold score value of 0.9 to provide a high level of confidence for protein interactions, and unconnected nodes in the network were concealed, as seen in GO enrichment analysis found that BP mainly included pathways related to cellular metal ion response, response to drugs, and response to radiation. CC included the endocytotic activation pathway of membrane rafts and membrane microfilms among other pathways. MF mainly included pathways related to DNA binding transcription factor and ubiquitinated protein ligase . PathwayJUN as a representative gene and focused on cancer types with co-occurring malnutrition to explore the association between them. Significant correlations (P ≤ 0.05) were found between JUN expression levels and survival in patients with liver hepatocellular carcinoma (LIHC), esophageal squamous carcinoma (ESCA) and rectal adenocarcinoma (READ), as shown in Based on these results, we selected JUN expression levels did not significantly correlate with tumor purity, but more likely affected prognosis by regulating the tumor microenvironment. For example, in ESCA, JUN expression was positively correlated with infiltration of CD8+ T cells and dendritic cells. In LIHC, JUN expression was positively correlated with infiltration of CD4+ T cells, macrophages, and neutrophils while in READ, JUN expression was positively correlated with CD4+ T cell, macrophage and dendritic cell infiltration.To further explore possible immunological mechanisms, we looked at the relationship between gene expression, tumor tissue purity, and the abundance of immune infiltrating cells in the microenvironment using the TIMER database. Tumor tissue purity is the proportion of cancer cells in a tumor sample, which is critical to selecting genes related to deconvolving immune cells in the tumor tissue. This was inferred from copy number alteration data using the R package CHAT . As seenTo verify the potential of the identified proteins as therapeutic targets, we analyzed the differential expression of proteins in cancer and cancer-adjacent tissues. IHC results from the HPA database showed that JUN expression in cancer tissues was higher than that in normal liver and rectum tissues. On the other hand, antibody staining levels of JUN in ESCA cancer tissues were lower than those in adjacent tissues .JUN was significantly overexpressed in normal tissues than in liver cancer tissues.The TCGA database was used to expand the sample size for validation and obtain expression data at the transcriptome level for this gene. The box plot of differential expression analysis  showed tA “C-T-P” network was constructed based on the 30 most relevant signaling pathways obtained in We performed molecular docking between the top 6 protein targets in the “active ingredient-target-disease” network and the top 10 active ingredients in the SLD using Autodock vina. Based on similar experiments, binding energy < –4.25 kJ-mol-1 was interpreted as indicating that the active ingredient had some binding ability for the target, binding energy < –5.0 kJ/mol indicated good binding activity, and binding energy < –7.0 kJ/mol indicated strong binding activity . AnalysiIncreased catabolism of fat, muscle, and other tissues is a clinical characteristic shared by malnutrition and cancer cachexia, a specific kind of CAM characterized by persistent inflammation . Notablyin vitro and in vivo. Flavonoids have been shown to lower triglyceride and cholesterol levels and reduce inflammatory mediators  family, plays an important role in processes including cell proliferation, differentiation, and tumor transformation . A studyin silico docking simulation determines the detailed mode of binding of kaempferol to PD-1/PD-L1 and these results suggest that kaempferol can potentially be developed as a potent small molecule inhibitor for PD-1/PD-L1 blockade  can be found in the article.SL and SJ wrote the manuscript, performed GO, KEGG. immune infiltration, survival analysis, expression differences, and molecular docking. HX modified the figures. PX edited and improved the manuscript. SL compiled the TCM database and target capture. SJ collated the disease target database. SZ directed the research and proposed changes to the manuscript. All authors reviewed the manuscript and approved the final version of the manuscript.


To identify the potential key genes and molecular pathways associated with keratoconus and allergic disease.  The pubmed2ensembl database was used to identify the text mining genes (TMGs) collectively involved in keratoconus and allergic disease. The GeneCodis program was used to perform the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of TMGs. The protein-protein interaction (PPI) network of the TMGs was established by STRING; the significant gene modules and hub genes of PPI were further performed using the Cytoscape software. The DAVID database was used to perform the GO and KEGG analyses of the significant module.  In total, 98 TMGs collectively involved in keratoconus and allergic disease were identified. 19 enriched biological processes including 71 genes and 25 enriched KEGG pathways including 59 genes were obtained. A TMG PPI network was constructed, and 51 genes/nodes were identified with 110 edges; 3 most significant modules and 12 hub genes were chosen from the PPIs. GO enrichment analysis showed that the TMGs were primarily associated with collagen catabolic process, extracellular matrix organization and disassembly, cell adhesion and migration, collagen-containing extracellular matrix, extracellular matrix, and structure organization. KEGG pathway analysis showed that these DEGs were mainly involved in the IL-17 signaling pathway, inflammatory bowel disease, rheumatoid arthritis, allograft rejection, T cell receptor signaling pathway, cytokine-cytokine receptor interaction, and TNF signaling pathway. IL10, IL6, MMP9, MMP1, HGF, VEGFA, MMP3, MMP2, TGFB1, IL4, IL2, and IFNG were potential key genes involved in keratoconus. IL-17 signaling pathway was the potential pathways accounting for pathogenesis and development of keratoconus. The results revealed that  Keratoconus (KC) is a corneal ectasia disease characterized by thinning and steepening, which would cause irregular astigmatism and progressive myopia, leading to further loss of vision . KC is cAt present, various treatments for KC exist, including spectacles, contact lenses, corneal collagen cross-linking (CXL), and corneal surgery. However, these treatments present several limitations. In its early stages, spectacles or rigid contact lenses only improve the visual impairment but cannot delay or prevent the degeneration of KC . CXL canKC is a multifactorial disease that involves several genes and environmental factors . AllergiCorneal stroma, which consists of keratocytes and extracellular matrix (ECM), is the main structural fraction of the cornea accounting for 90% of corneal thickness . And theText mining, an effective method to quickly extract critical information from a large amount of the biomedical literature, has been widely used to explore novel associations between genes and pathologies . In recehttp://pubmed2ensembl.ls.manchester.ac.uk/) [The pubmed2ensembl website (.ac.uk/)  is an onP value cut-off (P = 0.00001) was set.The GeneCodis  was usedhttps://string-db.org/), a database includes 3 billion interactions associated with 24.6 million proteins referred to 5090 organs [In order to assess functional associations among the products of the selected TMGs, the STRING  was used to visualize the PPI network . Then, tMoreover, to select hub genes from the PPI network, the cytoHubba plugin of Cytoscape was used, which was computed by four ranking methods: EPC (edge percolated component), MCC , MNC , and DMNC (density of maximum neighborhood component) . The tophttps://david.ncifcrf.gov/) was used to perform GO enrichment analysis and KEGG pathway enrichment analysis. The GO enrichment analysis includes biological process (BP), cellular component (CC), and molecular function (MF) [P < 0.05 was considered statistically significant.To clarify the functions of the significant genes module, the DAVID database (ion (MF) . The funion (MF) . P < 0.0Based on the data mining strategy that is described in The 98 TMGs were analyzed for GO biological processes (BP) using GeneCodis to identify the most enriched terms closely related to the pathology of keratoconus. As shown in Next, the 71 TMGs were further analyzed for KEGG pathways using GeneCodis. The KEGG pathway enrichment analysis identified 25 significant pathways that involved 59 TMGs. The top 10 KEGG pathway contained malaria, allograft rejection, rheumatoid arthritis, cytokine-cytokine receptor interaction, pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, graft-versus-host disease, type I diabetes mellitus, and inflammatory bowel disease .The PPI network of the 59 selected TMGs was constructed using the STRING database, which had a total of 51 nodes with 110 edges . FurtherIL10, IL6, MMP9, MMP1, HGF, VEGFA, MMP3, MMP2, TGFB1, IL4, IL2, and IFNG, were considered to be hub genes  family, which is composed of IL-17A–F, plays a crucial role in acute and chronic inflammatory reactions . IL-17 hd C/EBPs . Gomes ed C/EBPs . Karolakd C/EBPs . The prod C/EBPs . Therefoβ includes three isoforms in mammals, TGF-β1, TGF-β2, and TGF-β3, and is a key mediator of fibrogenesis [β markers in severe keratoconus patients [β contributes to upregulate the expression of MMP2 by modulating Smad2 [β. These genes are the potential key genes that may be involved in KC.Interleukin-10 (IL-10) is an anti-inflammatory cytokine with important roles in preventing T helper type 1 cells from producing cytokine. Several studies have shown that there was no obvious change in IL-10 in tears of keratoconus and control subjects –53, whilogenesis . A recenpatients . It was ng Smad2 . Interfeng Smad2 . It was Strengths of our study include the fact that it is one of the first to use text mining and bioinformatics analysis to identify the potential crucial genes and key pathways of KC based on the common genes involved in KC and allergic disease. Limitations of this study must also be acknowledged. Firstly, this study explored the molecular mechanism of keratoconus in the gene level using bioinformatics analysis; further experimental studies are required to verify the results. Secondly, adolescents' population is a high-risk group for KC, but we did not include the age factor in this research; future investigations taking the age into consideration may provide more accurate and comprehensive conclusions.IL10, IL6, MMP9, MMP1, HGF, VEGFA, MMP3, MMP2, TGFB1, IL4, IL2, and IFNG, that may be involved in the keratoconus as well as in allergic diseases. These genes were enriched in the HIF-1 signaling pathway, T cell receptor signaling pathway, and TNF signaling pathway. Extracellular matrix remodeling and immune inflammatory response may be the key alterations in KC. The absence of experimental validation is a limitation of this study, and further studies are needed.In conclusion, we identified 12 hub genes,