Salvia miltiorrhiza (SM) on diabetic nephropathy (DN) were examined using a systematic network pharmacology approach and molecular docking. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to screen active ingredients of SM. Targets were obtained using the SwissTargetPrediction and TCMSP databases. Proteins related to DN were retrieved from the GeneCards and DisGeNET databases. A protein–protein interaction (PPI) network was constructed using common SM/DN targets in the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The Metascape platform was used for Gene Ontology (GO) function analysis, and the Cytoscape plug-in ClueGO was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Molecular docking was performed using iGEMDOCK and AutoDock Vina software. Pymol and LigPlos were used for network mapping. Sixty-six active ingredients and 189 targets of SM were found. Sixty-four targets overlapped with DN-related proteins. The PPI network revealed that AKT serine/threonine kinase 1 (AKT1), VEGFA, interleukin 6 (IL6), TNF, mitogen-activated protein kinase 1 (MAPK1), tumor protein p53 (TP53), epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase 14 (MAPK14), and JUN were the ten most relevant targets. GO and KEGG analyses revealed that the common targets of DN and SM were mainly involved in advanced glycation end-products, oxidative stress, inflammatory response, and immune regulation. Molecular docking revealed that potential DN-related targets, including tumor necrosis factor (TNF), NOS2, and AKT1, more stably bound with salvianolic acid B than with tanshinone IIA. In conclusion, the present study revealed the active components and potential molecular therapeutic mechanisms of SM in DN and provides a reference for the wide application of SM in clinically managing DN.The mechanisms underlying the therapeutic effect of  Diabetic nephropathy (DN) is a serious complication that is common in diabetic patients. As the incidence and mortality of diabetes increase yearly, the prevalence of DN rises sharply  and DN hSalvia miltiorrhiza (SM) has a longstanding history of use for promoting blood circulation in TCM. Its main actions are reducing blood viscosity, improving hemorheological characteristics, accelerating fibrin degradation . We used pharmacokinetic information retrieval filters for absorption, distribution, metabolism, and excretion (ADME) screening based on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. Active compounds without potential target information were excluded. Some active compounds that were not predicted but reported in the literature were also included.SM active components were retrieved from the TCM Systems Pharmacology Database [TCMSP, http://www.Swisstargetprediction.ch). Then, the targets were standardized in the UniProt (https://www.uniprot.org) database with status set as ‘reviewed’ and species set as ‘human’ [Targets were obtained from the TCMSP and SwissTargetPrediction databases ( ‘human’ . After rhttps://www.genecards.org/) and the DisGeNET Database (https://www.disgenet.org/home/) using the search term ‘diabetic nephropathy.’Potential DN-related targets were retrieved from the Human Gene Database  for analysis, and common targets were identified as DN-related targets of the active components for further analysis.Screened targets of the active components and DN-related proteins were imported into a Venn diagram webtool (https://string-db.org/), and a protein–protein interaction (PPI) network was constructed. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) is a comprehensive multifunctional data platform [Homo sapiens,’ the confidence score cutoff was set at 0.4, and other settings were set to default.To study the interactions between the active components of SM and their target proteins, drug–disease intersection target genes were searched using the interaction database platform STRING v.11.0  has a comprehensive annotation function, and gene annotation data are updated on a monthly basis [The Metascape platform (ly basis . SM targP set to <0.01 and the κ score set to ≥ .53.Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted using the Cytoscape plug-in ClueGO. The candidate DN-related genes targeted by SM were entered into the ClueGO plug-in, with https://www.rcsb.org/) [Using KEGG pathway enrichment analysis, we identified the potential DN-related genes targeted by SM active components. These targets were confirmed by molecular docking with the experimentally verified SM active components. The verified components were tanshinone IIA, which can improve kidney hypertrophy and 24-h urine protein excretion , and salsb.org/) . The comFrom the molecular docking results, we selected the top five receptor proteins with the lowest energy value and the ligand that bound to these receptor proteins most stably and ran AutoDock Vina for docking. Pymol and LigPlos software was used for network visualization and construction, respectively.From the TCMSP database, we obtained 202 known active compounds of SM, which we screened based on OB ≥ 30% and DL ≥ 0.18, yielding 65 active ingredients that met the conditions. In addition, we also included the known active compound salvianolic acid B, which was screened out by ADME. Finally, 66 active components were selected for further analysis .Candidate targets of active components in SM were searched from the TCMSP and SwissTargetPrediction databases. After UniProt standardization, deduplication occurred. After the removal of duplicate data, 189 targets were identified (Supplementary Table S1). Next, we used CytoScape 3.6.0 software to build a ‘active ingredients–targets’ interaction network, as shown in We retrieved 1189 and 3084 DN-related disease targets obtained from the GeneCards and DisGeNET databases, respectively.Venn analysis was performed using the 189 targets of SM active components and 1189 and 3084 DN-related target genes, and 64 drug–disease intersection gene targets were obtained for further analysis, as shown in P-value of <1.0e-16  were selected for analysis. The results showed that BPs enriched in DN-related SM targets mainly included cytokine-mediated signaling pathway, apoptotic signaling pathway, positive regulation of cell migration, reactive oxygen species metabolic process, regulation of inflammatory response, regulation of cell–cell adhesion, response to oxygen levels, cellular response to growth factor stimulus, and regulation of protein serine/threonine kinase activity  terms were enriched, and the 20 most significantly enriched BP terms  GO terms were enriched, and the 19 most significantly enriched MF terms based on ve genes B.P<0.01 were selected for analysis. The results showed that the intersection genes were mainly enriched in membrane rafts, RNA polymerase II transcription factor complex, external side of plasma membrane, extracellular matrix, adherens junction, and glutamatergic synapse  GO terms were enriched, and the 13 most significantly enriched CC terms based on  synapse C. DetailP<0.01 and a κ score ≥ 0.53 in order to visualize the results of KEGG enrichment  were molecularly docked with the active component salvianolic acid B. The mechanisms of action of TCM therapeutics are complex, with multiple components and targets. When the pathogenesis of disease has not been clarified, it becomes more difficult to analyze the mechanism of action of a TCM therapeutic. Network pharmacology combines system network analysis and pharmacology. It allows systematic study of the effective components, targets, and pathways of drugs at the molecular level, improving our understanding of the interactions between components, targets, and pathways.In the present study, TCM active component–target network analysis revealed that luteolin, tanshinone IIA, salviolone, salvianolic acid B, dihydrotanshinlactone, and other active ingredients can act on multiple targets in the network. This finding suggests that these components may be important for the therapeutic effect of SM in DN and warrant further exploration. Luteolin has the most potential targets, followed by tanshinone IIA. According to previous reports, luteolin not only increases insulin-mediated glucose uptake and enhances insulin sensitivity  but alsoIn the PPI network of 64 targets of SM acting in DN, AKT1, VEGFA, IL6, TNF, MAPK1, TP53, EGFR, STAT3, MAPK14, and JUN were the top ten targets based on node degree. These proteins are regarded core proteins and may play important roles in the therapeutic effect of SM in DN. These proteins are involved in oxidative stress, inflammation, vascular permeability, and immune regulation. For example, the activation of AKT1 promotes cell proliferation and inhibits cell apoptosis. It is an important player in the immune inflammatory mechanism of DN . It is cTo predict the mechanism underlying the therapeutic effect of SM in DN, we performed GO enrichment analysis of the 64 potential targets. As shown in To further explore the potential mechanism of SM in treating DN, we conducted KEGG analysis of the 64 potential targets of SM acting in DN. As shown in To further explore the potential molecular mechanism of SM in the treatment of DN, we conducted molecular docking studies of 16 targets closely related to DN according to a KEGG-based screening, using the experimentally validated key components tanshinone IIA and salvianolic acid B as ligands. Results showed that the 16 potential targets had a good binding ability to salvianolic acid B, and the interactions were more stable than those with tanshinone IIA.The present study had some limitations. We only explored the effect of SM in DN at the network pharmacology level. However, current network information technology is not comprehensive, and the accuracy of database data and real-time updates needs to be improved. Therefore, the results obtained in the present study require verification in terms of pharmacodynamics, and mechanistic experiments are needed to explain the complex multitarget, multipathway, and synergistic interactions involved in the therapeutic effects of SM.The present study analyzed the mechanisms underlying the therapeutic effect of SM in DN using network pharmacology, by constructing an SM ingredient–target–DN-related pathway network . Our finClick here for additional data file.


Varecia variegata and Eulemur macaco, and one destructive flower feeder, Lemur catta. To study the tongue’s structure, we used direct observation together with different microscopy techniques, ranging from optical microscopy to electronic microscopy. We found differences in the size, shape, and distribution of the tongue’s papillae. Most notably, there are large distinct papillae present at the tip of the tongue in nectar-feeding species. In addition, histological images of the ventro-apical portion of the tongue show that tongue houses an encapsulated structure in all species studied. The non-destructive flower-feeding species share similar tongue and sublingua anatomy, suggesting that the observed features may be adaptive for flower feeding. These features were not observed in the destructive flower feeder, Lemur catta.Comparative studies of primate gustatory anatomy are sparse and incomplete. Here, we compare both the macro and micro anatomy of the tongue in three lemurid species. We included two non-destructive nectar-feeders, Eulemur rubriventer. We compare both the macro and micro anatomy of three lemurid species who died of natural causes in captivity. We included the following two non-destructive nectar feeders: Varecia variegata and Eulemur macaco, and the following destructive flower feeder: Lemur catta. Strepsirrhines and tarsiers are unique among primates, because they possess a sublingua, which is an anatomical structure that is located below the tongue. We include a microanatomical description of both the tongue and sublingua, which were accomplished using hematoxylin–eosin and Masson trichrome stains, and scanning electron microscopy. We found differences in the size, shape, and distribution of fungiform papillae, and differences in the morphology of conical papillae surrounding the circumvallate ones in all three species. Most notably, large distinct papillae were present at the tip of the tongue in nectar-feeding species. In addition, histological images of the ventro-apical portion of the tongue displayed that it houses an encapsulated structure, but only in Lemur catta case such structure presents cartilage inside. The presence of an encapsulated structure, coupled with the shared morphological traits associated with the sublingua and the tongue tip in Varecia variegata and Eulemur macaco, point to possible feeding adaptations that facilitate non-destructive flower feeding in these two lemurids.The mobility of the primate tongue allows for the manipulation of food, but, in addition, houses both general sensory afferents and special sensory end organs. Taste buds can be found across the tongue, but the ones found within the fungiform papillae on the anterior two thirds of the tongue are the first gustatory structures to come into contact with food, and are critical in making food ingestion decisions. Comparative studies of both the macro and micro anatomy in primates are sparse and incomplete, yet there is evidence that gustatory adaptation exists in several primate taxa. One is the distally feathered tongues observed in non-destructive nectar feeders, such as  Cheirogaleus major, Microcebus rufus [Eulemur mongoz [Eulemur rubriventer [Eulemur fulvus rufus [Eulemur fulvus sanfordi, Eulemur coronatus [Eulemur macaco [Varecia variegata variegata [Eulemur mongoz mongoz has been observed to spend 80% of its foraging time feeding from the nectar-producing parts of four species of plants [Ceiba pentandra) [Eulemur mongoz. Varecia variegata variegata spends approximately 25% of its feeding time exploiting nectar, making nectar the second most important food item in its diet [Ravenala madagascariensis) is in bloom. During this period, over 72% of Varecia’s feeding time is spent on this one plant; thus, Ravenala is Varecia’s primary source of caloric intake [Eulemur mongoz nor Varecia variegata are destructive to any part of the plant. Non-destructive nectar feeding, when combined with the morphology of some “lemur-loving” plants  [Coevolution has produced a codependency between primates and plants ,2,3,4. Pus rufus , Eulemurr mongoz ,18,19, Eriventer , Eulemurus rufus , Eulemurr macaco ,21,22, aariegata ,13. Eulef plants ,18,23. Eits diet ,13. Howec intake . It is iflowers) ,13, implflowers) ,23,24.The tongue is a highly mobile organ that occupies the lower half of the oral cavity. The mobility of extrinsic and intrinsic tongue muscles, combined with the rich innervation of the overlying mucosa, allows the tongue to not only reach and explore potential food resources, but it is central in formulating informed nutritional decisions. Across chordates, the tongue has been adapted and modified to carry out particular forms of feeding ,26,27. AThe tongue is divided in the anterior two-thirds, the body, and the posterior third, the root . It is composed of a series of intrinsic muscles and extrinsic muscles. The extrinsic muscles originate outside the tongue , and are used to protrude, retract, and laterally deviate the tongue, while the intrinsic muscle modifies the shape of the tongue. The intrinsic tongue musculature, located on the dorsum of the tongue, is covered by a topographic mucous membrane. The topographic appearance is due to the presence of a vast number of papillae, sorted as gustatory and non-gustatory. It is within these gustatory papillae that taste buds are found. Taste buds house a cluster of taste cells that transmit specific tastants to the central nervous system. The papillae can be observed on the dorsum of the tongue. The posterior one-third of the tongue, the root, is often referred to as the pharyngeal tongue. Beyond innervation, the most distinct feature of this region is the large cluster of lymphoid follicles, which, together, are referred to as the lingual tonsils. Although human anatomy  providedPapillae are raised regions within the mucosa of the soft palate, uvula, epiglottis, pharynx, larynx, esophagus, and tongue . There aVarecia variegata variegata (the black and white ruffed lemur), Eulemur macaco (the black lemur), and Lemur catta (the ring-tailed lemur). These species were selected, partially because of their reported feeding ecology. Varecia and Eulemur are specialized non-destructive flower and nectar feeders, while Lemur is more generalized in its feeding preferences and has not been documented to be a non-destructive flower feeder. Alport [Lemur and Eulemur as strepsirrhine species that lack foliate papillae, and nothing has been described for the nectar-feeding Varecia. We have carried out detailed histological and microanatomical examinations, with the aim of (1) completing the current knowledge of the morphology and location of the papillae on the tongue; (2) bringing useful data to light, to aid in taxonomical tasks; and (3) contributing to a better understanding of anatomical patterns, as they relate to diet and ecology.The comparative histomorphology and microanatomy of the tongue and sublingua of the following three Malagasy prosimians are examined here: . Alport  provided. Alport . However. Alport ,38. One . Alport  listed LLemur catta , five Varecia variegata  and two Eulemur macaco  specimens. The specimens were photographed with a Nikon digital camera COOLPIX D-3000. The lemurs were obtained through donations of naturally dead specimens from Spanish zoos  where the animals were fed with special commercial diets for lemurs, supplemented with fresh fruits. Immediately upon death, the lemurids were frozen at their respective zoos. They remained frozen upon arrival to the Anatomy and Radiology Department, Anatomical Museum, at the Universidad de Valladolid, where they were again placed in freezer to maintain specimen integrity. To obtain the tongues, the animals were allowed to thaw for 24 h in a refrigerator. The jaw was lowered, and the tongue was pulled with forceps. Once the tongue had been placed in antepulsion, it was sectioned at the level of the glosso-epiglottic fissure  using a scalpel. The samples obtained were immersed in 10% formaldehyde in a glass jar marked with data corresponding to species, sex and date, and kept in that way until their use. The animal was frozen again for future research.The tongue and sublingua were removed from seven Together with direct observation we used a Leica M205FA fluorescence stereo microscope. Semi-automatic stereo microscope for transmission (contrast and relief), reflection and fluorescence studies. Optics: 1× (working distance: 61.5 mm), 2× (working distance: 20 mm) and 5× (working distance: 19 mm). Zoomed from 0.7 to 20.5×. Reflection light by LED ring with 1/4, 1/2 or full-ring illumination. Digital color camera, Leica DFC310FX, maximum resolution 1392 × 1040 pixels (1.4 Mpixels CCD). Scientific-technical services, Universidad de Oviedo.®. The preparations were photographed under a Nikon Eclipse 80i light microscope attached to a Nokia DS-5M camera.Fixation was performed with 10% formaldehyde in 0.1 M phosphate buffer  for at least 24 h in agitation. Thereafter the pieces were dehydrated and processed for routinely embedding in paraffin. The paraffin-embedded samples were cut into 10-µm-thick sections parallel to the tongue’s frontal or sagittal plane and mounted on gelatin-coated microscope slides. The structure of the tongue samples was assessed using Mayer’s hematoxylin–eosin and Masson trichrome stain . The stained sections were washed in water, dehydrated in an ethyl alcohol battery in increasing concentrations, diaphased in xylol and mounted with Entellan® .Deparaffinized and rehydrated sections were processed for detection using the EnVision antibody complex detection kit , following supplier’s instructions. Briefly, the non-specific binding was blocked with 50% bovine serum albumin in TBS for 20 min. Sections were then incubated overnight at 4 °C with the primary antibody . Subsequently, the sections were incubated with the anti-rabbit EnVision system-labelled polymer (Dako-Cytomation) for 30 min, washed in buffer solution, and treated with peroxidase blocking buffer (Dako Cytomation). Finally, the slides were washed with buffer solution and the immunoreaction was visualized with diaminobenzidine as a chromogen, washed, dehydrated, and mounted with EntellanThis study was performed on the dorsal surface of the tongue. We used scanning electron microscopy and environmental scanning electron microscopy . The samples were immersed in a solution of hydrochloric acid 1 N concentrated, at room temperature, ranging from 10 to 20 min  in agitation. This treatment was performed to remove debris from sloughed cells. Then, after several steps of rinsing in tap water, the pieces were dehydrated in a graded acetone series, critical-point dried in a Balzers CPD 030, sputter coated with 3 nm gold in a Balzers BAL-TEC SCD 050 and examined under a conventional scanning electron microscope MEB JEOL-6100  or with an ESEM Quanta 200 FEG scanning electron microscope .Lemur catta, but slightly convergent in the forward direction in Varecia variegata and Eulemur macaco. Varecia variegata present regular transverse folds that are visible on the dorsal surface , while those in Eulemur macaco seem to be big flask-shaped formations, ended in a “filiform apex” that is forwardly oriented  on the tip of the tongue. The hammerhead papilla seems to be a modified form of fungiform papillae, and this modification may provide a wider sensory (gustatory) detection surface on the tip of the tongue. The presence of modified fungiform papillae has been described in humans [Nectar feeders are rewarded with a nutrient-rich liquid, containing proteins, sugars, and seven essential amino acids, if they feed non-destructively. The fungiform papillae are the first gustatory papillae that come into contact with a tastant and can be more sensitive to sweet tastants . We founn humans .Colobus guereza, the internal structure of the papillae varies with age, presenting, in the inner core, a shape that is more similar to a cactus in the most senescent than that in young specimens.Beyond the modified apical fungiform papillae, the overall histomorphology of the fungiform papillae among our captive lemurid population is in line with the reported observations by Takemurao  and otheEulemur fulvus rufus [Eulemur rubriventer [Eulemur macaco studied, we found these papillae located laterally on the tongue, just as they are described in humans. Although not systematically documented by Alport’s [Eulemur macaco.Regarding the foliate papillae, in former studies of the us rufus ,40 and Eriventer  tongue, riventer  review oAlport’s , similarLatin for wall), located within their chemosensory area of the posterior one-third of the tongue. The vallum limits and isolates the taste buds and pores. The taste buds/pores are located on the lateral surface of the central chemosensory papilla, which is located medial to the vallum. Just as with folate papillae, the taste buds are found within crypts—allowing tastants to pool within them, eliciting a gustatory response. Sonntag [Lemur catta, tastants are directed to the gustatory crypt surrounding the isolated papilla, by the undifferentiated conical papillae surrounding them. In Varecia, the vallate papillae are surrounded by flattened conical papillae and have several crests. In Eulemur, one  or two (those forming the posterior branch) vallate papillae are surrounded by a common structure that is similar to the “vallum” mentioned by Sonntag [One of the most interesting observations made in this study, from a morphological perspective, was the comparative analysis of circumvallate papillae. These papillae are named after a particular structure, the “vallum” ( Sonntag  studied  Sonntag . There a Sonntag . DemonstLemur catta appears to have a cartilaginous structure within this encapsulated organ (as S100 positive immunostaining and morphology shows), which, theoretically, would allow less mobility than the other two species studied. Additional behavioral and anatomical studies should be carried out before we can characterize this structure and substantiate our hypotheses.Nectar feeders are reported to have exceptionally mobile tongues . We founLemur catta, in contrast with a wider end for the tips in the nectar-feeding species. Wider and “feathered” tongues have been reported in nectar-feeding bats [Varecia variegata appears to be feathered or brushed, which is a feature observed on the tongue of nectar-feeding animals [Varecia and Eulemur). Additional anatomical and, more importantly, behavioral studies of the sublingua are needed to understand the significance of this anatomical trait.For completion, we included a brief comparison of the sublingua in all of the three species studied. We found a narrow tip in ing bats . Additio animals . The fun animals . Toothco animals . Althoug animals . We perfVarecia variegatta and Eulemur macaco when compared with Lemur catta, point towards the possibility of feeding adaptations associated with non-destructive flower feeding. We hope that the morphological characterization of the papillae in this species could aid in the taxonomical classification of extant lemurids. We also encourage behavioral research teams to work hand-in-hand with laboratories that can undertake ultrastructural studies, so as to offer a broader vision and to characterize, in a better way, the biological legacy that our planet offers us.The documented differences found in (1) papillae morphology and location, (2) the histomorphology of the lingual encapsulated structure, and (3) broader sublingua in


Currently, cardiovascular risk associated with COVID-19 has been brought to people’s attention, but the mechanism is not clear. The aim of this study is to elucidate the mechanisms based on multiple omics data.Weighted gene co-expression network analysis (WGCNA) was used to identify key pathways. Combination analysis with aneurysm and atherosclerosis related pathways, hypoxia induced factor-1 (HIF-1) signaling were identified as key pathways of the increased cardiovascular risk associated with COVID-19. ScMLnet algorithm based on scRNA-seq was used to explore the regulation of HIF-1 pathway by intercellular communication. Proteomic analysis was used to detect the regulatory mechanisms between IL18 and HIF-1 signaling pathway. Pseudo time locus analysis was used to study the regulation of HIF1 signaling pathway in macrophages and vascular smooth muscle cells (VSMC) phenotypic transformation. The Virtual Inference of protein-activity by Enriched Regulon (VIPER) analysis was used to study the activity of regulatory proteins. Epigenetic analysis based on methylation revealed epigenetic changes in PBMC after SARS-CoV-2 infection. Potential therapeutic compounds were explored by using Cmap algorithm.HIF-1 signaling pathway is a common key pathway for aneurysms, atherosclerosis and SARS-CoV-2 infection. Intercellular communication analysis showed that macrophage-derived interleukin-18 (IL-18) activates the HIF-1 signaling pathway through IL18R1. Proteomic analysis showed that IL18/IL18R1 promote NF-κB entry into the nucleus, and activated the HIF-1 signaling pathway. Macrophage-derived IL18 promoted the M1 polarization of macrophages and the syntactic phenotype transformation of VSMCs. MAP2K1 mediates the functional regulation of HIF-1 signaling pathway in various cell types. Epigenetic changes in PBMC after COVID-19 infection are characterized by activation of the type I interferon pathway. MEK inhibitors are the promising compounds for the treatment of HIF-1 overactivation.The IL18/IL18R1/HIF1A axis is expected to be an therapeutic target for cardiovascular protection after SARS-CoV-2 infection. MEK inhibitors may be an choice for cardiovascular protection after SARS-COV-2 infection COVID-19 is a global respiratory epidemic caused by SARS-CoV-2, currently infects at least 68 million people, and the number of cases is rising with the emergence of mutated strains such as Delta . At presClinical studies have shown that the severity of COVID-19 is positively correlated with levels of inflammatory factors , 7, whicClinical symptoms of COVID-19 are highly heterogeneous, ranging from asymptomatic infection to ARDS . MultiplIn this study, we explored key signaling pathways based on RNA-seq data of SARS-CoV-2 infection, aneurysms and atherosclerosis. The IL18/IL18R1/HIF-1 signaling axis was found to mediate an increased risk of peripheral vascular disease such as aneurysms and atherosclerosis after COVID-19. Epigenetic changes in immune cells after SARS-CoV-2 infection promotes the expression of IL18. We found that MAP2K1 plays a key role and that MEK inhibitors can be used as treatment options for cardiovascular sequelae after COVID-19.Data sets GSE120521, GSE156754, GSE7084, GSE57691, GSE98278, GSE157859, GSE152418, GSE171668, and GSE174818, derived from GEO database. Each dataset was designed as follows:GSE120521: Atherosclerotic plaques were obtained from 4 patients and dissected in stable and unstable areas.GSE156754: Pluripotent stem cells (iPSCs) were induced into cardiomyocytes, cardiac fibroblasts and endothelial cells, and the transcriptome changes were observed 48 hours after SARS-CoV-2 infection and simulated infection.GSE7084: Abdominal aorta (7 samples) and abdominal aortic aneurysm (6 samples) samples were obtained at autopsy and surgically, respectively, and transcriptome sequencing was performed to explore differences in expression.GSE57691: Genome-wide expression analysis of abdominal aortic aneurysm (AAA) and aortic occlusive disease (AOD) specimens obtained from 20 patients with small AAA (mean maximum aortic diameter=54.3 ± 2.3 mm), 29 patients with large AAA (mean maximum aortic diameter=68.4 ± 14.3 mm), and 9 AOD patients (mean maximum aortic diameter=19.6 ± 2.6 mm). Relative aortic gene expression was compared with that of 10 control aortic specimen of organ donors.GSE98278: The data set compares transcriptome of elective stable (n = 31) vs ruptured (n = 17) AAA and intermediate size (n = 15) vs large (n = 16) AAA.GSE157859: A total of 18 COVID-19 patients were included, including 6 mild cases, 7 moderate cases and 5 severe cases. PBMC samples from each sample were collected at the treatment stage, convalescence stage, and rehabilitation stage, respectively, and RNA sequencing was performed.GSE152418: RNAseq of PBMCs in a group of 17 COVID-19 subjects and 17 healthy controls.GSE171668: The dataset contained 420 autopsy samples from 17 COVID-19 patients, covering 11 organs. In this study, single-cell sequencing data of heart samples from patients with COVID-19 were included.GSE174818: Using Illumina HumanMethylation EPIC, methylation sequencing was performed on whole blood tissues from 102 patients and 19 non-COVID-19 patients to detect epigenetic changes in PBMC after COVID-19.http://cpdb.molgen.mpg.de/CPDB and https://clue.io/query. The rest of the data processing was supported by R (4.0.5) and Python (3.9.5).Proteomic analysis and matching of therapeutic potential compounds were performed by Weighted gene co-expression network analysis (WGCNA) explores regulatory modules through gene expression pattern and the correlation with relevant reference factors. Cardiomyocytes, endothelial cells, and fibroblasts in GSE156754 were induced by pluripotent stem cells. Unlike the original design of the dataset, this study does not consider the amount of virus used during infection. To identify specific transcriptome changes caused by SARS-CoV-2 infection, we performed WGCNA. By default, the soft threshold for network segmentation is calculated automatically. After mapping modules and groups, modules with positive correlation and P value less than 0.05 with COVID-19 infection group were selected for subsequent analysis. We did not consider the heterogeneity of cell types except for differentiation-related pathways. GSE7084, GSE57691 and GSE120521 were used to explore the key differential genes in aneurysm and atherosclerotic plaque, respectively. The results were shown in the volcano map. Only the upregulated genes were included in this study. . Through the enrichment analysis, the key pathways of COVID-19, aneurysms and atherosclerosis were obtained. In the enrichment results, we focus only on signal transduction pathways.ScMLnet is an R packet supported by Python for constructing intercellular/intracellular multilayer signal networks based on single-cell RNA-seq expression data. Based on specific gene expression, prior network information and statistical inference, scMLnet integrates intercellular pathways (ligand-receptor interactions) and intracellular subnetworks (receptor-TF pathways and TF-target gene interactions) to construct a multi-layer network. ScMLnet was implemented by using R (version 4.0.5) and Python (version 3.9.5).Due to SARS-CoV-2 infection, macrophages infiltrated in the cardiovascular system. In this study, macrophages were used as the ligand, and cardiac myocytes, endothelial cells and vascular smooth muscle cells were used as the receptor cells. After combined analysis with aneurysms and atherosclerosis transcriptome differences, key genes were identified and labeled. We focused on the regulation mechanism related to HIF-1 signaling pathway and obtained the IL18/IL18R1/HIF-1 signaling pathway axis. The expression distribution of IL18 IL18R1 and HIF1A were plotted to demonstrate the source and the scope of regulation.Consensuspathdb-human integrates the interaction network of Homo sapiens, including complex protein-protein, genetic, metabolic, signaling, gene regulation and drug target interactions, and biochemical pathways. Data were collected from 30 publicly available databases and interrelationships collated from the literature. Interactive data is integrated in complementary ways to form a seamless network of interactions involving different types of interactions. In this study, IL18R1 was used as the starting point of regulation and HIF1A was used as the endpoint of regulation. We only focused on direct interaction relationships, and all indirect regulatory relationships were filtered out.Gene set variation analysis (GSVA) is a nonparametric, unsupervised method used to estimate the degree of enrichment of relevant gene sets from expression data. In this study, we evaluated the expressions of NF-κB and HIF-1 signaling pathway in each sample derived from GSE57691 and GSE98278 by GSVA score. The genes contained in NF-κB and HIF-1 signaling pathway are provided by R program package KEGGREST.a priori defined set of genes shows statistically significant, concordant differences between two biological states. In this study, we divided the samples into high and low expression groups based on the expression level of IL18 in RNA-seq at bluk level for GSEA analysis to evaluate the expression of NF-κB and HIF-1 signaling pathway (GSE57691 and GSE98278).Gene Set Enrichment Analysis (GSEA) is a computational method that determines whether an We performed single-cell transcriptome analysis based on GSE171668.The data were filtered and standardized, and then clustered to explore cell subtypes and determine the characteristic expression genes among clusters. It is worth noting that the annotations of cells in this study follow the annotations of the original data set. Because the cell annotation of the original data set has been manually corrected, it had high reliability. UMAP plot showed that the clustering results of this study were consistent with the original data. To investigate the regulation of the IL18/IL18R1/HIF-1 signaling pathway on cell phenotypes, we performed a pseudo-time locus analysis. Macrophages were annotated as M1 and M2 polarization directions, while smooth muscle cells were divided into contraction and synthetic phenotypic transformation directions. CD163 was the marker of M2 polarization, but it was noteworthy that the expression of traditional M1 macrophage markers were low in the original data. Studies have shown that NAMPT promotes inflammation and M1 polarization of macrophages (http://dx.doi.org/10.6084/m9.figshare.695962).At present, studies on the mechanism mainly rely on RNA-seq, which represents the functional state of the corresponding biological process through the expression of mRNA. However, mRNA expression does not constitute a reliable predictor of protein activity because it does not capture the various post-transcriptional and post-translational events. Proteomics based on mass spectrometry technology can better directly detect protein abundance, but still cannot directly obtain the active status of proteins. The VIPER algorithm allows computational inference of protein activity by using the expression of genes that are most directly regulated by a given protein. Regulatory network described in Human breast Carcinoma Network was used in this study  and differential methylation region (DMR) were explored. DMP of logFC<0 and pvalue<0.05 is considered as the core difference site. The core DMP locus was enriched and analyzed to detect epigenetic changes of PBMC after SARS-CoV-2 infection. In this study, we only focused on signaling pathways, and other biological processes and compounds were filtered out.The Connectivity Map team at the Broad Institute has generated expression profiles from the gains and losses of thousands of chemical or genetic functional disruptors in a variety of cellular environments. A subset of these interferers is drugs, probes, or tool compounds with known activities and targets that have been well annotated in previous scientific literature. These perturbators have been systematically analyzed by CMap in nine cell lines. TAS is an aggregate measure of signature strength (SS) and replicate correlation (CC) which is intended to represent a perturbagen’s transcriptional activity. The more transcriptionally active a perturbagen, the higher its TAS. The CS score represents the similarity between the compound’s effect on the transcriptome and the reference transcriptome changes. In this study, we considered both the transcriptional activity score and CS score, and the total score was defined as the product of the two scores.As shown in In GSE156754, human induced pluripotent stem cells (iPSCs) and iPSCs-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells were infected with SARS-CoV-2 and compared with uninfected cells. We performed WGCNA on this dataset and identified 15 regulatory modules with a soft threshold of 18, R square reaches 0.9. At this point, the network conforms to the scale-free distribution.  showed that IL18/IL18R1 can promote NF-κB entry into the nucleus and activate related pathways through phosphorylation. Correlation analysis based on GSVA scores showed that NF-κB was positively correlated with the activation level of HIF-1 signaling pathway   and endothelial cells (ECs) annotated in the scRNA-seq data derived from the coronary system and were consistent with the compositions of the peripheral blood vessel walls, so as the pathological changes. In addition to macrophage infiltration , 25, pheWe found significant changes in epigenetic modification patterns of PBMC after SARS-CoV-2 infection, which may be related to the reprogramming of immune memory. By analyzing the differential methylation sites, we obtained the only signal pathway, namely the type I interferon signaling pathway. The type I interferon response is critical for providing effective protection against viral infection and is triggered rapidly by host sensors  that recognize pathogen-associated molecular patterns (PAMPs). Activation quickly induces the expression of hundreds of genes called interferon-stimulating genes (ISGs), which in turn control other downstream molecules, including pro-inflammatory cytokines ; these cIn this study, we found that MEK and mTOR inhibitors have potential therapeutic value in overactivation of HIF-1 signaling pathway. Especially MEK inhibitors, which were consistent with the results of VIPER analysis. Ras/Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways are responsible for regulating cell growth, proliferation, survival and apoptosis; and have been considered as promising targets for cancer therapy . ConnectInterestingly, we found that estrogen receptor activators antagonize transcriptomic changes resulting from overactivation of the HIF-1 signaling pathway. This is consistent with clinical observations of better outcomes in women with SARS-CoV-2 infection . In factIn this study, we found that macrophages increase cardiovascular risk through the IL18/IL18R1/HIF-1/MEK signaling pathway after SARS-COV-2 infection. MEK inhibitors may be an option for cardiovascular protection after SARS-COV-2 infection.The original contributions presented in the study are included in the article/LZ, and HB, conceptualization. LZ, methodology, software, formal analysis, and original draft preparation. ZW, ML, and PS, validation. SW and CZ, investigation and resources. HW, data curation. PS and SW, review and editing. LZ and PS, visualization. ML, supervision. HB, project administration and funding acquisition. All authors contributed to the article and approved the submitted version.This study was funded by the National Natural Science Foundation of China to Hualong Bai (Grant No: 81870369) and Key Projects of Medical Science and Technology in Henan Province (Grant No: SBGJ202002035).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.


Spectrophotometric methods were used to determine the total antioxidant status (TAS) values as well as superoxide dismutase (SOD) and glutathione reductase (GR) activities in the vitreous fluid samples. (3) Results: no significant differences in TAS values and antioxidant enzyme activities were observed among patient with RRD with and without PVR and with MH and ERM. The longer the duration of RRD leading to PVR and better postoperative visual acuity, the higher the TAS level. No significant differences were found between “macula on” and “macula off” subgroups within the RRD group and the RRD combined with PVR group. (4) Conclusions: The preliminary results do not support the thesis that the antioxidant status of vitrectomized eyes is different in patients with RRD with or without PVR in comparison to patients with MH and ERM. In patients with RRD, PVR presence and detached macula do not affect the values of TAS, SOD and GR in the vitreous fluid. The duration of the disease influences TAS in the vitreous in eyes with RRD complicated with PVR.  The most ROS are produced in the mitochondrial respiratory chain in both enzymatic and oxidation reactions [Oxidative stress has been implicated in the development of retinal cellular damages in many retinal diseases , such aseactions . Previoueactions .Rhegmatogenous retinal detachment (RRD) is a sight-threatening condition with an incidence of approximately 1 out of 10,000 people . RRD canRRD is an indication for prompt surgical management, and the pars plana vitrectomy (PPV) is the most commonly chosen procedure ,14. The Superoxide dismutase (SOD) and glutathione reductase (GR) are important antioxidant enzymes responsible for the scavenging free radicals .2−∙) into molecular oxygen (O2) and hydrogen peroxide (H2O2), using the redox properties of metals present in the enzyme’s active centers as a mechanism of catalytic activity [Superoxide dismutase is an enzyme that catalyzes the two-stage dismutation reaction of superoxide anion radicals (Oactivity . This practivity . Dependiactivity .Glutathione reductase (GR) occurs in the cytosol and mitochondria, and its main function is to maintain the proper concentration of glutathione in the cell by the regeneration of reduced glutathione (GSH) from glutathione disulfide (GSSG) with the participation of NADPH . GSH is The aim of the study was to assess the antioxidant status, on the basis of total antioxidant status (TAS) values and antioxidant enzyme—SOD and GR—activities, in the vitreous fluid of patients with RRD, MH and ERM, to test the hypothesis that oxidative stress is increased in RRD, particularly with concomitant PVR.The study was designed as a prospective observational study. The study protocol was approved by the Ethics Committee of Medical University of Lublin, Poland . Written informed consent was taken from each participant after explaining the nature of the study. The study was conducted at the Department of General Ophthalmology and Pediatric Ophthalmology Service, Medical University of Lublin, Poland, between the years 2018 and 2020. The study group consisted of 73 consecutive patients (39 women and 34 men), who underwent vitrectomy and fulfilled the inclusion criteria . One eyeEach patient was examined with a slit lamp, patients with RRD had additional B-mode ultrasound examination, while MH and ERM were examined with optical coherence tomography (OCT) of the macula to confirm the diagnosis. Best corrected vision acuity was examined with use of the Snellen charts before the surgery and two week after the surgical intervention All the patients with RRD and PVR had grade C PVR according to the classification of “Retina Society Terminology Committee (1983)”  and updaEach surgery was performed by the same surgeon (KN). Undiluted vitreous fluid was acquired from the vitreous cavity at the beginning of PPV in all procedures. The vitrector line was connected to a sterile syringe (5 mL). After the closure of the aspiration line, undiluted core vitreous (500–1000 µL) was aspirated into the syringe by active vitrector cutting and syringe suction . All vitreous samples were snap-frozen and then stored at −80 °C until assay.All the samples were submitted to the Department of Medical Chemistry, Medical University of Lublin, Poland, in order to determine the antioxidant parameters. The values of total antioxidant status and activities of antioxidant enzymes, i.e., superoxide dismutase and glutathione reductase, were measured in the vitreous fluid. TAS, SOD and GR were assayed with RANDOX kits according to the manufacturer’s instructions. All determinations were performed two or three times.®•+ is measured at 600 nm. Briefly, 20 μL of sample (the vitreous fluid), standard  or double deionized water (blank) was added to 1000 μL of the freshly prepared chromogen  in phosphate buffered saline . After mixing and pre-warming to 37 °C, the initial absorbance of the sample/standard/blank was measured. Next, 200 μL of substrate  was added, and after 3 min, the final absorbance was measured. Results are expressed in mmol of TAS/g of protein.TAS was determined spectrophotometrically using the ready-to-use diagnostic kit TAS . In this method, 2,2′-azino-di-(3-ethylbenzothiazoline-sulphonate) (ABTS®) incubated with a peroxidase (metmyoglobin) and hydrogen peroxide forms a relatively stable blue-green radical cation (ABTS). The low molecular weight antioxidants present in the added sample inhibit the formation of this colored product in proportion to their concentration. The color intensity of ABTS2•-), produced from xanthine in the reaction catalyzed by xanthine oxidase, reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride, resulting in the formation of a red formazan dye. The superoxide dismutase present in the added sample inhibits this reaction by converting the superoxide anion radicals into hydrogen peroxide and molecular oxygen. The color intensity of a red formazan dye measured at 505 nm is proportional to the SOD activity. Briefly, 30 μL of sample (the vitreous fluid) or standard (0.01 mol/L phosphate buffer pH 7.0 in redistilled water) was added to 1000 μL of the freshly prepared substrate  in buffer . After mixing and pre-warming to 37 °C, 150 μL of xanthine oxidase (80 U/L) was added. The initial absorbance was recorded after 30 s, and then, the absorbance was measured for a further 3 min. Results are expressed in U of SOD/mg of protein. The activity of superoxide dismutase (SOD) was determined spectrophotometrically using the ready-to-use diagnostic kit RANSOD . In this method, the superoxide anion radical  was mixed with 1000 μL of the freshly prepared substrate  in buffer . Next, 200 μL of NADPH (0.17 mmol/L) was added. The absorbance of the sample was measured every 1 min for 5 min. Results are expressed in U of GR/g of protein. The activity of glutathione reductase (GR) was determined spectrophotometrically using the ready-to-use diagnostic kit GLUT RED . The assessment of GR activity is based on the ability of glutathione reductase to catalyze the reduction of glutathione disulfide (GSSG) to glutathione (GSH) with a concomitant oxidation of NADPH to NADPThe concentration of protein was determined by the Bradford method  using boAll calculations were performed using STATISTICA 13.0  software. The values of the measurable parameters analyzed were presented using the mean value, median, quartiles, and standard deviation, and for the unmeasurable ones using the number and percentage. For measurable features, the normality of the distribution of the analyzed parameters was evaluated using the W Shapiro–Wilk test. A nonparametric statistical Wilcoxon signed-rank test was used to compare dependent variables of two related samples. A nonparametric test, the Mann–Whitney U test, was used to compare independent variables in two groups. Patient age was compared with the use of the he Kruskal–Wallis test. Spearman’s rank correlation coefficient was used to assess the relationship between the variables. A p value ≤ 0.05 was considered statistically significant. No statistical differences between groups in TAS values (p = 0.81) were found. However, TAS levels were the highest in the ERM and MH groups .SOD activity was the highest in the MH group (12.989 U/mg protein), while the differences in the activity of this enzyme between the groups were not statistically significant (p = 0.67) .Additionally, the activity of GR was slightly higher in the MH group in comparison to the RRD, RRD with PVR and ERM groups. These differences were not statistically significant (p = 0.80) .The analysis showed that the disease duration was significantly longer in the ERM with MH groups, compared to RRD and RRD combined with the PVR group (p = 0.0001), .The analysis showed a significant correlation between the disease duration and the TAS level in the PVR group between the duration of the disease and the TAS activity level (R = 0.48). The longer the duration of the disease leading to PPV, the higher the TAS level. There was no significant difference between the duration and the SOD and GR activity levels in the PVR and the RRD groups (p > 0.05) .There was no correlation found between the duration of the disease and antioxidant levels in the MH and ERM groups.Median postoperative visual acuity was 0.3 Snellen (0.09–0.4) in the group with RRD, 0.1 Snellen (0.06–0.3) in the group with RRD and PVR, 0.2 Snellen (0.1–0.4) in the group with ERM and 0.3 Snellen in the group with MH . There was a significant correlation between postoperative visual acuity and TAS levels in the group with PVR .“Macula off” was present in 72% of eyes with PVR and in 50% of eyes without PVR. Statistical analysis showed no significant differences (p > 0.05) between “macula on” and “macula off” subgroups in both groups in regard to TAS, SOD and GR.The present study did not support the hypothesis that oxidative stress is increased in the vitreous body of eyes vitrectomized due to RRD and PVR. However, it may expand the insight into the pathophysiology of RRD, which is very important as the incidence of RRD is increasing . We showIt has already been proven that high oxidative stress damages the retina, by the acceleration of photoreceptors’ and ganglion cells’ apoptosis. Most authors describe the influence of increased oxidative stress in the diabetic retinopathy and age-related macular degeneration. Recent investigations provided the information that metabolic processes in the pathological retina increase oxidative stress and lead to PVR formation . In thisThere are many methods of oxidative stress evaluation. In biological samples, oxidative status is assessed by direct ROS level measurement, biomolecule damage detection  or antioxidative status determination (enzymatic or nonenzymatic antioxidant levels) . As reco3+ to Fe2+ conversion in thiocyanate solution. Significantly increased oxidative stress was present in RRD compared to MH; only the extent of the detachment was significantly correlated with the BAP, with no significant relationships found for the duration, presence of PVR or VH, macular status or patient age. RRD exhibited a significantly lower BAP value than MH, while proliferative diabetic retinopathy (PDR) had a significantly smaller BAP than ERM and MH. However, there were no statistically significant differences in the BAP values among RRD, PDR, RVO and ERM. This is consistent with our results; however, different parameters of antioxidant status were assessed. Maeno et al. reported the outcomes of relatively small patient groups. Furthermore, the average age of the patients was statistically different. However, the authors detected no significant correlation between the BAP and the patients’ age, suggesting that the patient age is not likely to influence the BAP values in the disorders studied.Maeno and colleagues reported increased oxidative stress in vitreous fluid in patients with RRD . The stuMoreover, Maeno and colleagues reported that RRD itself had a much greater influence on vitreal oxidative stress than free hemoglobin in vitreal hemorrhage, previously described as the ROS production agent.Cederlund and colleagues as well as Takahashi and co-workers reported the correlation of oxidative stress with RRD severity 8311. Both teams expressed the severity of RRD in the presence or absence of vitreal haemorrhage, RRD extent (quadrants) and macular status.In the study of Cederlund and colleagues , levels In the present study, we categorized eyes vitrectomized due to RRD as without PVR and with PVR C, which means longer lasting RRD with a larger extent of RRD and with “macula off”. According to the definition of PVR, which is a complication of RRD , it can Takahashi and co-workers also reported no relationship among the RRD duration, macular status, PVR or HV presence or patients’ age . In a stWert and associates conducted a study assessing the effect of SOD3 on oxidative stress in the vitreoretinal interface. The study was carried out on both a human and a mouse vitreous model. The results shown that the regulation of SOD3 at the vitreous base and cortex may represent a future therapeutic target for vitreoretinal diseases. Material collected during the research from vitreous bodies of humans and mice showed higher levels of oxidative stress biomarkers (nitrates and/or 3-nitrotyrosine) suggesting SOD3 dysfunction. The dysfunction or complete lack of SOD3, and thus increased oxidative stress, causes internal damage to the inner retina, which can be part of DVR pathogenesis and other vitreoretinal diseases . In our Mancino and colleagues investigated total antioxidant capacity (TAC) in the blood, aqueous humor, and vitreous bodies of diabetic and nondiabetic patients . The totWe did not find any age dependence of antioxidants, and there were no significant differences in age between groups. The similar findings were reported by Brzović-Šarić and colleagues , who anaThe limiting factor for our study and its control group is that no vitreous of healthy subjects was included, as there were no indications for PPV in healthy subjects, and they were not performed. Thus, as a control group, MH and ERM eyes were included in the study, as conducted in previous studies ,31,39. BNew therapies decreasing harmful ROS formation will be the step forward in oxidative stress-derived ophthalmic diseases. Currently, there are only cell or animal model studies that demonstrate the beneficial use of antioxidants in the prevention of retinal damage. The treatment course may aim to both reduce ROS and promote the production of antioxidant enzymes.A longer duration of RRD leading to PVR is correlated with a higher TAS level in the vitreous.


Precision medicine tailors treatment for each patient by identifying the molecular drivers of their disease. This can allow more effective tumour targeting, avoid harmful standard chemotherapeutic side-effects, and offer savings to the healthcare system through not treating patients who are unlikely to respond to a specific agent. Treatment regimes are usually designed by identifying DNA-level alterations and selecting drugs tailored to that mutation. However, cancer is not a one-pathway disease and not all patients with particular mutations will respond to treatment, while patients without canonical pathway-activating mutations are excluded from potentially life-saving treatment. To address this, we have developed a NanoString assay combining proteomic and transcriptomic profiles of 4 key actionable, cancer-related pathways . We used RNA-Seq data from gold standard cell lines with defined pathway changes to identify minimal gene sets indicative of pathway activation, and integrated them with phospho protein measurements to generate a pathway activation score. The combined panel was run on isogenic cell lines as well as glioma samples with both known and unknown driving alterations. We found pathway activation to be more variable than expected based on DNA alterations alone, implying that consideration of proteomic and/or transcriptomic-level information is important for future therapeutic decision-making.


Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world with a highly variable clinical course. Its striking genetic heterogeneity is not yet fully understood. Although the CLL genetic landscape has been well-described, patient stratification based on mutation profiles remains elusive mainly due to the heterogeneity of data. Here we attempted to decrease the heterogeneity of somatic mutation data by mapping mutated genes in the respective biological processes. From the sequencing data gathered by the International Cancer Genome Consortium for 506 CLL patients, we generated pathway mutation scores, applied ensemble clustering on them, and extracted abnormal molecular pathways with a machine learning approach. We identified four clusters differing in pathway mutational profiles and time to first treatment. Interestingly, common CLL drivers such as ATM or TP53 were associated with particular subtypes, while others like NOTCH1 or SF3B1 were not. This study provides an important step in understanding mutational patterns in CLL. Chronic lymphocytic leukemia (CLL) is a genetically and clinically heterogeneous disease. The disease manifestations range from asymptomatic with no need for therapy to an aggressive disease associated with therapeutic resistance and overall survival of less than 3 years . CLL is Stratification of CLL patients based on the entire mutation profile could improve the accuracy of prognostication as it has been shown in the context of other diagnoses . In acutUnsupervised learning, also known as clustering, has been extensively used to gain insight into the underlying structure of complex biological data and has led to discoveries of various cancer molecular subtypes . HoweverIn the present work, we used 1,329 canonical pathway signatures  from the collection of curated gene sets  from the Molecular signatures database (MSigDB)  gatheredA set theory algorithms  aimed ton = 113) samples without mutation in any of these genes from our patient dataset (n = 506). Therefore, we decided not to subset genes in the next analyses. We cut the dendrogram at k = 2–7 which means that we grouped the patients into 2–7 groups containing cases with the most similar pathway mutation profiles. We removed clusters of size <20 and tested time to first treatment (TTFT) differences between the subtypes. We identified those solutions with significant differences bearing potential clinical relevance. These concerned k = 3 and 5 that, after filtering out clusters of size <20, contained only two clusters  for clustering solutions splitting data into 5 and 7 groups . We split data into 2–7 groups and evaluated differences in TTFT for the three best solutions selected based on the proportion of ambiguous clustering PAC; . We iden7 groups . Cluster7 groups .Since the multiclass classification that we subsequently performed was challenging, we further elaborated the solution with the fewer  groups in all downstream analyses. First, we evaluated the effect of each subtype characterized by distinct pathway mutation profiles on the TTFT. The subtype with the most favorable prognosis differed from the one with the worst outcome by 20 years in the median TTFT (3 vs 23.4 years) independently of the IGHV status . We alson = 100), i.e., samples that were not used in any step of the model development, thus representing new, unseen data. The final model used 84 pathway signatures and achieved high prediction performance . The 84 pathway signatures contained 1,504 mutated genes in the dataset. We analyzed protein–protein interactions of mutated genes from each cluster and described gene communities using the fast greedy community detection algorithm. To interpret gene communities, we performed text mining of the column with the description of gene function for each gene and visualized networks . All the P values were adjusted for multiple comparisons using the Benjamini–Hochberg correction. If more solutions differed in TTFT statistically significantly, the one with the least subtypes was chosen for further analysis. A Multivariate Cox regression model was fitted to assess the independent prognostic impact of IGHV somatic hypermutation status of each subtype in the outcome of the patients.Publicly available clinical data were downloaded from the ICGC Data Portal and information about TTFT as an important clinical parameter was extracted. A log-rank test was used to identify whether the found subtypes differed in TTFT (r > 0.7/r < −0.7) and clusters smaller than 10 patients were removed leaving us with 497 patients and 317 pathway signatures. Then, data were split randomly into a training set (80% of patients) and a test set (20% of patients). To find the best number of rounds for the algorithm, it was run with subsample parameter set to 0.25 and the following parameter settings of learning rate and depth of trees were tested: 0.01, 0.05, 0.1, 0.3, and 4, 6, 9, respectively. The algorithm was stopped after 100 rounds without improvement of multiclass Logarithmic Loss function (mlogloss), which was evaluated using a five-fold CV. The algorithm was run again with an optimized number of rounds and selected parameter setting, which minimized mlogloss. Feature importance was ranked by its information gain, which corresponded to the relative contribution of the feature to a prediction. The process of the parameter tuning was repeated with half of the most important features and then in the following repetitions with 3/4 of the most important features until mlogloss started increasing. The performance of the model with optimized parameters and extracted features was tested using mlogloss, multiclass auROC, and multiclass aucPR. An information gain of the features was extracted for each subtype separately.The Extreme gradient boosting algorithm  is a macThe original contributions presented in the study are included in the article/Ethical review and approval was not required for the study due to the secondary use of published data. The original written informed consents with the research use of the data were collected by the ICGC consortium.PT designed the research and performed the analysis. PT and KP analyzed the results and wrote the manuscript. KP and SP supervised the study and critically evaluated the manuscript. All authors contributed to the article and approved the submitted version.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.


The Venn analysis was contained 79 co-targets. Enrichment analysis showed that HQD played a role in TNF signaling pathway, IL-17 signaling pathway, Th17 cell differentiation, etc. IL6, TNF, IL1B, PTGS2, ESR1, and PPARG with the highest degree from PPI network were successfully docked with 19 core components of HQD, respectively. According to ZINC15 database, quercetin (ZINC4175638), baicalein (ZINC3871633), and wogonin (ZINC899093) recognized as key compounds of HQD on ulcerative colitis. PTGS2, ESR1, and PPARG are potential therapeutic targets of HQD. HQD can act on multiple targets through multi-pathway, to carry out its therapeutic role in ulcerative colitis.Huangqin decoction (HQD) is a Traditional Chinese Medicine formula for ulcerative colitis. However, the pharmacology and molecular mechanism of HQD on ulcerative colitis is still unclear. Combined microarray analysis, network pharmacology, and molecular docking for revealing the therapeutic targets and molecular mechanism of HQD against ulcerative colitis. TCMSP, DrugBank, Swiss Target Prediction were utilized to search the active components and effective targets of HQD. Ulcerative colitis effective targets were obtained by microarray data from the GEO database (GSE107499). Co-targets between HQD and ulcerative colitis are obtained by Draw Venn Diagram. PPI (Protein–protein interaction) network was constructed by the STRING database. To obtain the core target, topological analysis is exploited by Cytoscape 3.7.2. GO and KEGG enrichment pathway analysis was performed to Metascape platform, and molecular docking through Autodock Vina 1.1.2 finished. 161 active components with 486 effective targets of HQD were screened. 1542 ulcerative colitis effective targets were obtained with |Log Multiple pathogenesis, such as environmental factors, genetically susceptibility and gut microbiome dysfunction, have been implicated2. The mucosal inflammation initiating in the rectum and extending proximally in the colon, and limited to the mucosal layer, contrast to Crohn’s disease (CD) is involving all layers of the bowel wall1. Owing to intestinal homeostasis dysfunction and intestinal barrier disruption, may result in long-term relapsing and remitting mucosal inflammation3. Patients with ulcerative colitis experience unpredictable flares-up may present fever, tenesmus, anemia, and possibly intestinal perforation, which is an afflicted condition of an individual5. Despite 5-ASA, rectal corticosteroids, and biological drugs are the main treatment of ulcerative colitis, approximately 15% of patients may require restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for medically refractory disease or to treat colonic neoplasia7. However, with the incidence and prevalence of ulcerative colitis is increasing worldwide, governments should be informed of the management and economic burden of this disease. Therefore, it is urgent to seek more therapeutic armamentariums with fewer adverse side effects for ulcerative colitis.Ulcerative Colitis (UC) is one subtype of inflammatory bowel disease (IBD) of unknown aetiology that affects the colorectum, often leads to bloody diarrhoea and abdominal painShang Han Lun” written by Zhang Zhongjing in Han Dynasty, which is used for “heat-dampness” ZHENG  for over 1800 years. HQD consists of four herbs: Scutellariae Radix , Paeoniae Radix Alba , Jujubae Fructus , Glycyrrhiza glabra L. . Recently, some fundamental analysis has proved that HQD may regulate the gut microbiota, suppress Ras-PI3K-Akt-HIF-1α and NF-κB pathways8, target ESR1 and EGFR to relieve endothelial dysfunction9 to treat ulcerative colitis. However, pharmacology and molecular mechanisms remain unclear.Traditional Chinese Medicine (TCM) has almost 5000 years of history, which creates many treatments for disease. Huangqin decoction (HQD), a traditional herbal formula from “In this study, we combined network pharmacology and bioinformatics to reveal the core target and pathways that are participated in the pathogenesis of HQD against ulcerative colitis and screen for molecular targets of HQD for the treatment of ulcerative colitis. The workflow is shown in Fig. https://tcmspw.com/tcmsp.php10 were used to filter out active components, according to absorption, distribution, metabolism, excretion (ADME) criteria (OB ≥ 30% and DL ≥ 0.18)11. Confirmation of active components SMILES structures from PubChem (https://PubChem.ncbi.nlm.nih.gov) 12. Effective targets were selected from DrugBank (https://go.drugbank.com) and Swiss Target Prediction (http://www.swisstargetprediction.ch/). 13. Interaction of over 100 was recognized as an active component.Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  with 44 healthy individuals and 75 ulcerative colitis colonic mucosa samples. Based on the GEO2R web tool (http://www.ncbi.nlm.nih.gov/geo/geo2r/), differentially expressed genes (DEGs) with the cut-off criteria of |Log2FC|> 1 and adjusted P-value < 0.05 and visualized by volcano plot.Expression profiling data from GSE107499 (GPL15207 [PrimeView] Affymetrix Human Gene Expression Array) was obtained from the GEO database (http://www.uniprot.org), selecting “Homo sapiens”. Draw the Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) for co-targets between HQD and DEGs and shown as a heatmap. Built on the co-targets result, the PPI network was conducted by Search Tool for the Retrieval of Interacting Genes .Metascape (http://www.pdb.org/), using Pymol software to separate modified, ligand and remove water. 3D structure of core component was downloaded from PubChem, after adding hydrogen and calculate charges, then using Autodock Vina 1.1.2 software to dock with core targets, limited binding energy ≤ − 5.0 kcal·mol-1 as stable connection site.3D protein structure of core target was downloaded from RCSB Protein Data Bank , 13 in Paeoniae Radix Alba , 29 in Jujubae Fructus , 92 in Glycyrrhiza glabra L.   Fig. . Among 1According to the GEO2R web tool, a total of 1542 DEGs were identified, which consist of 1024 upregulated and 518 downregulated genes Fig. . Venn anIn the PPI network Fig. , after hP < 0.01. There were 818, 26, and 66 GO terms related to biological processes, cell components, and molecular functions, respectively  and all 19 active compounds of HQD , respectively. The binding energy score was all smaller than − 5.0 kcal·mol-1, TNF-baicalein and PPARG-baicalein were − 9.2 and − 9.3 kcal·mol-1, PTGS2-5,2′-Dihydroxy-6,7,8-trimethoxyflavone was − 9.5 kcal·mol-1, ESR1-wogonin was -8.5 kcal·mol-1, respectively  and IL1B (RCSB PDB: 1I1B) was MOL000098 quercetin, TNF (RCSB PDB: 1TNF) and PPARG (RCSB PDB: 1PRG) was MOL002714 baicalein, PTGS2 (RCSB PDB: 5F19) was MOL000552 5,2′-Dihydroxy-6,7,8-trimethoxyflavone, and ESR1 (RCSB PDB: 3OS8) was MOL000173 wogonin. The binding energy scores of IL6-quercetin and IL1B-quercetin were − 7.2 and − 7.6 kcal·molFurthermore, according to the ZINC15 database, quercetin ZINC4175638), baicalein (ZINC3871633), and wogonin (ZINC899093) as the stable compound recognized as key compounds. From Table 8, baical18. Profound knowledge of genetics, microbiota or cytokine imbalances in the bowel and its function in inflammation will sustain the outcome of possible therapeutic approaches if we act on those targets. In addition, the development of new ligands with therapeutic efficacy on the bowel would be a potential avenue in the search for treatments or prevention of ulcerative colitis. To date, with widely using TCM in different diseases, clinical effectiveness has been demonstrated. As for this concern, we made a combination of network pharmacology and bioinformatics might contribute to a further understanding of the disease aetiology and find novel potential therapeutic way.Given its unclear pathogenesis, potentially progressive, and debilitating disease course, the therapeutic goals for ulcerative colitis have changed from treating symptoms to mucosal healing. However, conventional treatments are mainly restricted to control inflammation and clinical symptoms, and hard to meet the expectations of long-term management. Therefore, combination therapy is the urgent strategy for ulcerative colitisIn this study, 20 core components with 486 targets of HQD and 1542 DEGs (GSE107499) of ulcerative colitis were examined. 79 co-targets which indicated that the targets of HQD against ulcerative colitis may be through IL6, TNF, IL1B, PTGS2, ESR1,PPARG, which have a high degree in PPI network. IL6, TNF, IL1B have been proved that have a strong impact on the pathological process, as for docking test, we found PTGS2 and PPARG have the lower energy than others, and we focus on more details in PTGS2, ESR1, and PPARG.19. Also, it can involve activation of NOS3 and endothelial nitric oxide production20. Dysregulation of estrogen receptors in the intestinal mucosa of ulcerative colitis indicates that estrogen signaling may play a role in the local immune response and maintain epithelial homeostasis in a gender- and age-dependent manner21, even though there is no evidence in difference prevalence in male and female.ESR1, a nuclear hormone receptor, is involved in the regulation of eukaryotic gene expression and affects cellular proliferation and differentiation in target tissues by the steroid hormones and their receptors. ESR1 has mutual transrepression with NF-κB, e.g. decreases NF-κB DNA-binding activity and inhibits NF-κB-mediated transcription24.PPARG (PPARγ), a new coming experiment targeting the treatment of ulcerative colitis, is one of the isoforms of nuclear PPAR receptors that belong to the steroid receptor superfamily. It can be bound to DNA-specific PPAR response elements (PPRE) and modulates the transcription of its target genes, so that controls the peroxisomal beta-oxidation pathway of fatty acids. In addition to playing a role in the regulation of cardiovascular circadian rhythms adipocyte differentiation, and glucose homeostasis, PPARγ also acts as a critical regulator of gut homeostasis by suppressing NF-κB-mediated proinflammatory responses28. However, disease onset of ulcerative colitis provokes inflammation of the colonic intestinal wall, showing significant downregulation of PPARγ, compared with remission mucosal tissue31. Increased transcriptional activity of PPARγ was linked to decreased intestinal inflammation in DSS-induced colitis mice32.Presence of PPARγ can be observed throughout the gastrointestinal tract: mesenteric adipocytes, macrophages, and epithelium, most prevalently in the more differentiated layer of epithelial cell, have the highest levels of PPARγ mRNA than other tissue33. PTGS2 (COX-2) is a key gene with a particular role in the inflammatory response and expresses in epithelial cells and mononuclear cells in IBD35. Prostaglandins are critical to the process of mucosal healing in the colonic tract36.Besides, a present study has proved that PPARγ can be activation by COX-2 products  to perform anti-inflammatory in ulcerative colitis37. Acacetin by inhibiting inflammation and regulating the intestinal microbiota in DSS-induced colitis mice38. Wogonin is quickly converted into baicalin and various flavones in metabolism and may act in the PI3K-Akt pathway, AMPK pathways, and inhibition of telomerase activity39. Baicalin has been confirmed to inhibit IL-33 and NF-κB p65 expression, increase IκB-α levels, regulate Th17/Treg balance and gut microbiota to act antiinflammation41. Oroxylin A as the main component extracted from Scutellariae radix can decrease the expression of inflammatory cytokines IL-6 and IL-1β, and attenuated IL-6/STAT3 signaling pathway in ulcerative colitis mice42. Norwogonin can increase Bax and decrease in Bcl-2 levels, induction of autophagy along with triggering G2/M phase cell cycle arrest to suppress the colon cancer43.Moreover, core components of HQD act in a multi-component and multi-target way. Quercetin, acacetin, wogonin, norwogonin, baicalein, salvigenin, oroxylin a were key active components of HQD in the treatment of ulcerative colitis. Quercetin has an antioxidant effect, suppresses proinflammatory mediators' release and expression of inflammatory proteinsIn GO and KEGG pathway enrichment analysis, HQD may through several cellular components, such as membrane raft, extracellular matrix, and receptor complex to active in molecular functions, including oxidoreductase activity, endopeptidase activity, and protein kinase activity to get involved in biological processes, such as TNF signaling pathway, IL-17 signaling pathway, Th17 cell differentiation, and NF-kappa B signaling pathway.44. Th17 is the most synthetic T cell, plays a vital role in gut between homeostasis and inflammation, and is thought to play a pathogenic role in IBD46. In a previous study, Th17 cell differentiation can be driven by lnc-ITSN1-2 and correlate with increased IBD risk, activity, and inflammatory cytokines47. NF-κB signaling is often dysregulated resulting in overzealous inflammation and distinctly into two pathways, canonical or non-canonical48. Different from rapid, acute inflammation is canonical NF-κB signaling pathway, non-canonical NF-κB signaling has become more evident in maintaining immune system homeostasis in the gut, which regulates T cells differentiation and function50. In sum, HQD mainly acts into multi-component, multi-target, multi-pathway in treating ulcerative colitis.Recover intestinal mucosal homeostasis faces a particularly challenging change, because it is against infection, commensal organisms, and unbalance T cells differentiationFurther pharmacological experiments are needed in order to validate the therapeutic mechanism of HQD on ulcerative colitis.In this study, we combined bioinformatics analysis, network pharmacology, and molecular docking to reveal the mechanisms of HQD against ulcerative colitis. HQD may be through multi-components and multi-pathway in treating ulcerative colitis.Supplementary Tables.


Houttuynia cordata Thunb. is a traditional Chinese herb widely used mainly because of the pharmacological effects related to heat clearance and detoxification. Emerging clinical evidence indicates that the efficacy of Houttuynia cordata Thunb. on RILI is upstanding. Nevertheless, its underlying therapeutic mechanism remains unclear and warrants further elucidation. Houttuynia cordata Thunb. were retrieved from the traditional Chinese medicine system pharmacology database (TCMSP) and literature review. The related targets of RILI were retrieved from the GeneCards database. Common targets among the active compounds and diseases were identified through Venn diagram analysis. Cytoscape was employed to construct and visualize the network relationship among the drug, active compounds, targets, and disease. The protein interaction network (PPI) was constructed by STRING. The reliability (the binding affinity) of the core targets and active compounds was verified by molecular docking.  The major active components and corresponding targets of Houttuynia cordata Thunb. against RILI. The core active compounds included quercetin, kaempferol, hyperoside, and rutin. Hub nodes including TP53, VEGFA, JUN, TNF, and IL-6 were identified in the PPI network. The GO categories were classified into three functional categories: 112 biological processes, 9 molecular functions, and 32 cellular components of the active compounds of Houttuynia cordata Thunb. The KEGG pathway enrichment analysis demonstrated the enrichment of target genes in several key cancer-related signaling pathways, including the cancer pathways, TNF signaling pathway, PI3K-Akt signaling pathway, and HIF-1 signaling pathway. Molecular docking analysis validated the effective binding capacity of the main active compounds with the core targets.  A search of the TCMSP database and related literature revealed 12 active compounds of Houttuynia cordata Thunb. have a potential pharmacological effect against RILI via the cancer pathways, TNF signaling pathway, and PI3K-Akt signaling pathway. The main active components of  Radiation-induced lung injury (RILI) is the most common and serious complication of radiotherapy for breast cancer, lung cancer, and esophageal cancer. Compelling evidence shows that the incidence of asymptomatic RILI is as high as 43%, whereas the incidence of clinically diagnosed asymptomatic RILI is 5–15% . RILI siHouttuynia cordata Thunb., one of the oldest traditional Chinese herbs in China, with a history of clinical application for more than a thousand years, has been included in the Chinese Pharmacopoeia since 1963. Houttuynia cordata Thunb., as a representative drug in the category of purging heat and detoxification, is a genus of Houttuynia in the family Sambucus, characterized by a pungent taste and a slightly cold nature, and belongs to the lung meridian. It is frequently clinically used in combination with other herbal medicines to manage lung-phase diseases, including asthma, pneumonia, and bronchitis [Houttuynia cordata Thunb., including antiinflammatory and antioxidant but also demonstrated its inhibitory effects against pulmonary fibrosis and tumors [Houttuynia cordata Thunb. on lung injury, but its specific active compounds and molecular mechanisms against RILI remain elusive.Mounting clinical evidence has demonstrated a certain degree of clinical efficacy and upstanding safety with the use of traditional Chinese medicine (TCM) to manage RILI . TCM belonchitis , 11. Modd tumors –14. SeveNetwork Pharmacology is pivotal in the study of the possible mechanisms of action of drugs in the following aspects: (i) mining drug and disease data in public databases; (ii) analysis of the active compounds of drugs and target genes of related diseases; (iii) exploring the possible mechanisms of action of drugs to reveal the pharmacological effects of multicomponent, multipathway, and multitarget therapeutic diseases of TCM . MoreoveHouttuynia cordata Thunb. in treating RILI and to use molecular docking for reverse verification. The findings provide objective evidence for preliminary elucidation of the pharmacological effects and molecular mechanisms of Houttuynia cordata Thunb. in treating RILI. The main framework of this research is shown in Therefore, this study aimed to use a comprehensive network pharmacology-based approach to investigate the potential effective components and molecular mechanisms of https://www.cnki.Net/), PubMed (https://www.Pubmed.ncbi.nlm.nih.gov/), TCMSP (https://tcmspw.com/tcmsp.php) [https://pubchem.ncbi.nlm.nih.gov/) [http://www.tcmip.cn/ETCM/index.php/Home/) [https://www.genecards.org/) [https://bioinfogp.cnb.csic.es/tools/venny/), Uniprot (https://www.uniprot.org/), String (https://string-db.org/) [https://david.ncifcrf.Gov) [https://www.omicshare.com/), and RCSB PDB (https://www.rcsb.org/) [https://pymol.org/2/).The databases used in this study include the following: CNKI (msp.php) , PubChemih.gov/) , ECTM (hp/Home/) , GeneCards.org/) , Venn (hdb.org/) , David (crf.Gov) , OmicShasb.org/) , and thesb.org/) , Open Basb.org/) , and PyMHouttuynia cordata Thunb. were obtained from the TCMSP database and literature. According to the pharmacokinetic characteristics, oral bioavailability (OB) ≥30% and drug similarity (DL) ≥0.18 were applied as the screening conditions to obtain the main active compounds [The chemical constituents of ompounds , 27. SubHouttuynia cordata Thunb. in RILI treatment. Next, the key targets of RILI were mapped to RILI-related genes and the common targets were screened to identify the key targets for RILI treatment. The web-based online mapping software Venny 2.1 was used to construct a Venn diagram of RILI mapping targets.The GeneCards database was searched using the keyword “radiation-induced lung injury” to collect the target genes related to RILI. This was followed by mapping the RILI-related genes to the drug target genes and screening the common targets to obtain the key targets of Houttuynia cordata Thunb. were input into Cytoscape software to construct a “Drug-Compounds-Targets-Disease” topological network. The important nodes in the network were selected based on the degrees of freedom of the topological structure of the network nodes. A higher degree value of a node implied that it is more important in the network. Such an observation indicated that the node is involved in more biological functions and, therefore, is more biologically important.The active compounds and related targets of Houttuynia cordata Thunb.-based treatment of RILI with the species restriction “Homospiece” and an Interaction Score >0.7 as the screening criterion. Topological analysis of the PPI network was conducted by the CytoHubba plug-in in Cytoscape (P < 0.01). The hub targets of Houttuynia cordata Thunb. for RILI treatment were further screened.The online STRING database was used to construct a PPI network for Houttuynia cordata Thunb. against RILI to understand the process of target action. Also, KEGG pathway analysis was performed to evaluate the distribution of targets in the pathway, with P < 0.05 and FDR < 0.05 as the screening criteria. Results were visualized via the OmicShare platform.The DAVID database was used to perform GO functional enrichment analysis on the core targets of Houttuynia cordata Thunb. using AutoDock Vina software. First, the 2D structures of the core compounds were downloaded from the PubChem database and optimized in Open Babel. Subsequently, the PDB ID and 3D structures of the target proteins were downloaded from the RCSB PDB. The original PDB file was processed with PyMOL software to remove water and original ligands. Using the AutoDock Tools, hydrogen, and charge were added to the protein structure. According to the position of the ligand, the coordinates of the protein active site were determined by AutoDock Vina for molecular docking studies. Lastly, the results were visualized in PyMOL.The core targets in the PPI network were molecularly docked to the four main active compounds of Houttuynia cordata Thunb. by screening, 7 active compounds were screened with OB ≥ 30% and DL ≥ 0.18 as the qualifying conditions. 95 targets were obtained. A literature search revealed the main pharmacological components of Houttuynia cordata Thunb., including methylnonane, cichorin, rutin, chlorogenic acid, and chrysin [Houttuynia cordata Thunb. were obtained  comprising 112 biological processes (BP), 9 cellular components (CC), and 32 molecular functions (MF). The top 10 entries of BP, CC, and MF are displayed in The 62 common genes for drugs and diseases were uploaded to the DAVID website for GO and KEGG analysis. GO enrichment analysis yielded GO terms , VEGFA (PDB ID:1KMX), JUN (PDB ID:1PXZ), TNF (PDB ID:4G4F), IL6 (PDB ID:1ALU), EGFR (PDB ID:2XKN), IL-1B (PDB ID:6Y8I), EGF (PDB ID:1EDM), PTGS2 (PDB ID:3NT1), and CAT (PDB ID:6GTK). Information about the affinity and hydrogen bond of compounds with key target protein is shown in mponents .Houttuynia cordata Thunb. has upstanding clinical efficacy for RILI but the specific molecular mechanism of action is elusive. Recently, network pharmacology has become an advanced method to analyze the complex mechanisms and collect active components of Chinese herbs or TCM formulas. This study indicated that the complex network relationship of multicomponent, multitarget, and multipathway of Houttuynia cordata Thunb. in RILI treatment.Compelling evidence shows that inflammation, oxidative stress, and fibrotic damage are the main pathological factors of RILI –32. In vHouttuynia cordata Thunb. through network pharmacology. At the same time, 26 core targets of Houttuynia cordata Thunb. against RILI and 4 associated pathways were predicted. More evidence from this study indicates that flavonoids , the main pharmacological active compounds of Houttuynia cordata Thunb., play a major inhibitory role against RILI. Flavonoids have previously been demonstrated to exert antiinflammatory, antioxidant, antitumor, antiradiation, and antifibrotic pharmacological effects [α protein content and decrease NF-kB protein level in the lung tissue of RILI mice. These events effectuated the regulation of the transcription of inflammatory factor genes, which successively improved the inflammatory exudation in mice with RILI and exerted a preventive effect on RILI [Houttuynia cordata Thunb. show a good affinity to bind to the core targets. These data revealed potentially the main active components of Houttuynia cordata Thunb. against RILI, which provides a basis for clinical application.In the present study, we identified 12 active compounds of  effects . In anim on RILI . In addi on RILI . Rutin e on RILI , 38. MorHouttuynia cordata Thunb. in RILI treatment include TP53, JUN, TNF, IL-6, IL-1B, PTGS2, and CAT, which are both the key drug targets and the core targets of the disease, suggesting that these targets may be the direct targets of Houttuynia cordata Thunb. against RILI. Combined with GO enrichment and KEGG pathway analysis, the targets of Houttuynia cordata Thunb. in RILI treatment were mainly enriched in cancer pathways, TNF signaling pathways, PI3K-Akt signaling pathways, and HIF-1 signaling pathways, similar to previous reports in radiation pneumonia. In view of these findings, the potential mechanism of Houttuynia cordata Thunb. in RILI treatment is related to the following aspects: (1) cancer pathways are the most enriched pathways for Houttuynia cordata Thunb.-based RILI treatment, with TP53, JUN, and VEGF as tumor-related bases, which are the core targets. Houttuynia cordata Thunb. may act directly or indirectly on tumor-related targets to exert anti-RILI effects. Radiotherapy for thoracic tumors causes radiation lung injury, so antitumor therapy is potentially among the main therapeutic approaches for RILI. For instance, VEGF is the main inducer of vascular neovascularization, and evidence shows that radiotherapy-induced pulmonary fibrosis is accompanied by vascular neovascularization. VEGF can promote endothelial cell proliferation and increase vascular permeability, and this successively exacerbates the inflammatory response [Houttuynia cordata Thunb. against RILI, among them, TNF, IL-6, and IL-1B, are mainly associated with the TNF signaling pathway. Therefore, the important anti-RILI mechanisms of Houttuynia cordata Thunb. are through antiinflammatory and antifibrotic effects. Of note, TNF is a key factor that initiates an inflammatory response in vivo and has been shown to play a critical role in early radiation pneumonia. Research using an animal model of pulmonary fibrosis also revealed that elevated synthesis and release of TNF stimulated the proliferation of fibroblasts and promoted collagen synthesis, events that caused pulmonary fibrosis. Inhibition of the TNF-a pathway demonstrated a significant protective effect on RILI in a mouse model of colon cancer lung metastasis [β a class of fibroblast growth factors, tend to increase after radiation and appear simultaneously with radiotherapy lung injury. Therefore, they are used frequently as a marker for the occurrence of lung injury after radiation therapy [Houttuynia cordata Thunb.-based prevention and treatment of RILI maybe through alleviating oxidative stress. Studies show that PTGS2 expression is positively correlated with the production of reactive oxygen species and inflammatory signals in tissues, and PTGS2 inhibition can reduce oxidative stress and inflammatory responses. The potential mechanism of the PTGS2 inhibitor, celecoxib, in RILI prevention and treatment is by reducing the toxicity of radiation to the lung [Houttuynia cordata Thunb. exert antitumor, antiinflammatory, antioxidative stress, antifibrosis effects and thus anti-RILI effects via the above pathways.Based on the PPI network, the core targets of response . (2) Inftastasis . Moreovetastasis , 42. Stu therapy . (3) PI3 therapy . (4) HIF therapy . Furtherthe lung , 47. TakHouttuynia cordata Thunb. treating RILI. However, a limitation is that further experiments are needed to demonstrate our findings.Collectively, the results in our study predict some potential therapeutic pathways and targets, and provide a reference for future research on Houttuynia cordata Thunb. for the treatment of RILI through network pharmacology and molecular docking. The results reflected the multicomponent, multitarget, and integrated regulation of TCM and provide a theoretical basis for the clinical application of Houttuynia cordata Thunb. on RILI treatment. However, this study did not consider the influence of drug dosage, drug ingredients, and drug delivery methods on the treatment results. Therefore, the results still need further verification (in vivo and in vitro) to provide the experimental basis for the future systematic study of Houttuynia cordata Thunb. on RILI treatment.In summary, this study preliminarily explored the active compounds, potential targets, and signaling pathways of