Reactome is a database of human biological pathways manually curated from the primary literature and peer-reviewed by experts. To evaluate the utility of Reactome pathways for predicting functional consequences of genetic perturbations, we compared predictions of perturbation effects based on Reactome pathways against published empirical observations. Ten cancer-relevant Reactome pathways, representing diverse biological processes such as signal transduction, cell division, DNA repair and transcriptional regulation, were selected for testing. For each pathway, root input nodes and key pathway outputs were defined. We then used pathway-diagram-derived logic graphs to predict, either by inspection by biocurators or using a novel algorithm MP-BioPath, the effects of bidirectional perturbations (upregulation/activation or downregulation/inhibition) of single root inputs on the status of key outputs. These predictions were then compared to published empirical tests. In total, 4968 test cases were analyzed across 10 pathways, of which 847 were supported by published empirical findings. Out of the 847 test cases, curators’ predictions agreed with the experimental evidence in 670 and disagreed in 177 cases, resulting in ∼81% overall accuracy. MP-BioPath predictions agreed with experimental evidence for 625 and disagreed for 222 test cases, resulting in ∼75% overall accuracy. The expected accuracy of random guessing was 33%. Per-pathway accuracy did not correlate with the number of pathway edges nor the number of pathway nodes but varied across pathways, ranging from 56% (curator)/44% (MP-BioPath) for ‘Mitotic G1 phase and G1/S transition’ to 100% (curator)/94% (MP-BioPath) for ‘RAF/MAP kinase cascade’. This study highlights the potential of pathway databases such as Reactome in modeling genetic perturbations, promoting standardization of experimental pathway activity readout and supporting hypothesis-driven research by revealing relationships between pathway inputs and outputs that have not yet been directly experimentally tested.www.reactome.org Reactome is a manually curated and peer-reviewed open-source database of human biological pathways . The patIn line with other manually curated biological databases , the accreaction, a biological event defined by a unique combination of inputs, outputs, catalysts and regulators. A reaction in Reactome is always supported by direct experimental evidence in a human research model or inferred from an orthologous reaction that is supported by direct experimental evidence in a model organism , representing an array of biological processes such as signal transduction, cell division, DNA repair and transcriptional regulation . These 1https://github.com/reactome/Release/blob/master/scripts/reaction_logic_table.pl). A detailed description of the conversion process is available in the Supplementary Methods.The 10 selected Reactome pathways were converted to logic graph formats  to facilitate semi-automated pathway analysis . The script used to convert pathways stored in the Reactome database into Logical Networks is available in GitHub  and output (O) node pairs and their connecting directed edges (described in Supplementary Methods). Root inputs (RI) in Logical Networks are input nodes (I) that never became outputs (O) in a pathway (RI = I—O), while terminal outputs (TO) are output nodes that never came to be inputs (TO = O—I). Curators used Cytoscape  to visuaWe selected RI nodes to use for perturbation predictions by intersecting the pathway gene set with the COSMIC database Cancer Gene Census (CGC) list  to find The selection of key outputs for each pathway was accomplished by finding all TO nodes indicative of pathway activation. TO nodes involved in negative feedback loops were excluded. Occasionally, nonterminal output nodes commonly used in biological assays to determine pathway activity were included in the testing. Key outputs could be individual proteins, mRNAs, small molecules, protein complexes, polymers, sets or, rarely, reaction nodes, when a TO was a part of a feedback loop. An example of RIs and key outputs is shown in A manual search of the PubMed database was conducted for each test case, using RI name, key output name and perturbation type as keywords. Gene name synonyms were included in the search. Search results were manually filtered based on the abstract content, and full text and figures of selected original research articles were inspected for relevant experimental findings and author statements. Various experimental designs and readouts were taken into account, as listed in Supplementary Table S1. In total, 531 papers  contained citable experimental evidence relevant for 847 test cases (the evidence from the retracted paper was excluded) (Supplementary Table S1). Of these, 158 papers were previously cited by the Reactome database in the context of the 10 selected pathways (Supplementary Table S1). Reactome-cited papers were not excluded from this study since these papers frequently contained the best available mechanistic evidence, were approved as relevant by external experts during Reactome peer review, and sometimes represented the only published experimental evidence of perturbation effects available.Inter-curator prediction agreement was determined between three curators on 100 randomly selected test cases by calculating percent agreement and the Fleiss Kappa statistic. Random selection of 100 test cases for inter-curator agreement determination was completed using Excel’s RAND function on 847 test cases with available literature evidence. Curators were trained on a pathway that was not used in the study to navigate Logical Networks in Cytoscape, apply the PathLinker tool to identify paths between RI and key outputs in Reactome Logical Networks, and predict effects of root input perturbations on key outputs  as depicted in Supplementary Figure S2. Curators were blinded to each other’s predictions. Percent agreement and Fleiss’ Kappa were calculated manually in Excel and using ReCal, an online tool .Each test case consisted of a unique combination of an RI, RI perturbation (upregulation or downregulation), a key output and the predicted change in the activity of the key output node  based on the Logical Network relationships. To predict the effects of a perturbation of the RI on the key output state, three Reactome curators applied the following rules (1): If an RI was connected with a key output through the edges of exclusively positive polarity or an even number of edges of negative polarity, the RI perturbation was predicted to result in the key output perturbation of the same directionality  (2); If a path from an RI to a key output contained an odd number of edges of negative polarity, the RI perturbation was predicted to result in the key output perturbation in the opposite direction  (3); If there was no path from an RI to a key output, or if an RI was connected to a key output through two paths of opposing polarity, the RI perturbation was predicted to cause no perturbation in the key output  (4). When an RI and a key output were connected through more than two paths, the polarity of each individual path was determined and the polarity of the majority of connecting paths was assigned as the combined polarity of the RI-key output relationship (not shown). The PathLinker Cytoscape app  was usedhttps://github.com/OICR/mp-biopath), to automate predicting the impact of RI perturbations on key outputs in Reactome pathways. This algorithm uses the Logical Networks to develop a non-linear mathematical model for each of the pathways. A high-level description of the MP-BioPath software can be found in the Supplementary Methods.In order to computationally simulate the impact of perturbations to RIs we developed a new computational algorithm, MP-BioPath  activity of the node, 0.01 represents a 100 times decreased activity compared to normal and 100 represents 100 times increase of the normal activity amount.Input nodes are related to output nodes via non-linear equations that take into account the logic and polarity of connecting edges. When inputs are connected to the output with positive polarity edges of AND logic, input node values are multiplied to determine the output node value. When an input is connected to the output with a negative polarity edge, a reciprocal value of the input is used in the multiplication equation. When inputs are connected to the output with edges of OR logic, the value of the output node is calculated as an arithmetic average of the values of input nodes. In this way, the value of each input node is considered when computing the value of the output node.The resulting system of equations is aggregated into a single optimization model for each pathway in order to calculate the magnitude and direction of the change of all nodes in the pathway that result from fixing the values for the known input perturbations. As it is possible for circumstances to arise where there is no solution to the system of equations based on the known inputs, flexibility is built into the model by having two values for each node: run time .MP-BioPath emits continuous values for the activity level of key outputs. In order to compare these values to literature-based perturbation experiments we needed to discretize these values to upregulation, downregulation, and no change. To establish appropriate cutoffs, we evaluated a range of cutoff values against curator-generated predictions across 18 539 test cases from 73 Reactome pathways (Supplementary Table S4).Treating the curator predictions as the ground truth, we evaluated cutoffs between 1% and 99% in 5% increments in order to construct an ROC curve and determine the accuracy, sensitivity, specificity, precision, false discovery rate (FDR) and F1 score at each cutoff . The F1 In total, 4968 test cases were analyzed over 10 pathways. Results of test cases for which experimental evidence was available were recorded and organized into a confusion matrix. Concordant test cases were scored either as true positives (TP) or true negatives (TN). A TP value was assigned when both Logical Networks and experimental evidence supported upregulation or downregulation of a key output as a consequence of an input perturbation. A TN value was assigned when the Logical Networks and experimental evidence supported no effect of an input perturbation on a key output. Discordant test cases were scored as either false negatives (FN) or false positives (FP). An FN value was assigned when, based on the Logical Networks, an input perturbation was predicted to not affect an output, but experimental evidence demonstrated that the output was affected. An FP value was assigned when, based on the Logical Networks, an input perturbation was predicted to affect the output, but experimental evidence showed that the output was not affected.As perturbations were bidirectional, a special case of FP was a false positive-wrong direction (FP-WD). An FP-WD value was assigned when, from the Logical Networks, an input perturbation was predicted to affect the output in the opposite direction from the experimental evidence .The resulting confusion matrix was used to calculate the average sensitivity and accuracy of Reactome pathway annotations. The comparison of accuracy, sensitivity, and specificity between curator-based vs. MP-BioPath based predictions on 847 test cases with published experimental results was done by running the Mann–Whitney test using the online tool Social Science Statistics .The 10 selected pathways, representing more than 1000 unique genes, ranged in size from 212 nodes and 246 edges  to 1106 nodes and 1276 edges , while the number of test cases per pathway ranged from 84 to 1836 . Some noOf the 4968 test cases, one-half of perturbations (2484) represented upregulation and the remainder represented downregulation. Among the 847 test cases supported by published experimental evidence, 54% of RI perturbations represented downregulation, while 46% represented upregulation. In contrast, RI upregulation was slightly more frequent than downregulation among 4121 test cases with no supporting experimental evidence . This is consistent with the observation that experimental studies preferentially use a loss-of-function approach to determine the roles of genes and their protein products, as previously reviewed .With respect to the status of key pathway outputs in 4968 test cases, perturbations of RIs were predicted to result in the upregulation of key pathway outputs in 1332 test cases (27%), downregulation in 1334 cases (27%), and to have no effect in 2302 cases (46%). No effect of RI perturbation on the key output status was expected when there was no path between an RI and a key output in Logical Networks (90% of cases) or when a negative feedback loop existed between an RI and a key output (10% of curator-predicted no-effect cases). While a plurality (46%) of the total test cases were predicted to have no effect on key outputs, just 161 (19%) of the 847 test cases with experimental evidence were ones predicted to have no effect, presumably reflecting the well-documented bias towards positive results in the published literature , 16.To measure inter-curator agreement on predicted perturbation effects, we randomly selected 100 test cases covering all 10 pathways as listed in Supplementary Table S3. Complete agreement was achieved on 81/100 predictions and there were no cases in which all three curators disagreed (Supplementary Table S3). The average pairwise percent agreement was 87.3%, with the Fleiss’ Kappa of 0.8 and the average pairwise Cohen’s Kappa of 0.801 . The KapAs the inter-curator agreement was substantial, we divided the workload of predicting the outcome of RI perturbations across the remaining 747 test cases among curators. We similarly divided the workload for prediction of effects for the 18 539 test cases across 73 pathways used to evaluate MP-BioPath cutoffs as described in the next section.Of the 847 test cases with experimental evidence, curator predictions based on Logical Networks agreed with published evidence in 670 cases (81% concordance), while MP-BioPath predictions were concordant with the published evidence in 625 test cases (75% concordance) , nor by the number of citations per pathway or node.We noted a modest positive correlation trend between the prediction accuracy and the number of citations per reaction-like event, but this effect did not achieve statistical significance . No significant correlation was found between prediction accuracy and the proportion of Reactome pathway citations that were also used as published evidence for effect of RI perturbations on the status of key outputs .When considering the types of reaction-like events present in different pathways, e.g. association, dissociation, conversion, posttranslational modification (PTM) and gene expression regulation (GER) , for theIn addition to analyzing the overall accuracy of curator-based and MP-BioPath-based predictions, we took a deeper dive into the ability of curators and MP-BioPath to predict the response to perturbations. In this analysis, we stratified test cases by their effect type  as shown in Supplementary Table S5. Curators were 88%, 90% and 84% accurate in predicting upregulation, downregulation and no change respectively. The respective accuracies of MP-BioPath were 84%, 86% and 79%. There was no significant difference in the accuracy, sensitivity and specificity between curators and MP-BioPath when predicting either upregulation, downregulation or no change .The distribution of confusion matrix categories with respect to the existence of a directed path between RI and key outputs in Reactome pathway diagrams is shown in For MP-BioPath-based predictions, all FP and FP-WD cases were due to calculated changes above the activity value cutoff. The plurality of FN cases were due to calculated changes in the right direction, but below the cutoff , followed by test cases with no directed path , calculated changes below the cutoff but in the wrong direction , and, lastly test cases with no change .The distribution of path lengths did not differ significantly between the concordant and discordant test cases for either curator-based or MP-BioPath-based predictions . When comparing path lengths between different confusion matrix categories, a significantly different distribution was detected between curator-derived TP and TN, TP and FN, FP and TN, and FP and FN cases. For MP-BioPath-derived confusion matrix categories, a significantly different distribution of path lengths was found between TP and FN, FP and FN, and FN and TN cases. The median and average path lengths for all confusion matrix categories, however, were similar, irrespective of whether they were curator- or MP-BioPath-derived . It should be noted that the number of test cases in the TP category surpassed the number of test cases in other categories on the order of 10–30-fold.Manual curation of the biological literature has long been considered to be the gold standard for pathway knowledgebases, and these curated knowledgebases are popular for data interpretation, analysis and hypothesis generation . MultiplWhile it is assumed that the knowledge gathered in curated pathway databases has utility for predicting the effect of naturally occurring and experimental perturbations, it is somewhat surprising that this assumption has never been empirically tested. In this study, we directly assessed the ability of Reactome, a large open access knowledgebase of curated human biological pathways, to predict the empirically-ascertained consequences of biological perturbations, by comparing Reactome pathway-based conclusions against published literature observations. Using a semi-automated process, we generated pathway Logical Networks for 10 cancer-related pathways covering ∼1200 unique genes and, from these graphs, we made nearly 5000 predictions of the downstream consequences of either increasing or decreasing the activities of pathway RI. The predictions were generated both manually, by curators, and computationally, using the novel pathway modeling tool MP-BioPath. We then mined the published literature to identify empirical studies that directly examined the effects of 847 of these perturbations and compared the empirical perturbation results to the predicted ones.https://github.com/dbtsai/libDAI)  algorithms that assume binary states for molecular participants in the pathway (e.g. affected vs. not affected)  and (ii)/libDAI) . PARADIGMP-BioPath applies a non-linear optimization model to a directed chain of reactions in a pathway, from root pathway inputs to terminal pathway outputs, through intermediary products. Thus, MP-BioPath algorithm represents an improvement over both a two-state or discrete state algorithm and provides a setup for the application of dynamic biochemical parameters.We found a high degree of concordance between the empirical and predicted perturbation effects. The concordance ranged from 56% (curator)/44% (MP-BioPath) to 100% (curator) /94% (MP-BioPath), with a mean accuracy of 81% (curator)/75% (MP-BioPath). There was no statistically significant difference in accuracy, sensitivity, and specificity between curator-based and MP-BioPath-based predictions . Our exaBesides the knowledge gaps that are intrinsic to any biological database and the disadvantage of comparing empirical data, which is usually generated in a small number of cell types, with in silico simulations in a generic cell, it should be noted that the methods used in this study have additional limitations. First, literature search itself is limited by the inconsistent use of gene and protein names, dependency on author-specified keywords, default settings of the search engine, and researcher bias, which increases the propensity for human-introduced errors when creating the ground truth for comparison. A more fundamental challenge arises from the arbitrary boundaries of biological pathways , 29. In For both curator and MP-BioPath predictions, we observed a significant increase in accuracy for the prediction of posttranslational modification events relative to prediction of GER events. We interpret this observation as reflecting Reactome’s better coverage of posttranslational modifications than transcriptional regulation. One factor explaining this is that GER is dependent on dosage of transcription factors and other quantitative parameters, such as the affinity of transcription factors for their different target sites , that ReIn addition, the fact that some of the literature references providing evidence of perturbation effects had been cited by Reactome during pathway curation did positively affect the accuracy of both curator and MP-BioPath predictions. Namely, 298/847 cases were supported by at least one of the 158 publications cited by Reactome. Although the fraction of published perturbation effect studies cited by Reactome does not significantly correlate with the predictive accuracy of individual pathways , when looking at these 298 test cases in isolation, their predictive accuracy calculated from the Supplementary Table S1, was 95% for curators and 91% for MP-BioPath. The future use of high-throughput perturbation datasets as experimental readouts will provide a more detailed evaluation of Reactome pathway-based predictive accuracy, removing some of the bias introduced by literature search.In summary, these results add to our confidence in the utility of pathway knowledgebases for predicting the effects of pathway perturbations and should be encouraging to ongoing efforts to create effective fully automated pathway-based predictive models for use in drug target discovery, mutation effect prediction, and synthetic lethality discovery , 31, 32.baac009_SuppClick here for additional data file.


Our results indicated good accuracy of the proposed mathematical algorithm in sifting the FCG from the TCM formula, which provided a methodological reference for discovering functional components and interpreting molecular mechanisms of the TCM formula in treating complex diseases.Depression, a complex epidemiological mental disorder, affects around 350 million people worldwide. Despite the availability of antidepressants based on monoamine hypothesis of depression, most patients suffer side effects from these drugs, including psychomotor impairment and dependence liability. Traditional Chinese medicine (TCM) is receiving more and more attention due to the advantages of high therapeutic performance and few side effects in depression treatment. However, complex multicomponents and multi-targets in TCM hinder our ability to identify the functional components and molecular mechanisms of its efficacy. In this study, we designed a novel strategy to capture the functional components and mechanisms of TCM based on a mathematical algorithm. To establish proof of principle, the TCM formula Danggui-Shaoyao-San (DSS), which possesses remarkable antidepressant effect but its functional components and mechanisms are unclear, is used as an example. According to the network motif detection algorithm, key core function motifs (CIM) of DSS in treating depression were captured, followed by a functional analysis and verification. The results demonstrated that 198 pathways were enriched by the target genes of the CIM, and 179 coincided with the enriched pathways of pathogenic genes, accounting for 90.40% of the gene enrichment pathway of the C-T network. Then the functional components group (FCG) comprising 40 components was traced from CIM based on the target coverage accumulation algorithm, after which the pathways enriched by the target genes of FCG were selected to elucidate the potential mechanisms of DSS in treating depression. Finally, the pivotal components in FCG of DSS and the related pathways were selected for experimental validation  Depression, a complex epidemiological mental disorder, stands at the 9th position behind prolific killers such as heart disease, stroke, and HIV, when ranked based on disability and death . A latesAngelica sinensis (Oliv.) Diels (Danggui), Poria cocos (Schw.) Wolf (Fuling), Alisma×bjoerkqvistii Tzvelev (Zexie), Paeonia lactiflora Pall (Baishao), Atractylis lancea var. chinensis (Bunge) Kitam (Baizhu), and Rhizoma Ligustici Chuanxiong (Chuanxiong) at a proportion of 3:4:8:16:4:3 (Danggui-Shaoyao-San (DSS), also called Toki-shakuyaku-san (TJ-23) or Dangguijakyak-san (DJS), is a widely used formula of traditional Chinese medicine derived from “Jin Kui Yao Lue” in the Eastern Han Dynasty of China . DSS is 8:16:4:3 . It has 8:16:4:3 . Recentl8:16:4:3 . DSS is 8:16:4:3 . The cli8:16:4:3  and expe8:16:4:3 . TerauchThe complex multicomponents and multi-targets in TCM hinder our ability to identify the functional components and molecular mechanisms of its efficacy. Systems pharmacology, a promising holistic strategy for the research of TCM, can comprehensively elucidate the synergistic mechanism of the TCM formula by generating complicated component–target and target–disease interaction networks . In recein vitro and in vivo.In this study, a novel computational systems pharmacology mathematical algorithm was designed and applied to detect the functional components group (FCG) and elucidate the therapeutic mechanisms of DSS in treating depression. First, all components in DSS were collected from databases and literatures. The active components were extracted from all components of DSS based on the criteria that combined Lipinski’s rule and Caco-2, and the component–target (C-T) network was constructed by predicting the targets of these active components based on published prediction tools. Second, the pathogenetic genes were collected from published databases and mapped to the protein–protein interaction (PPI) network to construct a weighted gene regulatory network of depression. The C-T network and pathogenetic gene regulatory network were combined to construct a C-T-P-D network. A network motif detection algorithm was designed and applied to capture the core intervention motif (CIM). The proteins in the CIM were defined as effective proteins, which could be used to screen effective components. Then the effective components in DSS were optimized and the FCG was obtained through the target coverage accumulation (TCA) algorithm, which would be selected to elucidate the underlining mechanisms of DSS in treating depression. Finally, a key component in FCG of DSS was used to perform experimental verification https://www.genecards.org). The genes with relevance scores higher than the average score were kept as high pathogenicity genes and then mapped to the PPI network, followed by the construction of the weighted gene regulatory network of depression. Cytoscape software (Version 3.7.0) was employed to visualize the network.A comprehensive weight gene network of depression was constructed by the PPI data acquired from public databases, including HPRD, MINT, STRING, Reactome, BioGRID, DIP, and intAct. Depression-related genes were extracted from GeneCards  were employed to acquire the targets of active components from DSS. The detailed method of chemical components collection and target prediction is described in the Supporting Information.Cytoscape software (Version 3.7.0) was used to create component–target (C-T) networks of DSS. Topological parameters of the networks were calculated by using NetworkAnalyzer.i and a to node ß.Mining motifs in complex networks can help us to have a deeper understanding of the treatment of diseases by drugs. In order to decode core intervention motifs of DSS in treating depression, refer to our previous mathematical algorithm with slight modification :qi↷=τn−t in CIM as In order to figure out the FCG in CIM, we assigned the target coverage of each component Set the sub-problems of the given question as follows:A GO analysis was performed by using the clusterProfiler package of R software. The KEGG database was used to perform KEGG pathway analyses. The detailed methods of GO and pathway enrichment analyses are described in the Supporting Information.2+ ([Ca2+]i).The neuroprotective effect of the top three components including vanillic acid, anisic acid, and ferulic acid (20 μmol/L) in FCG of DSS was assayed on corticosterone-treated PC12 cells by the MTT assay with desipramine (20 μmol/L) as the positive control group. Ferulic acid, which exhibited a potent neuroprotective effect on corticosterone-treated PC12 cells, was employed to evaluate the possible mechanisms by using apoptosis assay and Fluo-4/AM staining assay. Hoechst 33342 and PI double fluorescent staining were used to evaluate the anti-apoptotic activity. Fluo-4/AM staining was further performed to observe the effect of FA on the concentration of intracellular Can = 10): control group, FA (25 mg/kg) group, FA (50 mg/kg) group, FA (100 mg/kg) group, and venlafaxine (50 mg/kg) group. The drug was dissolved in normal saline with DMSO less than 0.1% [v/v] (vehicle). The solutions of drugs were administered to the mice via gastric intubation at a dosage of 0.2 ml/10 g (body weight) once daily between 9:00 a.m. to 10:00 a.m. for 14 days. Simultaneously, mice in the control group were orally given a vehicle at an equal volume. The study was approved by the Experimental Animal Ethical Committee of Modern Research Center for Traditional Chinese Medicine, Shanxi University. All experimental procedures in this study were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.In addition, the forced swimming test (FST) and the tail suspension test (TST) models were used to verify the antidepressant effect of FA. A total of 50 male ICR mice were randomly divided into five groups as follows , MAPT , AKT1 , and ALB  were probably the key targets of DSS in the treatment of depression.According to the random walk theory and Huffman encoding, the infomap algorithm was introduced to the network pharmacology model to extract critical information from the C-T-P-D network and explore the potential molecular mechanisms. The algorithm was performed to optimize the discovery of core intervention motifs in the C-T network through a reasonable global metric. As a result, six core intervention motifs were predicted in DSS . The detIn order to validate whether predicted CIM can represent full C-T network, two strategies were applied to certify the reliability and accuracy of CIM. First, the overlap of pathogenic genes’ number between CIM targets and C-T network targets were compared. We found that the C-T network contains 586 pathogenic genes. CIM contain 510 pathogenic genes . Second,One of the most important topological properties that could be employed to optimize the network is the importance of nodes in a network. In the current study, a novel approach to calculate the importance of nodes was designed, which takes into account the influence and connectivity of nodes. Based on our method, the pathway and GO term analyses of 580 effective proteins account for 94.24% and 86.69% of the intervention pathways and GO terms, respectively . Compare2+ could result in neuronopathy, which facilitates the development of depression , calcium signaling pathway (hsa04020), and PI3K-Akt signaling pathway (hsa04151) . Researcpression . In addipression .In the current study, we established a TCA algorithm to optimize CIM and obtain the FCG, which would be further employed to speculate the molecular mechanism of DSS in the treatment of depression. The contribution accumulation results demonstrated that the top five components including vanillic acid (DSS144), anisic acid (DSS59), ferulic acid (DSS17), phenylalanine (DSS297), and hexaphenone (DSS269) contribute to 51.03% target coverage of effective proteins. In addition, 40 components accounting for 90.00% targets coverage of effective proteins were grouped as FCG . Higher p < 0.05), and the number of pathways enriched by pathogenic genes was 208 (p < 0.05), which covered 81.25% of the pathogenic genes enriched pathways. The major targets of FCG were frequently enriched in the cAMP signaling pathway (hsa04024), calcium signaling pathway (hsa04020), MAPK signaling pathway (hsa04010), Ras signaling pathway (hsa04014), estrogen signaling pathway (hsa04915), and HIF-1 signaling pathway (hsa04066)  . The resIn order to further expound the mechanism of DSS, a GO enrichment analysis was performed to enrich all the targets interacting with FCG of DSS . The resDSS regulates the GO cellular component of depression, including synaptic membrane , ion channel complex , transporter complex , and neuronal cell body . Synaptic dysfunctions play important roles in the pathogenesis of depression, including the disruption in structural and molecular level . In addiDSS regulates the GO molecular function of depression including neurotransmitter receptor activity , ion channel activity , and passive transmembrane transporter activity . Our results indicated that DSS may affect autoreceptors and transporters of neurotransmitters to regulate neurotransmitter’s homeostasis in the synaptic cleft, which was closely related to the pathogenesis of depression .In order to illustrate the mechanisms of DSS in the treatment of depression at the system level, three important molecular pathways including the MAPK signaling pathway (hsa04010), cAMP signaling pathway (hsa04024), and HIF-1 signaling pathway (hsa04066) were used to construct a comprehensive signaling pathway . By searThe first five columns were considered as upstream and the rest columns were considered as downstream position of the pathway. Among these pathways, the MAPK signaling pathway (hsa04010) is regarded as one of the most important pathways in the treatment of depression with DSS. As demonstrated in The cAMP signaling pathway (hsa04024) and the HIF-1 signaling pathway (hsa04066) were also important pathways in the treatment of depression by DSS. A total of 47 targets were located in the cAMP signaling pathway, such as CSF1, GRB2, CREB3, and JUN. A total of 25 targets were located in the HIF-1 signaling pathway, such as TNF, EGF, HIF1A, and TRAF2. DSS may affect downstream of NFKB1 protein through the upstream IFNG and IFNGR1, followed by the regulation of downstream HIF1A cascade. In addition, TRADD could be regulated by DSS through the upstream TNF and TNFRSFIA, resulting in downstream TRAF2, TAB1, and MAP2K3 cascade amplification. The results suggested that DSS plays a therapeutic role by targeting different genes in the comprehensive pathway.P < 0.01, In order to further verify the results obtained by the network pharmacology analysis, the top three components including vanillic acid, anisic acid, and ferulic acid (20 μmol/L) in FCG of DSS were selected for experimental validation on PC12 cells by the corticosterone-induced damage model. As a result, desipramine (positive control), vanillic acid, anisic acid, and ferulic acid exhibited moderate neuroprotective activities to improve the PC12 cell survival rates from 56.34% ± 1.31% to 70.48% ± 1.02%, 59.68% ± 0.71%, 62.09% ± 2.53%, and 68.01% ± 1.72%, respectively. In addition, compared with vanillic acid and anisic acid, ferulic acid exhibited better neuroprotective activity (As demonstrated in 2+([Ca2+]i). The result is illustrated in 2+]i. However, pretreatment with FA could significantly reverse the enhanced green fluorescence, which indicated that the calcium signaling pathway was indeed related to the antidepressant mechanism of FCG in DSS.According to the results of the pathway enrichment analysis, the calcium signaling pathway was involved in the antidepressant mechanism of FCG in DSS. Fluo-4/AM staining was further performed to observe the effect of a representative component (FA) on the concentration of intracellular Cain vivo antidepressant effect of FA on two animal models of depression is shown in The At present, western medicines are the main antidepressants commonly used in clinics, but there are many problems, such as many adverse reactions, slow onset, and poor curative effect. The TCM formula can achieve the same curative effect as western medicines in treating depression . The TCMBy constructing a “drug–target–disease” network, systems pharmacology explores the interaction between biological functions and diseases at each node of the network, which is in line with the therapeutic principles of the TCM formula. Systems pharmacology is a powerful tool widely used to predict the potential mechanisms of TCM for treating diseases. However, there are few research reports on optimization of the TCM formula based on systems pharmacology. With the rapid development of computer technology, mathematical algorithms, and bioinformatics, our study proposes a comprehensive computational systems pharmacology model to optimize the TCM formula based on the mathematical algorithm. According to this model, we capture the CIM of DSS in treating depression and trace FCG from the CIM to speculate the possible mechanism of DSS in treating depression. This method provides a methodological reference for the interpretation of molecular mechanisms of TCM in the treatment of diseases and the development of new drugs.We used a network motif detection model to predict the CIM and performed a functional analysis and verification. The result demonstrated that the C-T network contained 586 pathogenic genes and the CIM contained 510 pathogenic genes. A further pathway enrichment analysis demonstrated that among the 198 pathways enriched by the target genes of the CIM, 179 coincided with the enriched pathways of pathogenic genes, accounting for 90.40% of the gene enrichment pathway of the C-T network. The result suggested that we have deleted the invalid or weak effect relationships and increased the functional coverage rate after optimization. Next, the TCA algorithm was applied to optimize effective ingredients and obtain the FCG. The FCG comprised 40 components from DSS, and the pathways enriched by the target genes of FCG were selected to elucidate the potential mechanism of DSS for the treatment of depression.Our proposed screening strategy possesses two advantages. The first level of design is to construct function motifs based on the infomap algorithm and verify that the information of disease-related genes will be preserved as much as possible in this step as the core protein or functional protein in the motifs. In the second step, potential FCG are reversely screened based on the core proteins or functional proteins, and further pathway enrichment analysis is performed on the functional component group for verification. The results of this dual model and verification fully demonstrate the reliability of our integrated analysis model and provide a methodological reference for other prescription optimization.Among the 40 key chemical compounds in FCG, the top five components including vanillic acid, anisic acid, ferulic acid, phenylalanine, and hexaphenone contribute to 51.03% target coverage of effective proteins, which play a critical role in the treatment of depression. Therefore, three accessible exogenous components including vanillic acid, anisic acid, and ferulic acid were used to evaluate the pharmacological effects in the model of corticosterone-treated PC12 cells. The results demonstrated that the neuroprotective activity of FA was superior to that of vanillic acid and anisic acid. Several studies have confirmed that FA is the major constituent in DSS, and the content of FA in DSS was much higher than that of vanillic acid and anisic acid . Therefoin vitro and in vivo.Ferulic acid  is a natural phenolic acid abundant in TCM, including Angelica Sinensis Radix, Chuanxiong Rhizoma, and Ferula teterrima Kar. Et Kir. . In addi2+ homeostasis  contributes to the pathogenesis of depression (Among the critical pathways enriched by FCG of DSS, the calcium signaling pathway is one of the most important pathways in the treatment of depression with FCG. A previous study has demonstrated that a dysregulation of the intracellular Capression . Thus, tIn addition, this study inserted the TCA algorithm to screen FCG of DSS in treating depression on the basis of the original CIM detection using the infomap algorithm. The reason of amend this part of the algorithm was for searching functional components, while the previous algorithm only looked for network motifs. The benefits of these changes are that one can more accurately obtain the FCG of the TCM formula. The similarity of the two algorithms is that they can all obtain the functional motifs in the formula. The difference of the two algorithms is that the previous algorithm can better reflect the functional motifs of “treating the same disease with different treatments” and the cumulative contribution rate of these functional motifs, while the current algorithm is more inclined to obtain the FCG in the TCM formula. The accuracy of the two algorithms changes according to different purposes, but the accuracy of the current algorithm is also high after verification. There are similar algorithms here, such as Louvain and label propagation. In this study, the TCA algorithm is inserted into these algorithms and optimized again to achieve accurate FCG search for DSS treatment of depression.in vivo antidepressant effect of these key compounds, several animal models based on different pathogenesis of depression should be employed in the future research. Finally, more study on the in vivo effect of FA on the calcium signaling pathway would be useful to understand the possible antidepressant mechanisms of FA. We hope, in the future, to explore the effect of FA on the calcium signaling pathway by knocking out genes or blocking pathways based on patients or animal models of depression.However, the limitations of this study exist. First, this algorithm cannot predicate the activation or inhibition effects of the targets. Second, to evaluate the Overall, a novel strategy to capture the functional components and mechanisms from TCM based on a mathematical algorithm was designed and applied to detect the FCG and decode the therapeutic mechanisms of DSS in treating depression. Therefore, the findings in this study provide a methodological reference for discovering functional components and interpreting molecular mechanisms of the TCM formula in treating complex diseases.


We assessed the relationship between acute primary angle closure glaucoma (APACG) severity and macular microcirculation, as well as the diagnostic ability of blood flow and macular structural parameters on optical coherence tomography angiography (OCTA) for APACG.APACG patients were assigned to mild, moderate, and severe groups in this cross-sectional study. Age-matched primary angle closure suspect (PACS) and healthy control groups were also recruited. The vessel density (VD) and foveal avascular zone (FAZ) in each macular superficial area were measured using OCTA. The retinal nerve fiber layer thickness (RNFLT) and ganglion cell complex thickness (GCCT) of the corresponding regions were measured using OCT.P < 0.05). VD and RNFLT showed high and moderate diagnostic ability, respectively, to distinguish moderate APACG from PACS, with significant differences (P < 0.05) in areas under the receiver operating characteristic curve (AUCs) for VD and RNFLT in six macular areas. The diagnostic abilities of VD and RNFLT for distinguishing severe APACG from PACS were increased, with significant differences in the AUCs for VD and RNFLT in five macular areas (P < 0.05). All macular VDs and GCCTs were similar among the three APACG groups (P > 0.05).All parameters in the control, PACS, and mild APACG groups differed significantly from those in the moderate and severe APACG groups (all Damage to the VD and FAZ in the macula increased with APACG severity. VD in the macular superficial layer showed a higher diagnostic ability than RNFLT, which was equivalent to that of GCCT. Glaucoma is defined as a multifactorial optic neuropathy. Increasing evidence shows that vascular factors play an important role in the pathogenesis of this disease , 2. HoweOptical coherence tomography angiography (OCTA) is a noninvasive imaging technique that allows reproducible quantitative assessment of retinal and choroidal vascular systems , which aRetinal ganglion cells (RGCs) are important damage markers in glaucoma, and are mainly present in the superficial retina . ApproxiThis study aimed to assess the relationship between the severity of APACG and macular microcirculation, as well as the diagnostic ability of blood flow and macular structural parameters, determined using OCTA, for APACG. To this end, we investigated changes in the macular microcirculation in eyes with different APACG severity.Patients with APACG who were hospitalized at the Tianjin Eye Hospital between January and April 2019 were consecutively enrolled. The study was approved by the ethics committee of Tianjin Eye Hospital  and followed the tenets of the Declaration of Helsinki for research involving human subjects. All participants provided written informed consent.All subjects were between 50 and 80 years of age. We recruited patients with APACG as well as age-matched patients with primary angle closure suspect (PACS) and individuals with healthy eyes.The inclusion criteria for APACG were as follows: (1) Patients with a previous history of APAC, within 6 weeks after onset, whose IOP was controlled within the normal range after drug or laser treatment. APAC was defined based on the following criteria: (i) at least two of the following symptoms: eyeball pain, nausea, vomiting, or both; history of intermittent visual blurring; and (ii) IOP exceeding 21 mmHg (based on Goldmann applanation tonometry [GAT]), with at least three of the following signs: conjunctival congestion, corneal edema, moderate pupil dilation, and shallow anterior chamber; and (iii) gonioscopy revealing angle closure , 26. (2)The inclusion criteria for individuals with PACS were as follows: (1) Static angle closure > 180°; and (2) IOP < 21 mm Hg without glaucomatous optic neuropathy .The exclusion criteria, for all subjects, were as follows: patients with high myopia or high hypermetropia (mean diopter ≤  ± 6D); patients with a history of any retinal diseases, non-glaucomatous optic neuropathy, uveitis, ocular trauma, or ocular diseases that seriously affected the turbidity of the refracting media of the eyeball; patients with a history of ocular surgery; and patients with moderate and above diabetes or hypertension .The age-matched healthy control group consisted of 33 eyes, PACS group consisted of 40 eyes and APACG groups consisted of 109 eyes. APACG groups were further divided into three subgroups according to severity. Using the grading method of the Glaucoma Staging System , APACG sAll participants underwent complete ophthalmological examinations 6 weeks after the onset of APAC, including best-corrected visual acuity and spherical equivalent (SE) by refractive error examination, slit-lamp examination, gonioscopy, and ophthalmoscopy, as well as IOP measurement by GAT during OCTA evaluation. Lenstar LS900  was used to measure axial length (AL), anterior chamber depth (ACD), and lens thickness (LT). Mean deviation (MD) values were measured for all subjects using standard automated perimetry .2 centered on the macula. The measurement area was divided into the macular fovea (F) and parafovea (PF), which were further divided into the superior hemiretina (SH), inferior hemiretina (IH), temporal (T), superior (S), nasal (N), and inferior (I) sectors, while the FAZ area was automatically measured. A light source with a wavelength of 840 nm was used for the OCTA examination, and the scanning speed was 70 kHz. Scanning was performed once in the horizontal and vertical directions. Patients were instructed to stare at the fixation point without blinking. A system with eyeball tracking and motion correction function was initiated, and the final image quality was checked by the operator. The vessel connection needed to be intact with no motion artifacts and vitreous floaters, and the signal strength index (SSI) needed to be greater than 60%; otherwise, the data were not used. The RNFLT in the corresponding region was obtained using the same protocol as that used for VD measurement. The thickness of the ganglion cell complex (GCCT)  was measured using the GCCT protocol of the system, and the average value of each subarea was automatically calculated  instruments, by two specially trained physicians. They measured the VD of the superficial retina  within 3 × 3 mmW test, calculated as mean and standard deviation, and categorical variables, such as the sex ratio, represented by the composition ratio. Using SPSS v18.0 statistical software , one-way analysis of variance was used to compare the differences in basic clinical characteristics, VD, and FAZ in each macular area among the five groups. If the variances of each group were homogeneous, Bonferroni-corrected post-hoc t-tests were used for each group comparison. Alternatively, Dunnett’s T3-corrected post-hoc t-tests were used for each group comparison. The chi-square test was used to determine the constituent sex ratio and the ratio of systemic diseases in the five groups. Using R 4.1.1 software, spearman correlation analysis was used for the correlation between visual field values and VD, FAZ, and R package GGplot2 was used to draw scatter graphs. MedCalc v18.2.1  was used for areas under the receiver operating characteristic curve (AUC) analysis to assess the diagnostic abilities of VD, RNFLT, and GCCT in the macula for APACG. Statistical significance was set at P < 0.05.The numeric variables were confirmed to be normally distributed using the Shapiro‒Wilks P > 0.05). As expected, compared to the healthy control group and the PACS group, the three groups of APACG had significantly more glaucoma medications, shorter AL, shallower ACD, thicker LT, and larger MD values (P < 0.05). However, there were no statistically significant differences among the mild, moderate, and severe APACG groups in the AL, ACD, LT, number of acute attacks, and the highest IOP during an acute attack (P > 0.05). However, the MD values, C/D ratio, and the number of glaucoma medications among the mild, moderate, and severe groups were significantly different (P < 0.05).The demographics and clinical characteristics of the experimental subjects are shown in Table P < 0.05). Further pairwise comparisons were performed for each group. Except for F-VD and FAZ, differences in other parameters were not significantly different among the healthy control, PACS, and mild APACG groups (P > 0.05). Compared with the healthy control and PACS groups, the F-VD in the mild group significantly decreased and FAZ significantly increased, and the difference was statistically significant (P < 0.05). Comparing the healthy control, PACS, and mild groups with the moderate and severe groups, differences in all parameters were statistically significant (P < 0.05), indicating that with the exacerbation of APACG, VD gradually decreased and FAZ gradually increased. Except for IH-VD, T-VD, and I-VD, differences in parameters were statistically significant between the moderate and severe groups (P < 0.05), which indicated that with the exacerbation of APACG, the VD decreased and FAZ increased . N-VD and I-VD were weakly correlated with MD values, while F-VD, PF-VD, SH-VD, IH-VD, T-VD, S-VD and FAZ were moderately correlated with MD values. The correlation between FAZ and MD value was the highest. The greater the FAZ value, the greater the visual field damage . For the moderate APACG versus PACS groups, the diagnostic ability of VD (AUC: 0.902–0.942) in the macular area was high, and that of RNFLT (AUC: 0.805–0.842) was moderate. The AUCs of SH-VD, IH-VD, T-VD, S-VD, N-VD, and I-VD were statistically significantly greater than those of RNFLT in the corresponding area, which means that the diagnostic ability was greater (P < 0.05). For the severe versus the PACS groups, the diagnostic abilities of VD (AUC: 0.900–0.958) and RNFLT (AUC: 0.828–0.887) in the macular area were both improved, and the difference between the two AUCs were reduced. The AUCs of PF-VD, SH-VD, IH-VD, S-VD, and N-VD were statistically significantly greater than those of RNFLT in the corresponding area, which means that the diagnostic ability was greater (P < 0.05). With APACG exacerbation, the diagnostic abilities of F-VD and RNFLT gradually improved. The diagnostic sensitivity of VD in the macular area for distinguishing moderate and severe APACG was higher than that of the structural parameter RNFLT  and RNFLT AUC: 0.613–0.672) in the macular area were both low, and the difference in AUCs between the two groups was not statistically significant (–0.672 inP > 0.05). For the moderate APACG versus PACS and severe APACG versus PACS groups, the diagnostic abilities of VD and GCCT improved, and the AUC values of SH-VD and IH-VD remained higher than those of GCCT in the corresponding area. Nevertheless, the difference was not statistically significant (P > 0.05; Table MedCalc software was used to compare the diagnostic abilities of VD and GCCT for APACG. For the mild APACG versus PACS groups, the AUC values of SH-VD and IH-VD were larger than those of GCCT in the corresponding area, but the diagnostic abilities of VD and GCCT were both low, and the difference was not statistically significant (In recent years, increasing attention has been paid to the role of abnormal intraocular blood flow or vascular regulation ability in the development and progression of various types of glaucoma. OCTA technology has mainly been used to study POAG, and the difference between POAG and primary angle closure glaucoma to date. The widespread use of OCTA technology in clinics has enhanced our understanding of the retinal microcirculation state in APACG patients with normal IOP after an acute attack. VD and structural lesions of the affected eyes continue to develop after an APAC attack  due to sIn the present study, no statistically significant difference in IOP was found among the five groups on OCTA examination, indicating that, other than IOP, the main influencing factor, the VD of each macular area decreased gradually and the FAZ increased gradually with APACG severity. Compared to the healthy control and PACS groups, the mild APACG group had a lower F-VD and larger FAZ, suggesting that these two parameters can be used as sensitive indicators of retinal microcirculation damage in patients with APACG. In the moderate APACG vs. PACS group, the diagnostic ability of VD was higher than that of the RNFLT in six macular regions, while in the severe APACG vs. PACS group, the diagnostic ability of VD was higher than that of the RNFLT in five macular regions. The diagnostic abilities of VD and the GCCT in each macular partition were similar in the three APACG groups. These results indicated that, compared with RNFLT, which is traditionally used as the structural indicator, VD had a higher diagnostic value for APACG, which was comparable to that of the GCCT, a classic indicator of glaucoma damage.Previous studies have found that the VD around the optic nerve head after an APAC was still low in cases with normal IOP, which was similar to the first conclusion in this study: VD in each macular sector after the acute attack was still low in cases with normal IOP, and decreased with increasing APACG severity . Due to Previous studies on POAG found that the retinal capillary density in a normal population was different from that in patients with moderate POAG, and that there was no difference between moderate and severe POAG patients . This waThe FAZ progressively increased under conditions of advancing age, diabetic retinopathy, retinal vein obstruction, and so forth –38. ThisPrior studies have shown that VD as measured by OCTA could be used to distinguish healthy eyes from glaucomatous eyes, as well as to distinguish between healthy people and those with suspected glaucoma , 46. Hen2 region in the macular area had greater diagnostic accuracy than that of the 3 × 3 mm2 region, because a smaller scan range might miss part of the GCCT area [2 region in This study had some limitations. The scan pattern of the 6 × 6 mmCCT area . HoweverIn conclusion, for cases of APACG with IOP that returns to normal after treatment, the VD was lower and the FAZ was smaller in the macular superficial layer than in healthy eyes, and the damage to the VD and FAZ was aggravated with APACG severity. VD in the macular superficial layer showed a higher diagnostic ability than RNFLT, and was equivalent to that of GCCT for detecting APACG. Further research is needed to establish whether a similar conclusions can be reached in a larger APACG population and in more types of glaucoma, and to understand the role of VD in the pathophysiological mechanism of glaucoma.


Sepsis-associated AKI (SA-AKI) may be characterized by damage to the renal tubular epithelium. In this study, the pharmacological mechanisms of Astragalus membranaceus and its active monomer Astragaloside IV (AS-IV) were predicted based on a network pharmacology approach and validated both in vivo sepsis model using a mouse cecum ligation puncture (CLP) and HK-2 cells were treated with lipopolysaccharide (LPS) to mimic Gram (–) induced sepsis to assess the renal-protective efficacy of Astragalus membranaceus and AS-IV.Method: We constructed an in vitro and in vivo, as shown by Western blot and immunohistochemistry analysis.Results: The findings demonstrated that Astragalus membranaceus and AS-IV attenuate renal tubular injury in mice with polymicrobial sepsis, including vacuolization, loss of brush border, mitochondrial ultrastructural changes, and increased staining of kidney injury molecule-1 (KIM-1). AS-IV protected human proximal tubular epithelial (HK-2) cells against LPS induced cell viability loss. Both Astragalus membranaceus and AS-IV activated the PI3K/AKT pathway both Conclusion: The findings demonstrate that Astragalus membranaceus and AS-IV protect against sepsis-induced kidney tubular injury by activating the PI3K/AKT pathway. Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated immune response to infection, which means that sepsis is diagnosed when the body’s response to infection results in life-threatening damage to its tissues or organs . Sepsis Astragalus membranaceus (AM) has historically been utilized primarily as a Qi-tonifying in traditional Chinese medicine. The active ingredients contained in Astragalus membranaceus and related plants have been investigated extensively . AstragaThe theory of network pharmacology was proposed by Hopkins in 2007 based on the scope of systems biology and pharmacology. The concept is based on multi-target research, which involves collecting and organizing data on target drugs, constructing bio-visualization networks, and analyzing the interactions between drugs and genes, targets, and diseases to reveal the mechanism of action from a systematic and holistic manner . The mecE. coli 0111: B4) was obtained from Millipore Sigma. Astragalus membranaceus was purchased from Kang Qiao Traditional Chinese Medicine Decoction Pieces Co. Ltd. Astragaloside IV (purity above 98%) was purchased from Xi’an Sobeo Pharmaceutical Technology Co., Ltd. The antibodies used for Western blotting, PI3K (#4255) and Phospho-PI3K p85 (Tyr458) (#17366) were purchased from Cell Signaling Technology. AKT (A17909) and Phospho-AKT (Ser473) (AP0637) were purchased from ABclonal.LPS  were weighed, distilled water was added in a ration of 1:10, soaked for 2–3 h, then decocted and extracted 3 times at 90°C for 1.5 h. The decoction was filtered twice with gauze and the filtrate combined. The filtrate was concentrated to a viscous liquid using a rotary evaporator at 50°C, poured into trays, and dried in a water bath set at 65°C to form an infusion. The infusion was frozen for two days at −80°C and then lyophilized to form a powder in a freeze dryer.Male C57BL/6J mice (18–22 g) were purchased from the Changzhou Cavens Laboratory Animal Co., China. All mice were housed in an automated 12-hour dark-light cycle at a controlled temperature of 22°C ± 2°C and relative humidity of 50–60%, with free access to standard dry feed and tap water. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals  and were approved by the Naval Medical University Committee on Animal Care (EC11-055).Sepsis was induced by CLP as previously described . IsoflurKidney tissues were cut into 5 μm sections and stained with H&E, PAS, and nitrotyrosine reagents, as well as KIM-1 antibody and Phospho-AKT antibody. The kidney injury score is as follows: 0, normal; 1, <10%; 2, 10–25%; 3, 25–50%; 4, 50–75%; 5, 75–100% of affected kidney areas obtained from mice exposed to sham or CLP surgery. Tubular atrophy or dilatation, loss of the brush border, vacuolization, epithelial cell shedding, and denuded tubular basement membrane were some of the criteria.2.HK-2 cells  were maintained in RPMI1640  supplemented with 10% v/v FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin  in humidified incubators at 37°C and 5% COThe TCMSP server consisted of a system-level pharmacology database with a flexible, user-friendly web interface. To gain a better understanding of the complex, network targets genes and diseases were constructed and analyzed using Cytoscape 3.0.The protein samples were electrophoresed in 8–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Amersham). The membranes were blocked with 5% BSA in PBS for 1 hour at room temperature and then probed overnight at 4°C with primary antibodies (1:1000 dilution). The membranes were then incubated with secondary antibodies  for 50 min at room temperature. Finally, Odyssey  and the relative protein expression were calculated from gray-scale blots using ImageJ software.t-test or ANOVA. P < 0.05 was considered statistically significant.GraphPad Prism 9.0.1 was used to perform statistical analysis and data were expressed as mean ± SEM. Statistical significance was determined using Student’s Our data are available. Please contact the corresponding author for requirement. More detailed Materials and Methods are in the Astragalus membranaceus protects against renal injury in CLP-induced sepsis. In this study, the CLP model, a widely accepted mouse model of sepsis, was used to induce SA-AKI in C57BL/6J mice. Histopathological examination revealed that 24 hours after CLP surgery, the kidney tissue interstitial edema in the CLP mice was obvious, with diffuse neutrophil infiltration and severe glomerular wrinkling, while the tubular lumen was occluded, some cells were swollen and vacuolated, and the tubular cystic lumen was dilated. Further treatment with Astragalus membranaceus (AM) improved focal tubular damage, tubular swelling, and vacuolar degeneration as well as inflammatory cell infiltration and interstitial edema to varying degrees in mice kidney tissues  and 1B. in vitro  induced sepsis-associated tubular injury. A total of 144 Astragalus-related compounds were identified through a database search. The TCMSP database was examined for OB and DL values of each compound, and 42 compounds (28.6%) had OB values greater than 30%, 50 compounds (40%) had DL values greater than 0.18, and 22 compounds (31.4%) had optimum OB and DL values. Astragalus saponins were excluded from the study due to their failure to meet the OB and DL thresholds. However, because these components are the most often reported active ingredients in the pharmacological action of Astragalus, they were included in this study. To screen for anti-SA-AKI activity, the final 26 monomers from Astragalus were collected . We discin vitro .P < 0.001). Astragaloside I (AS-I) and Astragaloside IV (AS-IV) demonstrated dose-dependent protective effects at 20 μM versus 50 μM concentrations . At 50 μM Iso and Cal showed significant protection (P < 0.001). In contrast, treatment of HK-2 cells with Kae, AS-II, Mairin, and Jaranol did not result in any significant changes in the LPS-induced cytotoxicity  <0.01 as the screening thresholds. A total of 686 entries for biological process (BP), 104 for cellular component (CC), and 121 for molecular function (MF) were obtained. The top 20 entries are displayed in P < 0.01 and FDR <0.01 to extract significant card values and discovered 2028 entries under BP, 63 entries under CC, and 132 entries under MF. P < 0.01 and FDR <0.01 as significant card values and obtained 1667 entries under BP, 43 entries under CC, and 89 entries under MF. The top 10 entries were selected for visualization, as shown in The therapeutic target database (TTD), Genecards, the Comparative Toxicogenomics Database (CTD), and the CTD were searched using the terms “sepsis” and “acute kidney damage (AKI). Genecards and the Comparative Toxicogenomics Database (CTD) were used to search for relevant disease targets, and the CTD database was used to identify experimentally validated disease targets designated as “marker/mechanism” or “therapeutic”. The CTD database selects targets with a high degree of confidence that has been experimentally validated with “marker/mechanism” or “therapeutic” as targets for research. Finally, 2498 sepsis-related genes and 6746 AKI-related genes were retrieved from the GeneCards database, while 48 sepsis-related genes and 120 AKI-related genes were retrieved from the CTD database. A total of 1938 targets were obtained after a comprehensive screening of all targets to eliminate duplicates, and the DAVID database validated a total of 1910 targets related to sepsis and acute kidney injury in humans. All genes were imported into the DAVID database, and the relevant genes were exported by selecting and clicking “show gene lis”. GO enrichment and KEGG pathway enrichment analyses were performed on the disease targets via the DAVID database, using Gene enrichment analysis revealed that the PI3K/AKT pathway was enriched for the most corresponding target genes, indicating that PI3K/AKT is an important intracellular pathway, and a previous study confirmed that PI3K/AKT plays an important role in SA-AKI , 16. WesChinese medicine is an important treasure of traditional Chinese medicine and is the material basis for the treatment of diseases in Chinese medicine. However, because of its complex composition and diverse effects, it cannot be precisely located in the body. Modern medical research has discovered that new drug development, is not only time-consuming and costly but also requires the application of network pharmacology and bioinformatics to significantly reduce the difficulties in new drug development. New drugs with significant efficacy against septic acute kidney injury and minimal adverse effects and cytotoxicity are urgently needed, and some monomers derived from Chinese herbal medicines are better choices than the herbs themselves because they can be administered in smaller doses, and have better-expected effects , 17.in vitro. Our findings indicated that that formononetin, AS-I and AS-IV, isorhamnetin, and Calycosin all exerted protective effects against LPS-induced cytotoxicity in HK-2 cells at 50 μM dosage, with AS-IV exerting the most prominent protective effect. Therefore, we investigated the mechanism of action of AS-IV against septic acute kidney injury both in vitro and in vivo.Astragalus membranaceus is a widely used Chinese herb known for its ability to tonify Qi and invigorate blood. In this web-based pharmacology study, a total of 22 preferred compounds with good oral bioavailability and drug-like coefficients were identified using the ADME parameters OB (>30%) and DL >0.18) reference values. Additionally, a review of the literature revealed that astragaloside analogues, the active components of Astragalus membranaceus, protect against sepsis-induced by cecum ligation perforation by inhibiting the inflammatory response and upregulation of the protein C pathway and that astragaloside has a protective effect on the kidney. Therefore, the present network pharmacology investigation included saponin components such as AS-I, AS-II, and AS-IV  referenc, kaempfein vitro and in vivo, including anti-oxidative stress, anti-inflammation, anti-apoptosis, and anti-fibrosis [in vivo and in vitro studies have demonstrated that AS-IV has a high level of biological activity and can protect cells by inhibiting the production of epithelial-mesenchymal transition-related proteins, attenuating oxidative stress damage, inhibiting the release of inflammatory factors and other mechanisms [in vitro studies, in septic rats, AS-IV therapies markedly decreased CLP-induced tubular damage and improved tubular pathology. PI3K is an important inositol and phosphatidylinositol kinase. Three subtypes of PI3K have been identified: class I, class II, and class III, with class I being the most extensively investigated. PI3K-IA is a receptor-mediated dimeric protein with dual protein kinase and lipid-like kinase activity [in vivo studies by decreasing oxidative stress and activating calpain-1, inhibiting the TBK1/PI3K/AKT signaling pathway, and several other additional mechanisms. Additionally, AS-IV can exert angiogenic and cardioprotective effects on the myocardium following myocardial infarction by activating the PTEN/PI3K/AKT signaling pathway and modulating the PI3K/AKT/GSK-3β pathway to alleviate the I/R effects caused by ligation of the left anterior descending coronary artery in rat hearts [AS-IV is the main active component of Astragalus and is frequently used in Chinese medicine as a quantitative and qualitative indicator of Astragalus. In recent years, various studies and reports have been published on the pharmacological effects of AS-IV fibrosis . Numerouchanisms . AS-IV hchanisms  and to echanisms . In the activity , while Pactivity . Proteinactivity . It alsoactivity . The PI3activity . Lysosomactivity . Wullschactivity . Inhibitactivity . AS-IV ht hearts . This stIn summary, our findings demonstrate for the first time that Astragalus membranaceus and AS- IV protect against sepsis-induced AKI in renal tubular epithelial cells by enhancing the PI3K/AKT pathway. This study provides evidence that Astragalus membranaceus and AS- IV may be a significant therapeutic approach in the treatment of sepsis-induced AKI.Supplementary Materials and MethodsSupplementary Figures 1-3


A non-significant reduction was found in the penetration depths and in a percentage of penetration around the canal perimeter between AH Plus and the tested calcium-silicate-based sealers (p > 0.05). Consequently, this reduction may not affect the three-dimensional seal of the root canal system in a negative manner. The penetration depth and percentage of penetration around the canal perimeter at both the root canal treatment and retreatment were significantly reduced from the coronal to apical points in all groups (p < 0.05).The aim of the present in vitro study was to evaluate the intratubular penetration of three bioceramic sealers in root canal retreatment. Here, 30 single-rooted human teeth were instrumented with the Protaper Universal system and filled with gutta-percha and the epoxy-resin-based sealer AH Plus mixed with rhodamine B. After two weeks in a humid environment, they were re-instrumented with Reciproc Blue and divided into three groups according to the endodontic sealer to be used in the re-filling (n = 10): G1: CeraSeal, G2: TotalFill BC Sealer, G3: TotalFill BC Sealer HiFlow. For the filling, a single cone technique was used, and the respective sealers were mixed with fluorescein. The roots were then sectioned at 2, 5, and 8 mm  from the apex, and the dentinal tubule penetration depth and percentage of penetration around the canal perimeter were evaluated by means of confocal laser scanning microscopy (CLSM). Penetration between groups was compared using the Kruskal−Wallis test, and within each group using the Wilcoxon test. Statistical significance was established at  The field of endodontics is constantly evolving as a result of the introduction of new techniques and advances in the development of dental materials. Numerous types of sealers have been used together with gutta-percha for root canal filling, resulting in the development of new sealers with enhanced properties. In this regard, calcium-silicate-based or bioceramic materials were introduced as a novel category of sealers . BioceraCurrently, calcium-silicate-based sealers such as MTA Plus , MTA Fillapex , iRoot SP , and EndoSequence BC sealer , also known as TotalFill BC Sealer  in Europe, have exhibited a low cytotoxicity, adequate bond strength, and adequate sealability. Additionally, TotalFill BC Sealer has been shown to enhance the osteoblastic differentiation of periodontal ligament cells, induce dentin remineralization, and possess antibacterial properties ,7. HowevRecently, a new calcium-silicate-based sealer has been developed for this purpose, TotalFill BC Sealer HiFlow , whose physicochemical properties remain stable at temperatures that correspond to those achieved inside the root canal during root canal treatment with heated gutta-percha techniques. It also presents a lower viscosity and film thickness, and a higher flowability when heated . Both ToOn the other hand, CeraSeal  is a recently introduced pre-mixed endodontic sealer containing calcium silicates, zirconium oxide, and thickening agents. However, a limited number of studies have been published to date on this sealer, and there is a lack of evidence among the literature on its in vitro and in vivo properties .One of the expected properties from these materials is their sealability, in other words, their potential to achieve a three-dimensional seal of the root canal system. As a means of assessing seal ability in vitro, the penetration depth inside dentinal tubules and the percentage of penetration around the canal perimeter can be evaluated. Confocal laser scanning microscopy (CLSM) is a widely used technique for the assessment of these parameters. In fact, several published studies use this method to evaluate the penetration of endodontic sealers ,13,14.However, to the best of the authors’ knowledge, there are no studies on the evaluation of the tubular penetration of bioceramic sealers in root canal retreatment. Root canal retreatment involves the removal of previous root canal fillings via chemical−mechanical disinfection through several methods, such as the use of solvents, Hedstroem files, ultrasonic tips, and/or rotary files. This procedure is considered as the treatment of choice in cases of inadequate initial root canal treatment, in the presence of signs and symptoms of periapical periodontitis , in the presence of recurrent carious lesions, and/or in the presence inadequate  provisional or definitive coronal restorations .E. faecalis and P. gingivalis, may influence the clinical outcome of root canal treatment. Thus, root canal retreatment should focus on the disinfection and three-dimensional seal of the root canal system [During this process, the three-dimensional cleaning of the endodontic space is the most critical phase, in order to reduce the bacterial load . The prel system .Achieving an adequate three-dimensional seal of the root canal system may be challenging in root canal retreatment, as remnants of the previous root canal sealer and or gutta-percha can remain both at a macroscopic and microscopic level, hindering the penetration of the new filling materials. The persistence of residual filling materials and the lack of penetration of the new filling materials may influence on the outcome of the root canal retreatment, due to the presence of persistent microbial populations and reinfection due to inadequate sealing .Accordingly, the aim of the present in vitro study was to evaluate the tubular penetration of three bioceramic or calcium-silicate-based sealers in root canal retreatments in vitro. The null hypothesis was that there would be no differences between the tubular penetration of AH Plus in root canal treatment and the tubular penetration of the three calcium-silicate-based sealers in the root canal retreatment.The protocol for this study was approved by the ethics committee from Universitat de València (ref. 85F5S1EEZ8322123). Thirty human maxillary incisors with mature apices extracted as a result of periodontal causes from healthy patients were selected for the present in vitro study, according to the following inclusion criteria: absence of carious lesions, root resorptions, pulpal calcifications, or other morphological anomalies. Prior informed consent was obtained from the donor patients. Two radiographic projections were performed for each tooth to confirm the presence of a single straight root canal. The teeth were stored in 0.1% thymol solution  until processing and were de-coronated using a tungsten carbide disc  at a low speed under refrigeration in order to obtain 16 mm long samples from the apex, which were stored in saline solution during the study.Patency was confirmed in all specimens by means of K8 and K10 files  through visual inspection of the file’s tip at the apical foramen after insertion. The working length was established at 16−1 mm. All root canals were prepared using the Protaper Universal system up to F3 file  with an X-Smart motor  following the manufacturers’ instructions regarding speed and torque. The shaping sequence was performed following the manufacturer’s instructions .During the instrumentation process, patency was confirmed between each file, and 2.5% NaOCl was used as an irrigant solution. Once the preparation was completed, the roots of the experimental groups were irrigated with a final sequence of 5 mL of 17% EDTA for 3 min and 2 mL of 2.5% NaOCl, separated by saline solution. Root canals were then dried using F3 absorbent paper tips .Root canal filling was performed by a previously trained single operator, via a single cone technique using F3 gutta-percha and epoxy-resin-based sealer AH Plus . Gutta-percha cones were introduced until working length, 0.5 mm, and confirmed with periapical radiography. AH Plus was mixed manually and transported into the root canal using the gutta-percha cones. Then, 0.1% Rhodamine B (red) dye was added to the AH Plus sealer to provide the fluorescence that would allow the posterior microscopic evaluation. The samples were stored for 2 weeks at 100% humidity and 37 °C.The specimens were subjected to root canal retreatment using Reciproc Blue system . The files were used with VDW Silver motor , following the manufacturers’ instructions regarding speed and torque, and were introduced into the root canals in three forward and backward movements with light apical pressure until working length was reached. The root canals were then re-prepared using F4 and F5 instruments from the Protaper Universal system, as described above. Irrigation procedures were kept the same as in the initial endodontic treatment.For root canal re-treatment filling, root samples (n = 30) were randomly allocated into the different experimental groups (n = 10 per group): CeraSeal , TotalFill BC Sealer , or TotalFill BC Sealer HiFlow . The composition of the tested sealers is presented in A diamond cutting disc  was used at low speed to obtain three 1 mm thick discs of each root at 2, 5, and 8 mm from the apex of the tooth . The equipment used for the microscopic study was a CLSM Leica TCS SP8  from the Microscopy Service at Universitat de València , with excitation and emission wavelengths for rhodamine B of 543 nm and 578 nm, and 514 nm for fluorescein staining. Sample analysis was performed at 10× magnification. The images were subsequently recorded and analyzed using ImageJ v1.53a software . The tubular penetration depth (in μm) and the percentage of penetration around the canal perimeter were measured from the images obtained by CLSM, based on the methodology of a similar study ,20 Figu.® SPSS V25.00 software . The measurements of the tubular penetration depth and percentage of penetration around the canal perimeter were performed once by a single investigator for each of the samples. The descriptive data of tubular penetration depth and percentage of penetration around the canal perimeter of the residual sealer of the first root canal filling  and of the sealer used in the retreatment  are presented. As the data did not possess a normal distribution, the mean and median values and the interquartile range (IQR) of the data are given. Both the percentages of penetration around the canal perimeter and the depth of penetration in micrometers for each sealer were compared between the different measurement points using the Kruskal−Wallis test. The Mann−Whitney U test was used to compare the groups globally and for each measurement point.The obtained data were analyzed statistically using IBMQuantitative results of the CLSM image analysis in terms of the tubular penetration depth and the percentage of penetration around the canal perimeter exhibited by AH Plus and CeraSeal are presented in p > 0.05), both in total and per third .No significant differences were observed between the tubular penetration depth and percentage of penetration around the canal perimeter between AH Plus sealer and CeraSeal (p < 0.05). The CeraSeal samples, however, only showed a significant reduction in the depth of penetration from the coronal to the apical measurement points (p < 0.05)  A.p < 0.05) and the coronal and middle points (p < 0.05), while the CeraSeal samples only showed a significant reduction in the percentage of tubular penetration from the coronal to the apical measurement points (p < 0.05)  B.The quantitative results of the CLSM image analysis in terms of the tubular penetration depth and percentage of penetration around the canal perimeter exhibited by AH Plus and TotalFill BC Sealer are presented in p > 0.05), both in total and per third .No significant differences were observed between the tubular penetration depth and percentage of penetration around the canal perimeter between AH Plus sealer and TotalFill BC Sealer (p < 0.05). The TotalFill BC Sealer samples, however, only showed a significant reduction in the depth of penetration from the coronal to the apical measurement points (p < 0.05)  A.p < 0.05)  B.Quantitative results of the CLSM image analysis in terms of tubular penetration depth and percent penetration of the dentinal tubule perimeter exhibited by AH Plus and TotalFill BC Sealer HiFlow are presented in p > 0.05), both in total and per third .No significant differences were observed between the tubular penetration depth and percentage of penetration around the canal perimeter between AH Plus sealer and TotalFill BC Sealer HiFlow (p < 0.05). The TotalFill BC Sealer samples, however, only showed a significant reduction in the depth of penetration from the coronal to the apical measurement points (p < 0.05)  A.p < 0.05)  B.The aim of this in vitro study was to assess the intratubular penetration potential of three calcium-silicate-based sealers  in root canals initially treated with an epoxy-resin-based sealer (AH Plus) via laser confocal optical microscopy. Currently, no endodontic sealer meets all of the expected ideal properties, such as dimensional stability, biocompatibility, and adequate seal . On the The penetration depth of the sealers was evaluated under laser confocal microscopy. It is an observational technique that allows for evaluating the penetration of sealers inside the dentinal tubule using fluorescent staining. This is possible because the microscope performs several slices of the sample at depths of 20–30 µm , and ovep < 0.05) in any of the cases. In other words, the reduction in the penetration of the tested calcium-silicate-based sealers in root canal retreatment is not significant and consequently may not affect the three-dimensional seal of the root canal system negatively.Regarding the results of the present study, AH Plus sealer showed a higher penetration compared with the three bioceramic sealers; therefore, the null hypothesis was rejected. This was expected, as it was the first sealer used, whereas bioceramic sealers were used for retreatment and thus may have found residual filling materials that hindered their penetration. In addition, one of the reasons for the popular use of AH Plus is its adequate flowability and viscosity, which may result in a higher penetration ,27. HoweIn parallel, in this study, it was observed that there was a large amount of residual AH Plus sealer inside the dentinal tubules after retreatment with the Reciproc Blue system. This is because, as previously mentioned, no retreatment system completely removes the root canal filling materials that have initially penetrated inside the dentinal tubules . FurtherInterestingly, the tubular penetration followed the same pattern in all teeth. Both in the depth of penetration in the dentinal tubules and in the percentage of penetration around the canal perimeter, the apical measurement point showed the lowest results, followed by the middle and coronal measurement points. This is in accordance with other authors ,29. ThisThis study followed a similar methodology to that of a recently published study by Eğemen et al. , in whicA limitation of this study lies on the standardization of the quantity of sealer used for the filling of each sample. Nevertheless, the anatomical differences in the root canal systems of the samples intrinsically hinders the standardization process. Another limitation of this study lies in the fact that the same filling method was used for both types of sealers, whereas the manufacturers’ recommendation is that the single cone technique should preferably be applied for calcium silicate sealers. However, this issue is under debate. For example, in the study by DeLong et al. in 2015 , the autThe results from the present in vitro study report a non-significant reduction in penetration depth and a penetration around the canal perimeter of the calcium-silicate-based sealers CeraSeal, TotalFill BC Sealer, and TotalFill BC Sealer HiFlow in the retreatment of teeth with a prior root canal treatment with the epoxy-resin-based sealer AH Plus. Consequently, this reduction may not affect the three-dimensional seal of the root canal system in a negative manner.Both the penetration depth and penetration around the canal perimeter were significantly reduced in a coronal−apical manner both in root canal treatment with AH Plus and in the root canal retreatment with the tested calcium-silicate-based sealers.


Objective: To explore the effect of Fasudil on HA spasm and its underlying mechanism.A, and ETB.Methods: Rabbits were divided into Sham, Fasudil, and Model groups for experiments. Fasudil was injected into the left medial lobe of the rabbit liver using a 16G lumbar puncture needle through the laparotomic route. The spasm model was established by inserting the catheter sheath into the femoral arteries of rabbits, followed by celiac artery angiography and left HA catheterization with a micro-catheter. Next, the GSE60887 and GSE37924 datasets concerning Fasudil treatment were analyzed. Moreover, immunofluorescence staining was conducted for YAP1 and α-SMA. Finally, Western blotting was performed to examine the expressions of YAP1, ROCK, ERK1/2, ETA and ETB. It was manifested in the results of immunofluorescence staining that the YAP1-positive fluorescence area was significantly decreased after Fasudil treatment. Moreover, Western blotting results showed that Fasudil decreased the expressions of YAP1, RhoA, ROCK, ETA, ETB, and p-ERK1/2. In addition, in-vitro Western blotting revealed that Fasudil suppressed the YAP/ERK/ETA/ETB signaling pathway in the case of HA spasm by inhibiting ROCK activation.Results: Fasudil could relieve HA spasm. The Go and KEGG pathway analyses revealed that the MAPK signaling pathway and the Hippo signaling pathway were enriched in vasospasm. Besides, GSEA revealed that ROCK was functionally enriched in the MAPK and Hippo signaling pathways. Co-expression analysis revealed that MAPK1 was significantly correlated with YAP1 and MYC, and YAP1 was significantly correlated with ETA/ETB signaling pathway and the ROCK activation.Conclusions: Fasudil ameliorates HA spasm through suppressing the YAP/ERK/ET Hepatic artery (HA) spasm is a potential complication of transcatheter arterial chemoembolization (TACE) for liver cancer , 2, and Rho-kinase (ROCK), a serine/threonine protein kinase, is one of the major downstream effectors of Rho GTPase , 7. The A) and ETB], can induce smooth muscle contraction and proliferation  was added to extract proteins, followed by quantitative determination by BCA assay . Then, 40 μg of proteins was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (12% separation gel and 5% concentrated gel) at 45 V for 150 min. After that, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane  through wet method and blocked with 5% non-fat dry milk diluted in phosphate-buffered saline (PBS) with Tween 20. Next, the membrane was incubated with primary antibodies  at 4° C overnight, and with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:5000) at room temperature for 60 min, followed by washing. Subsequently, images were visualized using an enhanced chemiluminescence kit, and BeyoECL Plus working solution was added for 2–3 min, followed by exposure and color development. Finally, the corresponding quantitative analysis was conducted using ImageJ software. All experiments were performed at least three times for the accuracy and stability of the results.B , YAP1 , and α-smooth muscle actin (α-SMA)  overnight at 4° C. After incubation with fluorescent dye-labeled goat anti-mouse and goat anti-rabbit secondary antibodies at room temperature for 1 h, the sections were washed again with PBS and sealed with 50% glycerol. Subsequently, the nuclei were stained with DAPI (1:1000) to display the position of the nuclei. Finally, the images of the tissues were observed and captured under a confocal fluorescence microscope.Immunofluorescence staining was performed on rabbit aortic tissue samples. The previously obtained tissue sections were fixed in 5% polyformaldehyde, washed with PBS and sealed with 100 μL blocking solution  at room temperature. Then the prepared sections were incubated with the primary antibodies against ETin-vitro HA spasm model was established as previously described [An escribed –31. The t-test was used for comparison between two groups. Data were expressed as mean ± SEM. P<0.05 suggested a statistically significant difference.Data were analyzed using one-way ANOVA, Tukey’s test was adopted for comparison among multiple groups, and Student’s in vivo and to ascertain the underlying action mechanism. Female NZW rabbits were randomly assigned into Sham group, Model group, and Fasudil group. Fasudil or normal saline was injected into the left medial lobe of the rabbit liver using a 16G lumbar puncture needle through the laparotomic route. Next, the catheter sheath was inserted into the femoral arteries of rabbits to simulate the spasm model, followed by celiac artery angiography and left HA catheterization with a micro-catheter. The celiac artery angiograms revealed that the rabbit models of HA spasm were successfully established . As shown in A) and EDN2 (ETB). According to the data from the GSE37924 dataset, MAPK1 was significantly correlated with EDN1 (ETA) and EDN2 (ETB) as well , and the antibody against ROCK used in this study could recognize both cleaved C-terminus of ROCK1 (30 kDa) and full-length protein (158 kDa) or activated form of ROCK. Therefore, the proteins related to ROCK shown in Western blotting were involved in the activation of ROCK.Additionally, the effect of Fasudil on the RhoA/ROCK signaling pathway in vasospasm was investigated. The mRNA data from the GEO database as well as the GSE60887 and GSE37924 datasets concerning Fasudil treatment were analyzed. The DEGs in both datasets and those identified by the GO and KEGG pathway analyses were determined. It was discovered that the MAPK signaling pathway and the Hippo signaling pathway were enriched in vasospasm. It has been evidenced that ROCK is an upstream regulator activating ERK cascades . The GSEYAP is a primary target of the Hippo signaling pathway, which plays a pivotal role in regulating vascular contraction. Moreover, ERK1/2 is an important player in the Hippo/YAP signaling pathway , 14. AccA and ETB receptors plays a critical role in vasospasm [B was decreased by Fasudil treatment in vivo. It was further discovered through Western blotting that Fasudil treatment inhibited the protein expressions of ETA and ETB in rabbit arterial smooth muscle tissues. Co-expression analysis revealed that YAP1 was significantly correlated with EDN1 (ETA) and EDN2 (ETB) based on the data from the GSE60887 and GSE37924 datasets.Additionally, various studies have confirmed that the activation of ETasospasm , 21. In In vitro, VSMCs were stimulated by mechanical pressure to mimic an in-vitro spam model. It was shown in Western blotting results that Fasudil injection restricted the expressions of ROCK, p-ERK1/2, YAP1, ETA, and ETB in VSMCs. Next, the effects of ROCK-OE Lentivirus (ROCK agonist) on VSMCs were also examined. Unexpectedly, the decreased expressions of ROCK, p-ERK1/2, YAP1, ETA, and ETB in Fasudil group was restored, suggesting that ROCK activation is involved in Fasudil-induced inhibition of HA spasm.A/ETB signaling pathway. These are summarized in a schematic diagram (In conclusion, the data of the present study suggested that Fasudil alleviates HA spasm by inhibiting ROCK activation to repress the YAP/ERK/ET diagram . Therefo


Microarrays can perform large scale studies of differential expressed gene (DEGs) and even single nucleotide polymorphisms (SNPs), thereby screening thousands of genes for single experiment simultaneously. However, DEGs and SNPs are still just as enigmatic as the first sequence of the genome. Because they are independent from the affected biological context. Pathway enrichment analysis (PEA) can overcome this obstacle by linking both DEGs and SNPs to the affected biological pathways and consequently to the underlying biological functions and processes.p value for each enriched pathways and limit the number of enriched pathways. This helps reduce the number of relevant biological pathways with respect to a non-specific list of genes.To improve the enrichment analysis results, we present a new statistical network pre-processing method by mapping DEGs and SNPs on a biological network that can improve the relevance and significance of the DEGs or SNPs of interest to incorporate pathway topology information into the PEA. The proposed methodology improves the statistical significance of the PEA analysis in terms of computed The proposed method provides two-fold enhancements. Network analysis reveals fewer DEGs, by selecting only relevant DEGs and the detected DEGs improve the enriched pathways’ statistical significance, rather than simply using a general list of genes.The online version contains supplementary material available at 10.1186/s12859-022-04936-z. The advent of microarrays  allowed polymerase biological process. All the cited methods will extend the genetic sequence of interest by adding on the complementary nucleotide from the template DNA strand. These methods allow a relative and accurate quantification of DNA and mRNA molecules with a sufficiently high reproducibility and low variability, and they are all well suited to study gene expression.In this manner, microarrays allow for screening of thousands of genes for a single experiment. Sanger-method, RT-PCR and microarrays rely on the extension of small segments of DNA through the However, after the initial fervor, it became apparent that even the lists of DEGs or SNPs were mainly as enigmatic as the first nucleotide sequence of the genome. The main reason being that these lists of DEGs and SNPs are independent from the affected biological context. To overcome this limitation, several statistical software tools –9 have bdisease, rather than a more specific one like cellular responses to external stimuli, a well-known pathway involved in the progression of colorectal cancer, for example. These biases prevent researchers from figuring out the proper affected biological pathways and the related functional interactions.Pathway enrichment analysis (PEA) can facilitate the interpretation of such a list of DEGs or SNPs, linking both to the affected biological pathways and consequently to the underlying biological functions and processes. Although, PEA can help figure out the affected biological pathways starting from the DEGs or SNPs of interest, poor quality and relevance of the employed input can produce pathways that are not directly related to the condition under investigation. This is due to the fact that, a poor quality list of DEGs or SNPs can enrich a general pathway such as p value (i.e. relevance) of the enriched pathways and reduce the number of enriched pathways, consequently improving their relevance with respect to the condition under investigation.To improve the enrichment analysis results, it is necessary to determine the relevant DEGs that can both improve the (i) DEG filtering relies on the Kruskal–Wallis test Metabolisuced. In  it is clented in .Cellular Senescence pathway Synthesisined in  .GSE16759 data set. In [PERK regulates gene expression pathway with AD. In [Signaling Pathways in the development of many human diseases including AD disease. The authors in [unfolded protein response (UPR) with onset of familial Alzheimer’s disease (Fig. Table h AD. In  the auththors in  characteComparing the enriched pathways in Figs. Finally, we performed PEA using the grouped and ungrouped DEG sets to assess the effectiveness of the proposed DGE preprocessing and selection method. Analyzing the obtained pathway enrichment results using both data sets highlight that DEGs critically impact PEA, since employing an ungrouped DEG set can lead to poor enrichment results. Also, the first ranked enriched pathway using grouped DEGs is related to the condition under investigation, which may induce new biological discoveries and simplify research.In this work, we proposed a new statistical network pre-processing approach to identify relevant DEGs that can improve PEA results, helping researchers identify the affected underlying biological functions and processes. The proposed method provides a two-fold improvement. First, network analysis yields fewer DEGs, choosing only relevant DEGs that directly involved with the condition under investigation. Second, the detected DEGs improve the enriched pathways’ statistical significance over a more general list of genes. As a drawback, the number of enriched pathways is still too large; thus, future research should be aimed at developing a method to further reduce the number of enriched pathways.Additional file 1: Provides detailed information about the other investigated GSE data sets.