Cyanotis arachnoidea C. B. Clarke is a traditional Chinese medicinal herb that has a limited clinical use in the treatment of diabetes mellitus (DM) in minority areas of Guizhou in China. However, few prior reports are available on the quality control of Cyanotis arachnoidea, and its quality markers and hypoglycemic mechanism are still unclear. The purpose of this study is to explore the quality markers (Q-markers) of Cyanotis arachnoidea and predict its hypoglycemic mechanism. In this study, ultra-high-performance liquid chromatography (UHPLC) fingerprint combined with chemical pattern recognition were performed, and four differential components were screened out as quality markers, including 20-Hydroxyecdysone, 3-O-acetyl-20-hydroxyecdysone, Ajugasterone C, and 2-O-acetyl-20-hydroxyecdysone. Network pharmacology analysis revealed 107 therapeutic target genes of Cyanotis arachnoidea in DM treatment, and the key targets were Akt1, TNF, IL-6, MAPK3, and JUN. The hypoglycemic mode of action of Cyanotis arachnoidea may be mediated by tumor necrosis factor (TNF) signaling, cancer, insulin resistance, and JAK-STAT pathways. Molecular docking analysis disclosed that the foregoing quality markers effectively bound their key target genes. An in vitro experiment conducted on pancreatic islet β-cells indicated that the forenamed active components of Cyanotis arachnoidea had hypoglycemic efficacy by promoting PI3K/Akt and inhibiting MAPK signaling. UHPLC also accurately quantified the quality markers. The identification and analysis of quality markers for Cyanotis arachnoidea is expected to provide references for the establishment of a quality control evaluation system and clarify the material basis and hypoglycemic mechanisms of this traditional Chinese medicine (TCM). Diabetes mellitus (DM) is a complex and prevalent chronic metabolic condition. Its global incidence is increasing annually. DM is described as a disorder of glucose metabolism resulting from absolute or relative insulin insufficiency . AccordiCyanotis arachnoidea is a traditional Chinese herbal and ethnic medicine that is widely distributed in India, Sri Lanka, and the Yunnan Province of China. Ancient Chinese medicine texts reported that it has been used to treat nephritic edema, diabetes, rheumatoid arthritis, and other diseases , thereby improving their typical symptoms. Nevertheless, active ingredients in Cyanotis arachnoidea and their hypoglycemic mechanisms remain unclear, and the adoption of this herb in clinical practice is severely limited. Furthermore, differences in the sources, harvest seasons, and processing methods of Cyanotis arachnoidea result in variability of its components, and effective quality control methods for Cyanotis arachnoidea are lacking.Chinese herbal medicine has been administered to prevent and treat DM for thousands of years and has attracted research attention as it has multi-target and multi-pathway characteristics. The glycosides , polyphediseases . Modern ecdysone . This coynthesis , has phaynthesis , and hasynthesis , antioxiynthesis , neuroprynthesis , and antynthesis  activityynthesis  showed tynthesis  demonstrCyanotis arachnoidea, and examine their hypoglycemic molecular mechanisms of action through the analysis of Q-markers and biomarkers, chemical pattern recognition and network pharmacology were performed to screen differential components, identify active ingredients and predict their potential targets. The TCM fingerprint is a comprehensive and quantifiable identification method that has been widely used for TCM quality control in combination with chemical pattern recognition techniques, such as hierarchical cluster analysis (HCA) and principal component analysis (PCA)  herbs have complex compositions and multiple components, targets, and pathways. Thus, it is difficult to elucidate their modes of action. In 2016, quality markers (Q-markers) were proposed to evaluate and control TCM quality . A Q-maris (PCA) . Networkis (PCA) .via calculations, simulations, and predictions (Molecular docking technology is a type of computer-aided drug design (CADD). This new drug research and development technology has rapidly developed to explore rational drug design. It uses information technology and exploits existing knowledge of related drugs and their targets to guide research in drug discovery and drug action mechanisms dictions . Digitaldictions . Moleculdictions .Cyanotis arachnoidea. We screened potential quality markers and identified them by chemical pattern recognition technology. We used network pharmacology to predicte the hypoglycemic mechanisms of the components based on their quality markers and verified the latter by molecular docking and in vitro experiments. Finally, we quantitatively analyzed the quality markers by UHPLC. In conclusion, the present work devised a quality control evaluation method for Cyanotis arachnoidea and provided a reference for clarifying the pharmacodynamic mechanisms of the active ingredients of this TCM in DM. The experimental design is shown in In the present study, we used ultra-high-performance liquid chromatography (UHPLC) to establish a fingerprint reflecting the integrity of the chemical composition of Cyanotis arachnoidea were purchased from China Shineway Pharmaceutical Group Ltd., Shijiazhuang, Hebei, China and authenticated by Professor Huixiang Yu of the Traditional Chinese Medicine Hospital of Chuxiong Yi Autonomous Prefecture, Yunnan, China. The dried roots were used in the experimental research, and the samples were deposited at the Hebei Traditional Chinese Medicine Formula Granule Engineering & Technology Innovate Center of the Hebei University of Chinese Medicine, Shijiazhuang, China.Fifteen batches of O-acetyl-20-hydroxyecdysone (PS210615-78-0005MG), and 2-O-acetyl-20-hydroxyecdysone (JOT-12130-0020MG) were obtained from Chengdu Push Biotechnology Co., Ltd. and Chengdu Pufei De Biotech Co., Ltd. . The purity of all the chemicals were greater than 98%. HPLC-grade methanol was obtained from Thermo Fisher Scientific . Ultrapure water was generated by a Millipore Milli-Q Reagent Water System . Chromatography-grade acetonitrile and formic acid were acquired from Sigma-Aldrich Corp. .20-Hydroxyecdysone (PU0362-0025MG), Ajugasterone C (PS0716-0010MG), 3-MIN6 islet β-cells (Lot No. CL-0674) were purchased from Procell Life Science & Technology Co., Ltd., .The Roswell Park Memorial Institute (RPMI 1640) culture medium, the glucose assay kit and the radioimmunoprecipitation assay (RIPA) lysis solution were bought from Beijing Solarbio Science & Technology Co., Ltd., . Protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit . Anti-P-PI3K, anti-P-Akt, anti-P-MAPK and anti-GAPDH primary antibodies were acquired from bioWorld Technology Inc., . Secondary antibodies were purchased from Zhongshanjinqiao Biotech . Polyvinylidene fluoride (PVDF) transfer membranes were obtained from EMD Millipore.Cyanotis arachnoidea sample batches are listed in The herbs were pulverized and sifted, and the 15 The reference substances were accurately weighed and dissolved in 10 mL chromatographic methanol to prepare stock solutions, and then were mixed at concentrations of 0.1723, 0.0251, 0.0539, and 0.0256 mg/mL. All solutions were stored at 4 °C until further use.Chromatographic separation was performed in a Phenomenex Kinetex 2.6-μm C18 chromatographic column  . The mobile phases were pure comprised pure acetonitrile (A) and 0.1% (v/v) formic acid in water (B), and the flow rate was 0.3 mL/min. The gradient elution program was as follows: 0–3 min, 5–13% A; 3–8 min, 13–15% A; 8–20 min, 15–35% A; 20–25 min, 35–95% A; 25–27 min, 95% A; 27–30 min, 95–5% B, and 30–33 min, 5% B. The column temperature was kept at 35 °C; the injection volume was 2.0 μL, and the detection wavelength was 248 nm.The same sample solution (S1) was measured sixfold in parallel to determine instrument precision. The relative standard deviation (RSD) values of the relative retention times and the peak areas under the common peaks were calculated using the peak of 20-Hydroxyecdysone as the reference. Repeatability was evaluated by analyzing nine independently prepared triplicate sample solutions at low (0.1 g), medium (0.2 g), and high (0.3 g) concentrations, each in triplicates. The stability of the method of analyzing the sample solutions was assessed at 0, 2, 4, 8, 12, and 24 h on the same day. Each sample solution was simultaneously assessed twice.Test solutions were prepared from powdered materials derived from the 15 sample batches and subjected to UHPLC analysis. The chromatograms generated were exported as CDF (computable document format) files and imported to the Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2012) . The chrHCA was performed using the Ward method in SPSS 23.0 . The areas of the common peaks of the 15 batches were the variables and the Euclidean distance  was the https://umetrics-simca.software.informer.com/14.0/) to distinguish the quality of the materials from different regions and was based on the peak areas of the common peaks of the fifteen batches. Sample quality and discrepancy were judged according to the distances between sample points (PCA was performed in SIMCA v. 14.1 (e points . PLS-DA https://www.ncbi.nlm.nih.gov/pubmed/). Standard 3D molecular structures were exported in structure-data file (SDF) format. Component targets were predicted using the BATMAN-TCM database (http://bionet.ncpsb.org.cn/batman-tcm/) (P < 0.05). BATMAN-TCM is a bioinformatics analysis tool that elucidates the molecular mechanism of TCMs. It simultaneously analyzes multiple medicines and components and clarifies their mechanism at the molecular and systemic levels. Cytoscape v. 3.7.2 (https://cytoscape.org/download.html) was used to construct a component-target network.Components contributing to the regional difference in material quality were identified by PLS-DA and used as candidate quality markers. The “SMILES” and “InChI” representations of the components were retrieved from the PubChem Compound database (an-tcm/)  and basehttps://www.genecards.org/), DrugBank (https://go.drugbank.com/), DisGeNET (https://www.disgenet.org/), and OMIM (https://omim.org/) databases by using the keyword/search term “diabetes mellitus”. The target genes were standardized by the UniProt database (https://www.uniprot.org/). The intersection target genes of component and DM were obtained on the Draw Venn Diagram platform (http://bioinformatics.psb.ugent.be/webtools/Venn/).Diabetes mellitus (DM)-associated target genes were collected from the GeneCards  (https:/DrugBank  (https:/DisGeNET  (https:/and OMIM  (https:/database  (https:/https://cn.string-db.org/). The confidence level for the interaction analysis in the network was >0.9. A topological analysis of the PPI network was performed using the “Analyze Network” function in Cytoscape v. 3.7.2, and the core targets were identified based on the degree, betweenness centrality (BC) and closeness centrality (CC) of each node.A PPI network analysis of the intersecting target genes was constructed on the STRING database platform  (https:/https://david.ncifcrf.gov). The biological processes and pathways potentially involved in the treatment of DM with Cyanotis arachnoidea were obtained at P ≤ 0.01 and Benjamini-Hochberg correction ≤0.01. Data for the active compounds and their core targets were imported into Cytoscape v. 3.7.2 to build a “drugs-active compounds-target genes-pathways” network.GO annotation and KEGG pathway enrichment analyses were performed by importing the core targets to the DAVID v. 6.8 database  (https:/https://www.rcsb.org/) and saved in protein databank (PDB) file format. The PDB IDs of the top five target proteins  were 4EKL, 2AZ5, 4NI9, 2ZOQ, and 5T01. Solvents and water molecules were removed with PyMOL software (https://www.pymol.org/). AutoDockTools v. 1.5.6 (https://autodocksuite.scripps.edu/adt/) was used to add hydrogens, calculate the atomic charges for the target proteins, and export the results in a pdbqt file format. Molecular docking was performed in AutoDock Vina (http://vina.scripps.edu/) after adjustment for the binding pocket radius size and the corresponding coordinate site to obtain reliable docking models for the core targets. Forty was the grid size of the docking box in blind docking, and the center X, Y, and Z axes of the top five key targets were , , , , and , respectively.Molecular docking was performed on the four potential quality markers and the top five core targets according to their degree values. The crystal structures of the targets proteins were downloaded from the PDB database  (https:/2 atmosphere and at 37 °C. Cells in insulin solution with the concentration of 1 × 10−7 mol/L comprised the model group. Cells in buffer solution (no insulin) comprised the control group. The cells in both groups were subjected to 0.1 mL of 10 mM glucose solution. After continued culture for 24 h, the glucose concentration in the supernatant of the model group was significantly higher than in that of the control group. Hence, the cellular insulin resistance model was successfully established. The cells were then collected and their density was adjusted to 1 × 106/L. Then 0.1 mL cell suspension was added to a 96-well culture plate. Chinese herbal extract (64 mg) was weighed out and dissolve in 10 mL of serum-free medium to obtain a 6,400 mg/L storage solution. The latter was diluted to 100, 200, and 400 mg/L for administration to the low, medium and high dose groups, respectively. Each group had triplicate wells. 0.1 mL of 10 mmol/L glucose was simultaneously added to all cells in each group. The cells were then incubated for 24 h, and the glucose concentrations in the cell culture media were measured by the glucose oxidase method (Recovered MiN6 islet β-cells were inoculated into RPMI 1640 medium and incubated under a 5% COe method .The expression levels of the phosphorylated PI3K (p-PI3K), Akt (p-Akt), and MAPK (p-MAPK) proteins were detected by western blot. After 24 h cell culture, the supernatants were removed, 200 μL cell lysate was added, and the cells were incubated at 4 °C overnight and centrifuged at 4 °C and 12,000 rpm for 10 min. The UV method and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to determine the protein content in the supernatants. to determine the protein content by UV method, and carried out 10% SDS-PAGE electrophoresis. The polyvinylidene fluoride (PVDF) membrane was then subject to a constant 250 mA for 60 min, and blocked with skim milk at 37 °C for 2 h. The membrane was then washed with phosphate-buffered saline (PBS), and a mixture of PBS-diluted primary antibodies  was added. The membrane was incubated overnight at 4 °C, washed again, and subjected to horseradish peroxidase (HRP)-labeled secondary antibody. Then the enhanced chemiluminescence (ECL) method was used to expose and develop the membrane with a gel imager. Finally, GAPDH was used as the internal reference and the protein bands were scanned and analyzed in Tanon Gis software to determine their grayscale values. The relative protein expression level equals the grayscale values of the target protein bands divided by those of the GAPDH protein bands.n = 6). The mixed reference solutions  were placed in 10-mL volumetric flasks, diluted to the mark with 70% (v/v) methanol, and shaken. In this manner, a concentration series of mixed reference solutions for the four components were obtained. Two microliters of the reserve solution were subjected to UHPLC analysis as previously described. The reference substance concentrations were plotted on the abscissa  while the peak areas were plotted on the ordinate . Standard curves were then drawn and the regression equations and linear ranges were calculated for each component.The mixed reference solutions were serially diluted to obtain gradient samples at six different concentrations  methanol. Then 2 μL diluted mixed reference solution was accurately pipetted and injected into the UHPLC. The chromatographic analysis was performed in duplicate. The precision, repeatability and stability of the determination method in methodological validation was refered to the UHPLC fingerprint. The peak area of each compound was recorded and the results of the RSD were calculated to determine sample precision.The 15 sample batches were accurately weighed and prepared as test solutions. UHPLC was then performed to establish the peak areas. The contents of the four potential quality markers were calculated and determined by measuring the peak areas.P < 0.05 was considered statistically significant.All data were expressed as (Cyanotis arachnoidea samples batches were collected in five different regions of Yunnan Province including Anning (S1–S3), Menghai (S4–S6), Jinning (S7–S9), Jinping (S10–S12), and Jinghong (S13–S15), and their fingerprints were established by UHPLC. Twenty common characteristic peaks were automatically selected as the fingerprints. Peak-6 served as the reference for a standard R chromatogram that was determined by the median method and superimposed on the fingerprints (O-Acetyl-20-hydroxyecdysone (peak-9), Ajugasterone C (peak-12), and 2-O-Acetyl-20-hydroxyecdysone (peak-13) (The fifteen erprints . Comparipeak-13) .Cyanotis arachnoidea was relatively stable, but the content of each component might be different. For this reason medicinal materials from different sources might vary in terms of their quality stability and homogeneity. Systematic studies of the chemical components of TCM indicate that their fingerprints could be used in their analysis and the determination of their overall quality control.According to the Similarity Evaluation System for Chromatographic Fingerprint of TCMs (Version 2012), the similarity values of the S1–S15 samples were in the range of 0.996–1.000 and generally remained at high levels . Hence, Cyanotis arachnoidea.The precision, repeatability, and stability of the foregoing method were validated, and the RSDs of the average retention time and the peak area were used to evaluate it. According to the precision, repeatability, and stability tests, the RSD values for the average retention times and peak areas were all <0.2% and <2.89%, <0.14% and <2.81%, and <0.28% and <2.96%, respectively . ValidatCyanotis arachnoidea samples were different although there were high similarities among the samples collected in various areas.The twenty common characteristic peaks were subjected to HCA in SPSS v. 23.0 to validate the results of the similarity assessment. The average distance between groups served as the measurement standard. At a distance of five, 15 sample batches were divided into two main clusters . S1–S4, Cyanotis arachnoidea samples. The areas of the twenty common characteristic peaks generated by the fifteen sample batches were used as the original variables.Principal component analyses (PCA) and partial least squares discriminant analyses (PLS-DA) were conducted in Simca-P v. 14.1 to screen components as potential quality markers and identify the differences in the chemical constituents and quality of the Cyanotis arachnoidea. Loading plots showed that the similarities between the variables and their effects on the scores. Loading and influence on the scores increase with the distance of the variables from the origin  for mapping and intersection. A total of 107 intersection target genes were obtained and they were considered to be the potential targets of Cyanotis arachnoidea in DM treatment  network. There were 107 nodes (target proteins) and 878 edges (interactions) in the network . Cytoscachnoidea .Cyanotis arachnoidea for DM treatment, GO analysis and KEGG pathway enrichment analyses of 27 core targets were conducted using the DAVID database. For the GO analysis, a total of 295 GO entries with P < 0.05 were obtained, including 230 biological process (BP), 42 cell component (CC), and 23 molecular function (MF). The top 10 BP, CC, and MF entries were selected for visualization , TNF (PDB ID: 2AZ5), IL-6 (PDB ID: 4NI9), MAPK3 (PDB ID: 2ZOQ), and JUN (PDB ID: 5T01). The binding energy docking results were listed in P < 0.05). It shows that the Cyanotis arachnoidea extract increased the glucose uptake and utilization by the islet β-cells, effectively lowered glucose concentration, alleviated insulin resistance, and exhibited hypoglycemic activity.It can be seen from P < 0.05). p-MAPK/GAPDH was 0.77 ± 0.06 which was significantly higher than that of the blank control group (P < 0.05). Compared with the insulin resistance group, the p-PI3K and p-Akt protein expression levels significantly increased while that of p-MAPK significantly decreased with increasing Cyanotis arachnoidea extract dose . The glucose concentration was significantly lower in the cell culture medium than in the insulin resistance group. Thus, Cyanotis arachnoidea upregulates PI3K and Akt proteins, strengthens the signal transduction barrier of the PI3K/Akt signal pathway, inhibits the MAPK signal pathway, enhances glucose intake, improves insulin resistance, and play a hypoglycemic role in insulin-resistant pancreatic islet cells.p-PI3K/GAPDH and p-Akt/GAPDH were 0.15 ± 0.01 and 0.18 ± 0.02 in insulin resistance group, and were significantly lower than those in the blank control group  and simultaneously quantified the four quality markers in the samples  for the precision, repeatability, and stability analyses of the four potential quality markers were all <3%. The result indicated that the instrument precision, method repeatability, and sample stability were good. Recovery of all four components was in the range of 98.72–101.44% which proved that the method was accurate .Cyanotis arachnoidea was 20-Hydroxyecdysone (18.47–27.99 mg/g) followed by 3-O-Acetyl-20-hydroxyecdysone (5.30–8.54 mg/g), Ajugasterone C (2.84–5.16 mg/g) and 2-O-Acetyl-20-hydroxyecdysone (2.16–3.54 mg/g)  . The relCyanotis arachnoidea. We explored its quality markers and predicted the molecular mechanism in treating diabetes mellitus. UHPLC was used to characterize the chemical constituents of Cyanotis arachnoidea and analyze 15 sample batches collected from five different origins in Yunnan Province, China. The fingerprints of medicinal materials were established. The chromatographic peaks, HCA, PCA, and PLS-DA identified and screened 20-Hydroxyecdysone, 3-O-Acetyl-20-hydroxyecdysone, Ajugasterone C and 2-O-Acetyl-20-hydroxyecdysone as potential quality markers of Cyanotis arachnoidea. We predicted their biological activity via network pharmacology and molecular docking. We determined that Cyanotis arachnoidea may mediate its hypoglycemic efficacy by regulating its target genes encoding Akt1, TNF, IL-6, MAPK3, and JUN. The hypoglycemic property might also be explained by its impact on the PI3K/Akt, MAPK, TNF, TLR, and estrogen signaling, cancer, insulin resistance, and JAK-STAT pathways. In vitro experiments on pancreatic islet β-cells confirmed that Cyanotis arachnoidea enhances glucose uptake, improves insulin resistance and play a hypoglycemic role by promoting the PI3K/Akt and inhibiting the MAPK signal pathways. The foregoing results suggest that the quality markers in Cyanotis arachnoidea may act on the entire biological network rather than a single target gene. The present work demonstrated the integrity and complexity of Cyanotis arachnoidea function and also explained the mechanism of Cyanotis arachnoidea in treating diabetes mellitus with multiple components, gene targets, and signal pathways.In this study, we used chemical pattern recognition and network pharmacology to design and test a method of evaluating the overall quality of the TCM Cyanotis arachnoidea. Nevertheless, the hypoglycemic efficacy and modes of action of each active ingredient in this TCM must be explored and elucidated. This information will facilitate the utilization and optimization of Cyanotis arachnoidea for the clinical treatment of diabetes mellitus.This study preliminarily identified the quality markers and potential pharmacological components of 10.7717/peerj.15948/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15948/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15948/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15948/supp-4Supplemental Information 4Click here for additional data file.


Panax notoginseng, vanillic acid, scopoletin, physcion in Gardneria angustifolia, 3,5-dimethyl-4-hydroxybenzaldehyde in Zaocys dhumnades, kaempferol in Radix Paeoniae Alba, and protocatechuic aldehyde in Cibotium barometz were the main active chemical components in the composite target network. Topology analysis yields core key targets, such as TP53, INS, IL6, VEGFA, MYC, CASP3, ESR1, EGF, CCND1, PPARG, ERBB2, NFKBIA, TLR4, RELA, and CASP8, and the results of KEGG enrichment analysis showed that JSQS mainly works through pathways in cancer, phosphatidylinositol-3-kinaseRAC–serine/threonine-protein kinase signaling, and mitogen-activated protein kinase (MAPK) signaling pathway, and aryl hydrocarbon receptor nuclear translocator signaling pathway. Molecular docking results showed that the binding fraction of PPARG, VEGFA and the effective active ingredients of ridged snake dispelling capsule was >70. In the clinical trial, morning stiffness, joint pain, and VAS scores of post-treatment in the treatment group were lower than those in the control group (P < .05). Additionally, ESR, CRP, RF, anti-CCP, TNF-α, IL-6, IL-17, and Th17/Treg were lower in the treatment group than in the control group (P < .05). JSQS exert multicomponent, multipathway, and multitarget synergistic actions in RA treatment. It can significantly improve the clinical symptoms and quality of life and delay the progression of RA disease.The study aimed to elucidate the effective chemical composition and molecular mechanism of rheumatoid arthritis (RA) treatment with Jishe Qushi capsules (JSQS) and perform clinical validation. The effective chemical components were screened by a database. We used Cytoscape software to construct the key target-RA composite target network of JSQS. Gene Ontology biofunctional analysis and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for the key targets, followed by molecular docking validation of core key targets. Ninety-nine patients chosen were divided into 49 cases in the treatment group and 50 cases in the control group according to the random number table method. The control group was treated with the combination of methotrexate (MTX) plus Glucosidorum Tripterygll Totorum. The treatment group was treated with MTX plus JSQS. The treatment effects of the 2 groups were evaluated. A total of 118 key anti-RA targets were obtained for JSQS. Quercetin in  The prevalence of RA in China is 0.32% to 0.36%, and RA incidence is on the rise. If not thoroughly treated, RA gradually leads to cartilage erosion and, ultimately, joint deformity, causing great pain to patients and seriously affecting their quality of life. Thus, RA has gradually become one of the serious diseases affecting people health. RA is still the number one joint disease causing disability in China; hence, RA prevention and treatment have been an important research topic. Nevertheless, the specific pathogenesis of RA is not fully clarified. At this stage, there is still a lack of ideal drugs and methods for RA treatment. The first-line antirheumatic agents, such as methotrexate (MTX), iguratimod, and leflunomide, can delay RA progression but still lack ideal effects on the progression of bone destruction. Moreover, biologics and targeted drugs are more expensive and cause certain serious adverse effects; therefore, most patients cannot adhere to them.Rheumatoid arthritis (RA) is a common immune disease characterized by the symmetrical destruction of multiple joints.Paeonia, which are modern drugs developed based on the traditional theory of Chinese medicine—have definite efficacy for patients who cannot tolerate immunosuppressants or the poor effect of biologics and are very important and effective complementary alternative medical treatments. Jishe Qushi capsules (JSQS) (Qian Medicine System Z20160028) were developed by the Miao medicine Jin Wu Jian Gu founded by Professor WuKai Ma of the Department of Rheumatology and Immunology of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine. The results of a randomized controlled trial have shown that JSQS effectively relieved and improved the symptoms of RA and slowed down disease progression, with definite clinical efficacy. Additionally, the annual clinical reception of related patients was more than 50,000, with the annual sales of the in-hospital preparation of JSQS exceeding 5 million yuan.,9 However, the drug is an empirical formula, and laboratory studies are lacking. This study used a combination of network pharmacology and clinical control trials to elucidate the material basis and molecular mechanism of JSQS in RA treatment.Chinese traditional medicine has a curative effect in the treatment of RA. These medicines—such as Glucosidorum Tripterygll Totorum and total glucosides of Paeonia lactiflora, Panax notoginseng, Homalomena occulta, Curcuma longa, and Caulis Sinomenii for the collection of active ingredients and protein targets using oral bioavailability of ≥30% and drug similarity (DL) of ≥0.18 as screening conditions. Because the TCMSP database did not provide the chemical components of Zaocys dhumnades, Cibotium barometz, and Gardneria angustifolia, we searched for them in the literature, and the protein targets of the compounds were collected using the ChemMapper database. After the targets retrieved from both data platforms were merged and de-duplicated, the protein target names were normalized using the Uniprot protein database, setting human as the species parameter.The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database was used to retrieve all chemical components of the Chinese herbal medicine composition of Using “rheumatoid arthritis” as the keyword, we searched the GeneCards and Online Mendelian Inheritance in Man human gene platforms for confirmed genes related to RA, downloaded the database into Excel, and de-duplicated the genes. The cross-targets of diseases and drugs were obtained by using Weishengxin.The targets of RA intersection with JSQS were imported into the String database, and the species was set as “homo sapiens.” Additionally, the minimum interaction score correlation was set as “highest confidence” (≥0.400). The drug–disease protein–protein interaction (PPI) network map was initially obtained by downloading the TSV file and processing it with Cytoscape 3.90 software. The core targets were selected by analyzing the network degree and other parameters with the help of the plug-in Network Analyzer.The compounds and related targets obtained from JSQS were categorized, as well as the targets that intersected with the diseases. The active ingredient-target network of the capsules for RA treatment was constructed by Cytoscape 3.90 software. The network topology parameters, such as degree and betweenness, were used to screen the key active ingredients of the drug using the built-in tools of the software.The drug-disease intersection targets were imported into the Database for Annotation, Visualization and Integrated Discovery (DAVID), and the species was set as human species. The kyoto encyclopedia of genes and genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were submitted to establish a “core target-biological pathway” network to screen out the major pathways. The KEGG enrichment bubble chart and GO term enrichment were created online using the results of bioinformatics analysis to reflect the interactions between drug targets and RA target-related pathways.http://www.rcsb.org/) protein crystal structure database or the Uniprot database. Then, we obtained the 3D structure of the compound corresponding to the protein we wanted to analyze from the TCMSP database, followed by importing the protein and compound into Discovery Studio 2016 Client software for molecular docking.We obtained the 3D crystal structure of the target protein from the RCSB PDB ; patients diagnosed for the first time, patients who did not receive formal drug treatment after diagnosis, or patients who received formal treatment but stopped taking drugs for more than 3 months; and patients with 2.6 ≤ Disease Activity Score using 28 joint counts <5.1, joint function classification of I to III, and X-ray stage I to III of both hands.Exclusion criteria were: patients aged <18 years or >60 years; patients with grade IV joint function and/or X-ray stage IV of both hands; patients with a history of hormone and/or biological treatment within 1 month; patients with severe cardiac, hepatic, renal, or another important organ insufficiency; patients with other serious diseases, such as malignancy, active gastrointestinal ulcer, and infectious diseases; patients with severe extra-articular manifestations, such as peripheral neuropathy, proteinuria, and interstitial pulmonary fibrosis; and patients with allergy to the drug of our study.The control group received MTX  10 mg once a week and Glucosidorum Tripterygll Totorum  20 mg thrice daily for 4 consecutive weeks. The treatment group received MTX, 10 mg once a week, combined with JSQS  4 capsules each time, thrice daily for 4 consecutive weeks. Both groups took folic acid tablets 10 mg  on the next day during the MTX administration. Patients of both groups were allowed to take celecoxib capsules  orally at a maximum dose of 2 capsules per day during the study period due to joint pain, and the number of taken capsules and the withdrawal time were recorded separately for both groups.The criteria for determining efficacy were formulated with reference to the Guiding Principles for Clinical Research on New Chinese Medicines  2020. Efficacy was defined as the overall improvement rate of patients’ main symptoms and signs of ≥75%, while erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were normal or significantly improved. Progress was defined as the overall improvement rate of patients’ main symptoms and signs of ≥50%, while ESR and CRP improved. Effectiveness was defined as the overall improvement rate of patients’ major symptoms and signs of ≥30%, while ESR and CRP were improved or not improved. Ineffectiveness was defined as the overall improvement rate of patients’ main symptoms and signs of <30%, while ESR and CRP did not improve. The total effective rate was calculated as follows:Total effective rate = (significant + improvement + effective)/ total number of cases.The joint pressure pain, swollen joint count, time of morning stiffness, and visual analog scale (VAS) of the 2 groups were compared before and after treatment. As VAS, a 10-cm scale was selected, with 0 representing a completely pain-free state and 10 representing intolerably severe pain. Patients chose the corresponding scale according to their pain level, and the physician in charge chose the corresponding score.First, 5 mL of fasting venous blood was collected from patients in both groups before and after treatment. After centrifugation at 3000 r/min for 10 minutes, the supernatant was pretreated and stored at low temperature and used to detect the relevant indexes of patients. Blood ESR was measured by the Westergren methods. CRP was measured by immunodiffusion. Rheumatoid factor (RF) was measured by latex agglutination test. Anti-cyclic citrullinated peptide (anti-CCP) was measured by enzyme-linked immunosorbent assay. The reagents were purchased from Shanghai Chemical Biotechnology Co., and the operation procedure was performed strictly according to the manufacturer instructions.Five mL of fasting venous blood was collected from patients in both groups before and after treatment. After centrifugation at 3000 r/min for 10 minutes, the supernatant was pretreated and stored at 4°C and used to detect the relevant indexes of patients. The levels of serum interleukin-6 (IL-6), serum interleukin-17 (IL-17), and tumor necrosis factor (TNF-α) in the 2 groups were measured by enzyme-linked immunosorbent assay. The respective kits were purchased from Shanghai Enzyme Link Biotechnology Co., and the operation procedure was performed strictly according to the manufacturer instructions.First, 5 mL of fasting venous blood was collected from patients in both groups before and after treatment. After centrifugation at 3000 r/min for 10 minutes, the supernatant was taken for pre-treatment. The changes in the expression levels of helper T cells 17 (Th17) and regulatory T cells (Treg) were detected using a flow cytometer , and the operation procedure was performed strictly according to the manufacturer instructions.We observed the adverse reactions during treatment in both groups. No obvious clinical signs and symptoms of discomfort were found.P value was below .05. All analyses were performed using IBM SPSS Statistics, version 23.0 . The t test was used for measurement data, expressed as ‾x ± s. The χ2 test was used for count data.Statistical differences were considered as significant if the Paeonia lactiflora, Panax notoginseng, Homalomena occulta, Curcuma longa, and Caulis Sinomenii through the TCMSPS platform with settings of oral bioavailability of ≥30% and DL of ≥0.18, as shown in Table Zaocys dhumnades, Cibotium barometz and Gardneria angustifolia—in ChemMapper , as shown in Table A total of 33 active compounds were collected from A total of 4881 and 28 RA-related targets were obtained from the GeneCards and Online Mendelian Inheritance in Man databases, respectively. The RA targets collected from the 2 databases were integrated and de-duplicated, providing a total of 4791 targets. The disease and drug targets were imported into Weishengxin to obtain the Venn diagram, as shown in Figure A total of 118 targets based on the intersection of JSQS and RA were imported into the String platform. The PPI network map was initially obtained after setting the corresponding parameters, and the downloaded TSV file was opened in Cytoscape 3.90 software to draw the PPI network map. The larger the value of Degree and Betweenness Centrality, the larger the shape of the node, and the larger the value of Betweenness Centrality, the closer the color of the node was to dark green, as shown in Figure One hundred eighteen common targets were used to construct a “Chinese herbal medicine-active ingredient-target” network containing 166 nodes and 423 edges, using Cytoscape software, as shown in Figure The targets were imported into the DAVID data platform for GO annotation analysis using “homo sapiens” as the study context. A total of 210 entries were obtained for GO enrichment analysis, including 159 entries for biological process (BP), 12 entries for cell composition (CC), and 39 entries for molecular function (MF). The top 10 entries were visualized (Q < 0.05) and ranked by gene size. BP was mainly involved in the positive regulation of gene expression, positive regulation of transcription from RNA polymerase II promoter, positive regulation of cell proliferation, positive regulation of transcription, DNA-templated, negative regulation of transcription from RNA polymerase II promoter, response to drug, negative regulation of apoptotic process, regulation of transcription from RNA polymerase II promoter, positive regulation of mitogen-activated protein kinase (MAPK) cascade, and response to estradiol. CC was mainly involved in cytoplasm, nucleoplasm, cytosol, nucleus, chromatin, macromolecular complex, receptor complex, transcription factor complex, perinuclear region of cytoplasm, and death-inducing signaling complex. MF was mainly involved in protein binding, identical protein binding, transcription factor binding, enzyme binding, macromolecular complex binding, transcription factor activity, sequence-specific DNA binding, RNA polymerase II core promoter proximal region sequence-specific DNA binding, RNA polymerase II transcription factor activity, sequence-specific DNA binding, DNA binding, and ubiquitin protein ligase binding. All 3 processes with respective involvement are shown in Figure 98 pathways were obtained by KEGG enrichment analysis through the DAVID data platform, and the top 20 pathways were screened for visualization, which showed that the key targets were mainly involved in Pathways in cancer, phosphatidylinositol-3-kinaseRAC–serine/threonine-protein kinase signaling pathway, Kaposi sarcoma-associated herpesvirus infection, Human cytomegalovirus infection, Measles, Hepatitis C, Hepatitis B, Epstein-Barr virus infection, Proteoglycans in cancer, Lipid and atherosclerosis, MAPK signaling pathway, Prostate cancer, aryl hydrocarbon receptor nuclear translocator signaling pathway, Alcoholic liver disease, Salmonella infection, Human papillomavirus infection, Bladder cancer, Legionellosis, Pancreatic cancer, Small cell lung cancer as shown in Figure TP53, INS, IL6, VEGFA, MYC, CASP3, ESR1, EGF, CCND1, PPARG, ERBB2, NFKBIA, TLR4, RELA, and CASP8 were selected to find the corresponding effective chemical components for molecular docking, and the docking scores were used to determine the affinity. The docking results are shown in Figure P < .05) in Table After treatment, the total effective rate of the control and treatment groups was 87.69% and 96.92%, respectively, and the difference between the 2 groups was statistically significant (P < .05), as shown in Figure After treatment, the time of morning stiffness, swollen joint count, joint pain count, and VAS score were significantly reduced in both groups compared with those before treatment. The improvement in these indexes was significantly bigger in the treatment group than in the control group (P < .05). The levels these indexes in the treatment group were significantly lower than those in the control group (P < .05), as shown in Figure After treatment, the levels of ESR, CRP, RF, and anti-CCP in both groups were significantly lower than those before treatment (P < .05). The levels of these indicators in the treatment group were significantly lower than those in the control group (P < .05), as demonstrated in Figures After treatment, the levels of TNF-α, IL-6, and IL-17 in both groups were significantly lower than those before treatment (P < .05). The improvement in these indexes was significantly bigger in the treatment group (P < .05), as shown in Figure After treatment, the Th17 ratio and Th17/Treg value decreased in both groups, while the Treg cell ratio increased in both groups  significantly reduces the expression of Cat-G, Cat-S, and autophagy marker LC3 in synovial tissues using immunohistochemistry and enzyme-linked immunosorbent assay in an equal volume emulsion of collagen-induced arthritis rat model. Physcion has been found to present a quantitative and temporal relationship in inhibiting the metastasis and proliferation of RA–fibroblast-like synoviocytes cultured in vitro. At the cellular level, kaempferol exerts anti-inflammatory effects, reducing the production and mRNA expression of pro-inflammatory cytokines, such as thymic stromal lymphopoietin, TNF-α, and interleukin-18.Quercetin has an anti-inflammatory effect on RA by reducing the ratio of MMP-13/TIMP-1, promoting the inhibition of cartilage extracellular matrix degradation, and protecting articular cartilage.The results of KEGG enrichment analysis showed that JSQS were mainly used to treat RA through the pathways in cancer, phosphatidylinositol-3-kinaseRAC–serine/threonine-protein kinase signaling pathway, MAPK signaling pathway, and aryl hydrocarbon receptor nuclear translocator signaling pathway, among others. The topological analysis of this study showed that TP53, INS, IL6, VEGFA, MYC, CASP3, ESR1, EGF, CCND1, PPARG, ERBB2, NFKBIA, TLR4, RELA, and CASP8 are the core key targets. Molecular docking was performed according to the corresponding active chemical components, suggesting binding fractions of >70 between PPARG and VEGFA and 3,5-dimethyl-4-hydroxybenzaldehyde, protocatechuic-aldehyde, vanillic acid, which had good binding energy. It indicated that these active chemical components might play an important role in RA treatment. However, further in vivo and in vitro studies are needed to confirm the possible existence of a therapeutic axis. belongs to the nuclear hormone receptors and consists of 3 different isoforms, namely PPARα, PPARγ (PPARG), and PPARβ/δ. PPARγ activators or compounds that positively regulate PPAR gene expression may represent a new class of nonsteroidal anti-inflammatory drugs for local or systemic treatment of many inflammatory conditions. PPAR activation has been reported to reduce the level of Th2 immune response, and high expression of PPARG inhibits IL-6 and TNF-α by suppressing nuclear factor kappa B activity, which, in turn, suppresses the overexcited inflammatory response. Additionally, PPARG activates osteoclasts, which cause bone destruction in RA patients, preserving the patient joint structure. The active osteoclasts cause bone destruction in RA patients, resulting in abnormalities in joint structure, resulting in morning stiffness, reduced motion, and long-term joint deformation.PPAR, a member of the nuclear receptor superfamily that regulates lipid, glucose, and amino acid metabolism,,20 Furthermore, it is important in promoting the formation and development of inflammation, especially for the formation of chronic inflammation.Synovitis and vasculitis of joints are the basic pathological factors of RA. Neovascularization is considered to be one of the main factors in the formation and maintenance of pannus in RA, and some studies have shown that VEGF can increase vascular permeability and play a very critical role in the formation and development of pannus in RA synovitis.This study identified the possible material basis and molecular mechanism of JSQS in RA treatment. Thus, our team conducted a controlled clinical trial to verify the therapeutic effect of JSQS in RA treatment. The results showed that the capsules could effectively improve morning stiffness, joint pain, VAS score, other indicators of RA, clinical symptoms, and patient quality of life. The results of laboratory tests, such as ESR, CRP, RF, and anti-CCP, also indicated effective disease control, reduced levels of inflammation-related factors, such as TNF-α, IL-6, and IL-17, and significantly improved Th17/Treg balance. In conclusion, the multicomponent, multipathway, and multitarget synergistic interventions of JSQS can significantly improve clinical symptoms and quality of life of RA patients and delay the progression of the disease. Insufficient sample size is a regret of this study due to time and financial constraints. After obtaining the preliminary results, we will carry out a multicenter study, and at the same time, we will optimize the design of the study protocol on the line, in order to provide a theoretical basis and guidance for the clinical application of Chinese medicine.Data curation: Nannan Zhang.Formal analysis: Xin Peng.Funding acquisition: Xueming Yao.Project administration: Cong Huang.Software: Nannan Zhang.Supervision: Wukai Ma, Xueming Yao.Writing – original draft: Yujie Li, Xin Peng.Writing – review & editing: Yuanxing Qin, Xudong Zhang.


The in vitro study showed significant antiparasitic activity, with 50% effective concentration (EC50) and 90% effective concentration (EC90) values of 8.2 to 10.8 and 13.7 to 19.3 μM, respectively. NFZ also affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg of body weight) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden (~40%). In patent infection, NFZ achieved a high reduction in the number of eggs (~80%), but the drug caused a low reduction in the egg burden of animals with prepatent infection. Finally, results from in silico target fishing methods predicted that serine/threonine kinases could be one of the potential targets for NFZ in S. mansoni. Overall, the present study revealed that NFZ possesses antischistosomal properties, mainly in terms of egg burden reduction in animals with patent S. mansoni infection.Schistosomiasis is a parasitic disease that afflicts approximately 250 million people worldwide. There is an urgent demand for new antiparasitic agents because praziquantel, the only drug available for the treatment of schistosomiasis, is not universally effective and may derail current progress toward the WHO goal of eliminating this disease as a public health problem by 2030. Nifuroxazide (NFZ), an oral nitrofuran antibiotic, has recently been explored to be repurposed for parasitic diseases. Here, IMPORTANCE The increasing recognition of the burden imposed by helminthiasis, associated with the limited therapeutic arsenal, has led to initiatives and strategies to research and develop new drugs for the treatment of schistosomiasis. One of these strategies is drug repurposing, which considers low-risk compounds with potentially reduced costs and shorter time for development. In this study, nifuroxazide (NFZ) was evaluated for its anti-Schistosoma mansoni potential through in vitro, in vivo, and in silico studies. In vitro, NFZ affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden and egg production. In silico investigations have identified serine/threonine kinases as a molecular target for NFZ. Collectively, these results implied that NFZ might be a potential therapeutic candidate for the treatment of schistosomiasis. Schistosoma, is a debilitating disease with a serious global burden  strategy for schistosomiasis morbidity and transmission focuses on large-scale treatment (preventive chemotherapy) with praziquantel (PZQ). For example, in 2019, an estimate of at least 236.6 million people required preventive treatment for schistosomiasis –7. The W–9–Strategies that have been applied successfully to expedite the discovery process for drugs against parasitic diseases include drug repurposing , 13. Kno14–S. mansoni. Based on the phenotypic assay, NFZ was first tested against adult parasites ex vivo, and subsequently, the 50% effective concentration (EC50) and 90% effective concentration (EC90) values were determined. The motility and morphology of the schistosomes were also monitored using light and scanning electron microscopy. Furthermore, NFZ was administered orally in a murine model infected with either immature (prepatent infection) or adult (patent infection) S. mansoni to characterize the full spectrum of activity of this drug. Finally, target fishing simulations were applied to predict the potential targets and pathways for NFZ in schistosomes.Nitrofuran derivatives have been used for decades to treat infectious diseases. In the early 1960s, nitrofurans were used to treat patients with schistosomiasis in China, but toxicity and suboptimal activity led to the abandonment of these compounds  and 21. in vitro antiparasitic potential of NFZ was assessed initially at different concentrations on S. mansoni worm pairs . For comparison, the known antischistosomal drug PZQ was used as a positive control, whereas vehicle-treated parasites  were used as a negative control. In vitro EC50 and EC90 values (72 h of incubation) of NFZ and PZQ obtained against adult worms of S. mansoni are summarized in 50 values of NFZ were 8.28 and 13.79 μM at 72 h against male and female worms, respectively. The EC90 values of NFZ were above 10 and 15 μM for male and female schistosomes, respectively. In comparison, PZQ has an EC50 value below 1 μM against adult worm pairs. As expected, 0.5% DMSO had no toxic effect on adult S. mansoni.The Assays regarding the survival times of adult worm pairs were also performed with NFZ to understand the kinetics and mode of action. As shown in P < 0.001).The capacity of NFZ to affect worm pairing and egg production was evaluated, and the number of eggs within 72 h is shown in Scanning electron microscopy examination revealed a normal tegument topography of control group schistosomes, with the typical preservation of tubercles and spines  and B. I50) and the selectivity index . As shown in A cytotoxicity assay was performed to evaluate the 50% cytotoxic concentration  to mice harboring either juvenile (prepatent infection) or adult (patent infection) S. mansoni. Results were compared with that of the control infected but untreated animal. Of note, all drugs were well tolerated, and all mice survived until the end of the experimental work.Considering the P < 0.01) in both prepatent and patent S. mansoni infections compared with control S. mansoni-infected mice (P < 0.05) (P < 0.001) was observed when the mice were treated with PZQ  . In contwith PZQ .The effect of NFZ on egg production was evaluated using the Kato-Katz method (eggs in the feces) and the oogram technique (eggs in the intestine). Results are summarized in P < 0.001) in the patent infection, whereas NFZ did not cause a significant reduction in the fecal egg burden of animals with prepatent infection. For comparison, PZQ achieved a fecal egg reduction of 89.3% and 28.6% in animals with patent and prepatent S. mansoni infection, respectively . S. mansoni and three putative targets  were also predicted to interact with NFZ.Unfortunately, Leishmania and Trypanosoma species . Since NFZ did not cause a high reduction in the number of worms in patent S. mansoni infections, these results show that the drug interferes directly with egg production, similar to what was observed in our in vitro experiments. Consistent with the results obtained, a significant egg burden reduction, even when worm reduction was low, has been reported with other antischistosomal compounds  for NFZ is achieved typically within 2 to 3 h after oral administration. The specific Cmax can vary depending on factors such as the formulation, dose, and individual patient characteristics, but information regarding the Cmax of NFZ is scarce. In mice treated intraperitoneally with NFZ at a dose of 15 mg/kg three times per week for 2 weeks, the mean levels of NFZ in the plasma corresponded to 8.5 μM  are putative proteins. In other words, these proteins are probable proteins still to be isolated and characterized within the worm proteome, and their existence is attested only by similarity to genome sequences. NFZ acts by interfering with the activity of enzymes, such as NADH dehydrogenase and certain enzymes involved in electron transport chains within bacterial and protozoal cells , 46. By elopment . Carboniscaffold . This enIt is important to note that the precise mode of action of NFZ may vary depending on the specific microorganism being targeted . FurtherS. mansoni. We demonstrated that NFZ affected parasite viability and egg production, and it induced severe tegumental damage in schistosomes. In an animal model of schistosomiasis, we further demonstrate that NFZ is orally effective in both prepatent and patent infections. Moreover, target fishing investigations have identified molecular targets for NFZ, a finding that needs to be validated in experimental target deconvolution studies. Therefore, these results implied that NFZ might be a potential therapeutic candidate for the treatment of schistosomiasis.Many studies have revealed that NFZ is well tolerated and safe , 50. Advin vitro experiments, compounds were solubilized in DMSO.RPMI 1640 medium, Dulbecco’s modified Eagle medium (DMEM), heat-inactivated fetal calf serum, and penicillin G-streptomycin solutions  were obtained from Vitrocell . HEPES buffer, dimethyl sulfoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma . Praziquantel was kindly provided by Ecovet Indústria Veterinária Ltda . NFZ was synthesized as describe in the published protocols , 51. In S. mansoni (BH strain) is maintained by routine passage through Biomphalaria glabrata snails and Swiss mice at Guarulhos University . Both rodents and snails were kept at 25°C and 50% humidity with an artificial 12-h/12-h day/night cycle and provided with water and food ad libitum. The 4-week-old mice were infected subcutaneously with S. mansoni cercariae, which were collected from S. mansoni-infected snails , and 10% heat-inactivated fetal bovine serum and were kept at 37°C in a humidified atmosphere containing 5% CO2. Cells were maintained in 25-cm2 culture flasks  and harvested using 0.25% trypsin in 0.2 g/L EDTA solution  were obtained from the American Type Culture Collection . Cells were cultured in DMEM containing 2 mM solution .S. mansoni were collected from mice by dissection at 42 days postinfection and were maintained in RPMI 1640 culture medium supplemented with 5% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO2. Compounds were diluted to 50 μM in supplemented RPMI medium in 24-well plates , to which one worm of each sex was added per well  for 72 h at 37°C and 5% CO2. After the addition of MTT solution, the cells were maintained at 37°C for 4 h. Absorbance was measured at 595 nm using a spectrophotometer , and the percentage of viable cells was determined concerning the control wells. At least two independent experiments in triplicate were carried out for each test compound. The selectivity indices (SIs) were calculated by dividing the 50% cytotoxic concentration (CC50) obtained on cells with 50% effective concentration (EC50) values determined on schistosomes  guidelines. For experimental protocols, 30 Swiss mice, at 3 weeks old, were infected subcutaneously with 80 S. mansoni cercariae each. Animals were then divided randomly into six experimental groups (five mice per group), and drugs (NFZ and PZQ) or a vehicle (2% ethanol in water) was administered for 21 days  or 42 days  postinfection by oral gavage using a single oral dose of 400 mg/kg  measurements were performed by different people (by at least two different investigators). To eliminate bias in interpretation, the manipulators of the experiments were not the same researchers as the data analysts .ab initio method HF/6-31G* with energy calculated considering ChelpG charges (Gaussian 09W). A mol2 file was constructed within an open babel converter, considering ChelpG as the atomic charges, and was input into the PharmMapper Web-based software . ECe curves . For thee curves . The levgy field .Animal studies are reported in compliance with the ARRIVE guidelines. The protocol for experimental design was reviewed and approved by the Committee for the Ethical Use of Animals in Experimentation of Guarulhos University  in conformity with the Brazilian law for Guidelines for Care and Use of Laboratory Animals.The raw data that support the findings of this study are available from the corresponding author upon reasonable request.


A lower GC/IPL VD ratio was associated with lower RNFL VD (p = 0.017) and systemic hypertension (p = 0.03) in multivariate analysis. Patients with a reduced GC/IPL VD ratio were more prone to poor visual field defects (p = 0.022) and paracentral scotoma (p = 0.046) and more likely to be on treatment for systemic hypertension (p = 0.024). Therefore, glaucoma patients on systemic hypertension treatment and reduced GC/IPL VD require cautious management.Superficial and deep macular vessel density (VD) is decreased in eyes with glaucoma. Superficial VD comprises both the retinal nerve fiber layer (RNFL) and ganglion cell/inner plexiform layer (GC/IPL), and various terms have been used previously to describe the layers of macular VD. In our study, we readjusted the macular segmentation. We obtained RNFL and GC/IPL VDs separately to evaluate VD changes of axon versus soma/dendrite of the retinal ganglion cells (RGCs) in detail. We included 66 eyes of normal tension glaucoma patients with inferior localized RNFL defects solely impacting the inferior hemiretina. Macular VD was measured as RNFL VD and GC/IPL VD. VD ratio was calculated by dividing the VD from the affected hemiretina by the VD from the unaffected hemiretina. RNFL VD ratio was related to RNFL and GC/IPL thicknesses ( Glaucoma is pathologically characterized by retinal ganglion cells (RGCs) loss. The optic nerve head (ONH) is thought to be the primary site of glaucoma pathology ,2,3. NumThe importance of ocular blood flow in glaucoma has been emphasized in numerous studies. With advances in optical coherence tomography angiography (OCTA), ocular blood flow has been quantified as vessel density (VD) at various locations within the eye, including the ONH, peripapillary (PP), and macular region, and obtaining the VD of a specific layer has become possible. A reduction in VD results from the loss of vessels within the inner retinal tissues, along with a loss of RGCs. However, electrophysiological studies have shown that vessel loss may precede RGC loss or VD reduction at the stage of RGC dysfunction in glaucoma suspects or early pre-perimetric glaucoma . TherefoMacular VD is associated with glaucoma. A decrease in superficial and deep macular VDs is observed in glaucomatous eyes compared to healthy and glaucoma-suspect eyes . A correWe aimed to determine the factors and functional outcomes associated with the vascular changes of each compartment of RGCs in the pathology of glaucoma. Hence, we evaluated the VD changes in the soma and dendrites (GC/IPL VD), axons in the macula (RNFL VD), and axons around the ONH (PP VD) of RGCsSixty-six eyes of normal tension glaucoma (NTG) patients with localized RNFL defects in the inferior hemiretina who only visited the glaucoma clinic of Seoul St. Mary’s Hospital between January 2022 and September 2022 were included in the study.p-value < 5%.As described in previous studies, NTG was defined as intraocular pressure (IOP) < 21 mmHg without the use of IOP-lowering eye drops at the first clinic visit, accompanied by the presence of open-angle and glaucomatous optic nerve damage ,23: diffThe inclusion criteria were as follows: (1) A best corrected visual acuity of 20/40 or better, (2) a localized wedge-shaped RNFL defect in the inferior hemiretina detectable on red-free RNFL photography, (3) asymptomatic to mild glaucomatous VF loss (mean deviation [MD] > −6 dB), (4) age > 18 years, (5) open angles on gonioscopy, and (6) well-controlled disease status with IOP-lowering eye drops. Patients who had undergone intraocular surgery other than cataract surgery were excluded. Moreover, patients with known retinal diseases were also excluded because of their possible impact on macular VD measurements.Paracentral scotoma was defined as VF defects on a pattern deviation probability map, with clusters of three or more test points with a <5% probability or two or more points with a ≤1% probability within the 12 points of central 10° .Microvascular dropout was defined according to the criteria used in a previous study  as focalOn the first day of the visit, all participants underwent ophthalmic examinations, including slit-lamp examination, Goldmann applanation tonometry, central corneal thickness , red-free fundus photography , RNFL and GC/IPL thickness measurement using OCT , and Humphrey VF examination using the Swedish Interactive Threshold Standard (SITA) 24-2 algorithm . All patients underwent OCTA  for VD measurement during the follow-up period. Detailed assessments of patient history and review of medical/ocular records and prescriptions were performed to determine the history of systemic hypertension and diabetes mellitus. All disc hemorrhages that occurred during follow-up were recorded. The mean IOP was the average of the measurements obtained during the last three visits.A Swept-source OCTA device with a scanning speed of 100,000 A-scans per second using a wavelength of 1050 nm was used to measure the vascular status of the ONH and macula. Scans of 4.5 × 4.5 mm were obtained. The PP and RNFL VD extended from the ILM to the RNFL. The GC/IPL VD extended from the outer border of the RNFL to the inner border of the INL. This segmentation was different from that used in our previous studies ,22,26, wVD measurements from OCTA images were performed as described in previous studies ,27. Onlyt-test and the chi-square test were used to compare variables between the higher and lower macular VD ratio groups. Pearson correlation analysis determined factors correlated with RNFL and GCIPL VD ratio. Univariate and multivariate linear regression analyses were used to identify the factors associated with various macular VD ratios. All statistical analyses were performed using SPSS . Statistical significance was set at p < 0.05.All values were presented as means ± standard deviations. Student’s p = 0.015), a lower ratio of RNFL VD (p < 0.001), worse VF parameters (lower MD [p = 0.022] and higher PSD [p = 0.035]). It was more likely to be on treatment for systemic hypertension (p = 0.024) and also exhibited paracentral scotoma (p = 0.046). There was no significant difference between the groups with respect to the prevalence of diabetes mellitus  and GC/IPL thickness .We also evaluated the correlations between the RNFL and GC/IPL VD ratios and other ocular parameters . Both RNp = 0.005), lower MD of the VF , and more frequent treatment for systemic hypertension . Whereas, in the multivariate analysis, a lower GC/IPL VD ratio was associated with a lower RNFL VD  and more frequent treatment for systemic hypertension .Regression analyses were performed to determine the factors associated with the GC/IPL VD ratio . In the p = 0.021), PSD of the VF , and the presence of paracentral scotoma  showed significant associations. In the multivariate analysis, the presence of paracentral scotoma  was the only associated factor.Further analysis was conducted to determine the significance of changes in the GC/IPL VD independent of a decreased PP VD or RNFL VD . The ratp = 0.001).These results indicated that the damage location in the macular region was associated with the RNFL VD more than it was associated with PP VD; therefore, the ratio between the GC/IPL VD and PP VD in the affected hemiretina was calculated and analyzed . A lowerRepresentative cases are shown in Analysis of VD of the superficial capillary plexus of the macula and PP region has been the subject of previous studies ,29,30. IWe demonstrated an association between impaired GC/IPL VD and systemic hypertension in NTG patients. The decrease in RNFL VD was related to structural deterioration and thinning of the RNFL and GC/IPL due to glaucomatous damage. This agrees with previous studies ,28,29,30The radial peripapillary capillary plexus (RPCP) supplies the NFL, which is dense in the PP region . StudiesFurthermore, GC/IPL VD was significantly associated with systemic hypertension, independent of the RNFL VD. The GCL and IPL are the layers where the soma and dendrites of RGCs are located. These layers may be vulnerable to blood flow instability due to systemic hypertension because they are located at the junction of the inner retinal blood supply and choroidal blood supply. Additionally, soma/dendritic regions are known to have high metabolic demands . TherefoPenteado et al.  evaluateGC/IPL VD represents the blood supply of retinal layers where the soma of RGCs reside, and the dendrites synapse with the bipolar and amacrine cells . The SynThe association between systemic hypertension and glaucoma has been investigated extensively. Systemic hypertension influences elevation in IOP ; howeverThe present study had several limitations. First, we evaluated only NTG patients. As these patients are known to have more vascular abnormalities ,37, the In summary, by readjusting the OCTA segmentation into RNFL and GC/IPL VD, we have demonstrated that glaucoma patients with vascular insufficiency within the GC/IPL were more likely to be on treatment for systemic hypertension. Patients with reduced GC/IPL VD were more likely to have worse VF defects and paracentral scotoma. These findings were independent of the RNFL and GC/IPL structures, the main structural parameters associated with the progression of glaucoma. This indicates that VD changes within the GC/IPL are independent of the glaucomatous process or result from axonal damage but may reflect the functional status of the soma/dendrites of the RGCs. Glaucoma patients on systemic hypertension treatment and reduced GC/IPL VD require careful management, as they are more likely to have worse VF defects and paracentral scotoma.


Systems characterization of immune landscapes in health, disease and clinical intervention cases is a priority in modern medicine. High-throughput transcriptomes accumulated from gene-knockout (KO) experiments are crucial for deciphering target KO signaling pathways that are impaired by KO genes at the systems-level. There is a demand for integrative platforms. This article describes the PathwayKO platform, which has integrated state-of-the-art methods of pathway enrichment analysis, statistics analysis, and visualizing analysis to conduct cutting-edge integrative pathway analysis in a pipeline fashion and decipher target KO signaling pathways at the systems-level. We focus on describing the methodology, principles and application features of PathwayKO. First, we demonstrate that the PathwayKO platform can be utilized to comprehensively analyze real-world mouse KO transcriptomes (GSE22873 and GSE24327), which reveal systemic mechanisms underlying the innate immune responses triggered by non-infectious extensive hepatectomy (2 hours after 85% liver resection surgery) and infectious CASP-model sepsis (12 hours after CASP-model surgery). Strikingly, our results indicate that both cases hit the same core set of 21 KO MyD88-associated signaling pathways, including the Toll-like receptor signaling pathway, the NFκB signaling pathway, the MAPK signaling pathway, and the PD-L1 expression and PD-1 checkpoint pathway in cancer, alongside the pathways of bacterial, viral and parasitic infections. These findings suggest common fundamental mechanisms between these immune responses and offer informative cues that warrant future experimental validation. Such mechanisms in mice may serve as models for humans and ultimately guide formulating the research paradigms and composite strategies to reduce the high mortality rates of patients in intensive care units who have undergone successful traumatic surgical treatments. Second, we demonstrate that the PathwayKO platform model-based assessments can effectively evaluate the performance difference of pathway analysis methods when benchmarked with a collection of proper transcriptomes. Together, such advances in methods for deciphering biological insights at the systems-level may benefit the fields of bioinformatics, systems immunology and beyond. For instance, to reduce the high mortality rates of patients in the intensive care unit (ICU), insights into the complex mechanisms underlying systems immunology triggered by infectious or non-infectious traumatic surgical treatments must be further investigated through both bench-experimental and computational analyses –4. High-p-value  or fold-change  or their conjunction  with septic wild-type mice, GSE24327_B (septic KO MyD88 vs. untreated WT) for comparing septic null (Myd88–/–) with untreated wild-type mice, and GSE24327_C (septic WT vs. untreated WT) for comparing septic wild-type with untreated wild-type mice  that was recovered from mouse spleens 12 hours after a colon ascendens stent peritonitis (CASP)-model surgery coupled with 4327 thatThe present article aims to describe the PathwayKO platform, focusing on its methodology, principles and application features. We applied the platform to analyze a real-world transcriptome GSE22873) as a case study to elucidate the mechanisms underlying systems immunology triggered by non-infectious extensive hepatectomy in single- and double-null mice  are statistically determined from data and are ready for pathway analysis under the same context;The ROC curves and key metrics are simultaneously calculated, both across methods and across data, in a pipeline fashion;The target KO signaling pathways are significantly identified and differentially marked by key regulators, which are coordinately, significantly and differentially expressed at the systems-level;The ROC curve-based statistics analysis is conducted under the same conditions to evaluate the performance difference of methods;Both interactive and pipeline modes can be chosen for desired computations.Moreover, we demonstrate the application of the PathwayKO platform model-based assessments, and the results suggest that the PathwayKO platform can effectively evaluate the performance difference of pathway analysis methods when benchmarked on a collection of proper transcriptomes.2The PathwayKO platform Figure 13https://ncbi.nlm.nih.gov/geo/. The 333 mouse KEGG signaling pathways were downloaded  from https://www.kegg.jp/. Users should install the PathwayKO platform and utilize this platform to complete tasks following the tutorials step-by-step, provided as online supplemental materials  stored in the assigned working directory  by utilizing the oligo  and limm3.23.2.1The main module pathwayko can automatically select DEGs through enabling the HES (high-edge-score) approach , in addiXFC and YFC are the values of the expression fold-change (FC) of X and Y, respectively, while pX and pY are the probability of observing XFC and YFC just by chance, then the edge score between X and Y is calculated by the following formula (1):The principles behind the high-edge-score (HES) approach and the change-point analysis method for statistically selecting differentially expressed genes (DEGs) were modified from the literature , 23, 24,XFC (or YFC), is calculated by comparing the expression values of KO samples versus normal samples in each data. The p-value for gene X (or Y), pX (or pY), is calculated between the same groups  by using a moderated t-test , d t-test . The FC  package . Third,  package , where a package . Fourth,3.2.2The main module pathwayko can conduct the desired computations in a pipeline fashion, i.e., across methods over one data see Figure 1p-value < 0.001). (ii) A false positive KO (FPKO) signaling pathway is a pathway that does not contain the KO gene, and was not significantly impacted by that KO gene, but was still identified to be significantly impacted. A true negative KO (TNKO) signaling pathway is a pathway that does not contain the KO gene and was not significantly impacted by that KO gene; thus, it was not reported to be significantly impacted. A false negative KO (FNKO) signaling pathway is a pathway that contains the KO gene and was significantly impacted by that KO gene, but was not reported to be significantly impacted. (iii) For such a true-false case, the response versus prediction with a probability allows us to employ the external pROC package  is a p-value that defines an optimal point  on an ROC curve  for multiple testing , i.e., an adjusted Fisher’s product of pPERT and pNDE  and on the values of AUC, pAUC_SP and pAUC_SE (via the bootstrap method). The output files are stored in a new directory (roctest) automatically generated and named after the module.The internal roctest module adapted from the pROC package  can cond3.3.3The internal combineresult module can combine individual results of each data across methods, and format them to be a summary output file (STATS.RData). Thereby, the internal violinplot module and the internal wilcoxtest module can conduct the ROC curve-based statistics analysis, comparing the individual key metrics Table 1 3.43.4.1The internal filtertrue module can extract the signaling pathways, and the internal pathwayview module adapted from the Pathview package  can rend44.14.1.1The work-flow logic of utilizing individual modules of the PathwayKO platform is indicated below Figure 34.24.2.1After a number of data having been preprocessed in a step-by-step manner and stored in the user’s working directory (4.34.3.1Once a collection of benchmark data have been previously preprocessed and stored in a user’s working directory (55.15.1.1Myd88 (Myd88–/–) mice had greatly reduced survival rates; (iii) double null Myd88_ Ager (Myd88–/–/Ager–/–) mice had even more greatly reduced survival rates; but (iv) single null Ager (Ager–/–) mice had drastically increased survival rates, which suggested opposing roles between MyD88 (encoded by Myd88) KO and RAGE (encoded by Ager) KO (Myd88 vs. WT), GSE22873_MA (KO Myd88_Ager vs. WT), GSE22873_A (KO Ager vs. WT), GSE22873_MAvM (KO Myd88_Ager vs. KO Myd88), GSE22873_MAvA (KO Myd88_Ager vs. KO Ager) and GSE22873_MvA (KO Myd88 vs. KO Ager). The results are presented as follows.The original bench-experiments suggested that (i) wild-type (WT) mice had a high mortality rate after sterile 85% liver resection; (ii) single null Ager) KO . The GSEAger) KO . These dAger) KO  with PatAger) KO . To inveAger) KO  from the5.1.2The distribution of edge scores varied drastically among data Figure 45.1.3Myd88, Cxcl1, Fos, Nfatc3, Ccr1l1, Cxcr2, Ccr4, Rela, Jun and Xcr1); GSE22873_MA had 130 DEGs, 57 DEGs and 2 DEGs ; and GSE22873_A had 135 DEGs, 27 DEGs and 2 DEGs . Similarly, by the HES1 threshold, there were 55 DEGs (with top-ranked H2-Bl and H2-T24) in GSE22873_MAvM (Myd88 and Cxcl1) in GSE22873_MAvA  in GSE22873_MvA  Ager itself was not yet significantly differentially expressed as early as 2 hours post-surgery when comparing RAGE-null against wild-type mice in GSE22873_A (KO Ager vs. WT), born by the transcriptome, in our current analysis; and (ii) RAGE (Ager)-mediated MHC/antigen-related genes play the most important roles, implying epigenetic effects.DEGs were statistically selected by using an HES threshold, as described in Methods see 4. By th5.1.4Myd88 and Cxcl1) connected the last edge, as highlighted by the HES3 threshold  connected the last edge  were marked on each ROC curve, indicating an optimal point  for a method. (ii) The 95% CIs of both specificity and sensitivity were marked on each ROC curve, indicating the deviation for a method on one data point. For instance, the drastic variation in 95% CIs shown in GSE22873_A (KO Ager vs. WT) data suggested weak signals over noise, which might impact the statistical hypothesis testing  T5.1.6vs. GSEA) had a significant difference  when benchmarked on GSE22873_M and GSE22873_MA, respectively, in view of both ROC curves themselves (via the venkatraman method) and the values of AUC, pAUC_SP and pAUC_SE (via the bootstrap method). SPIA was better than GSEA in these two cases (vs. GSEA) showed no significant difference  when benchmarked on GSE22873_A , 23 from GSE22873_MA (KO Myd88_ Ager vs. WT), 3 from GSE22873_A (KO Ager vs. WT) and 24 from GSE22873_MvA (KO Myd88 vs. KO Ager) , including 21 from GSE22873_M , while the composite signaling pathways were the remaining Myd88-containing signaling pathways , while the composite signaling pathways were the remaining Ager-containing pathways . Key regulators included ligands, receptors, adapters, transducers, transcription factors and cytokines, which were coordinately, significantly and differentially expressed at the systems-level. Such key regulators may constitute a signaling-axis, as marked on a target TPKO signaling pathway. Some top-ranked target TPKO signaling pathways are highlighted below as examples  and cytokines) on orchestrating innate inflammatory responses at the systems-level. (i) Key regulators in GSE22873_M , but up-regulation of IRF5, IL6 and MIP-1α (CCL3); these factors further regulated the proinflammatory and chemotactic effects.The Toll-like receptor signaling pathway (mmu04620) was the target basal signaling pathway that was solely responsible for the  mutants Figure 95.1.9.2Ager-null phenotype in both single Ager-null and double Myd88_Ager-null mutants ) on orchestrating innate inflammatory responses at the systems-level. (i) Key regulators in GSE22873_MA  and the calcium signaling pathway (mmu04020), induced inflammation, atherosclerosis and thrombogenesis, in addition to RAGE-mediated positive feedback. Secondly, key regulators including significantly up-regulated STAT5 and PIM1 were part of the AGEs-RAGE-JAK2-STAT3-PIM1 axis. This axis, coupled with the embedded JAK-STAT signaling pathway (mmu04630), regulated vascular remodeling. Alternatively, the AGEs-RAGE-JAK2-STAT5-CycD1/CDK4 axis, coupled with the cell cycle signaling pathway, regulated cellular proliferation, leading to renal hypertrophy. Finally, the key regulator AT1R was significantly up-regulated and part of the AGT-AT1R-TGFβ-TGFβR-SMADs-p27/ECM axis. This axis, coupled with the embedded TGFβ signaling pathway (mmu04350), regulated cell hypertrophy and mesangial matrix expansion. Strikingly, RAGE (encoded by Ager) itself was not significantly up- or down-regulated in both cases when comparing the indicated null mutants against wild-type mice as early as 2 hours post-surgery in our current analysis, which implies that RAGE likely has an indirect effect of unclear epigenetics on regulating the single Ager-null (KO RAGE) phenotype at the systems-level. In addition, the subtractive difference between the two cases  and NFκB, but up-regulation of p21RAS, which further up-regulated the production of IL6, which is responsible for inflammation.The AGE-RAGE signaling pathway in diabetic complications (mmu04933) was the target basal signaling pathway that was solely responsible for the  mutants Table 2.5.1.9.3Myd88 vs. KO Ager) for a contrast between the Myd88-null and Ager-null mutants was interesting and plausible to elaborate the subtractive effects of KO Myd88 against KO Ager. We identified 22 out of 23 significant TPKO signaling pathways (pGFdr < 0.001 or 0.005) from it  are suitable for assessing the performance difference of the pathway analysis methods under comparison . However5.2.4p-Threshold, the key metrics  reflect the local properties of the ROC curves and are suitable for a local comparison among methods  significantly decreased the survival rate. (iii) Deletion of RAGE gene (Ager null) significantly increased the survival rate. (iv) Deletion of RAGE gene modulated NFκB activation, up-regulated PIM1 expression and increased phospho-STAT3. These results suggest that blockade of RAGE might rescue liver remnants from the multiple signals that preclude adaptive proliferation triggered primarily by MyD88 signaling pathways. We hypothesize mechanisms underlying systems immunology triggered by the sterile 85% liver resection as early as 2 hours post-hepatectomy in the liver remnants of wild-type mice  were significantly increased at 6 hours when comparing hepatectomy wild-type mice to sham-surgery wild-type mice (https://ncbi.nlm.nih.gov/geo/query/acc.cgi/GSE22873) produced from tissues sampled at 2 hours post-hepatectomy (Myd88 vs. KO Ager), that reflects a virtual comparison between resected wild-type mice and untreated wild-type mice  assessing target chemicals  in hepatocyte remnants 2 hours post-injury affirmed the observation that AGE-RAGE suppressed the IL6-induced phosphorylation of STAT3 and the up-regulation of PIM1 expression (ex vivo bench-experiments in which hepatocytes isolated from both WT and RAGE-null mice 2 hours after 85% liver resection were incubated under the indicated conditions (Ager) itself was not significantly up- or down-regulated in our current analysis when comparing deficient mice to wild-type mice (KO Ager vs. WT) (see via unclear epigenetics) regulate the KO RAGE (null Ager) phenotype and that more differentially expressed genes (DEGs) may be targeted and involved  enhanced the levels of AGEs and fueled a mechanism suppressing IL6 action . The RAGlved see S6; furt6.2Strikingly, our data indicate that sterile extensive hepatectomy  and septFurthermore, our results favor recent proposals that TLRs, MyD88, RAGE and AGEs should have complex interactions to coordinate diverse mechanisms underlying innate immune responses at the systems-level , 40–42. 6.3The PathwayKO platform has currently incorporated several eminent methods that have been widely used by the community see Figure 1p-value < 0.0001). We further defined a set of terms modified from the literature  is a p-value that defines an optimal point  on an ROC curve  with local properties can be used to conduct a local comparison, while others  with global properties can be employed to perform a global comparison  approach and the change-point analysis method , 26 are 6.4via updating the platform in the future.We note some limitations in the current status of the PathwayKO platform. Firstly, the complete resulting output files may be further integrated for more displaying ways, e.g., integrating the results into one output after applying a voting or merging mechanism , 8. Seco7This article exemplified case studies associated with systems immunology to elaborate the methodology, principle and application features of the PathwayKO platform. The PathwayKO platform can comprehensively analyze gene-knockout (KO) transcriptomes to uncover mechanisms underlying systems immunology triggered by non-infectious extensive hepatectomy born by GSE22873 in the present study and by infectious CASP-model surgery born by GSE24327 in our previous publication. The PathwayKO platform model-based assessments on the performance of pathway analysis methods can also effectively evaluate the performance difference of methods when benchmarked on a collection of KO transcriptomes. A proper method toward data can be inferred. Taken together, we recommend the PathwayKO platform be applicable to broad fields  as long as KO transcriptomes are available.The PathwayKO platform is suitable for systems characterization of immune molecular landscapes of innate inflammatory responses in health, disease and clinical intervention cases through analyzing high-throughput transcriptomes from gene-knockout (KO) experiments. Real-world case studies suggest that both cases (GSE22873 and GSE24327) in the study share the same core set of 21 KO MyD88-associated target signaling pathways, including the Toll-like receptor signaling pathway, the NFκB signaling pathway, the MAPK signaling pathway, and the PD-L1 expression and PD-1 checkpoint pathway in cancer, alongside the pathways of bacterial, viral and parasitic infections. These findings suggest common fundamental mechanisms between the two cases and offer valuable insights into a better understanding of mechanisms underlying the innate inflammatory responses triggered by the non-infectious extensive hepatectomy (2 hours after 85% liver resection surgery in GSE22873) and the infectious CASP-model sepsis (12 hours after CASP-model surgery in GSE24327). Our results thus provide novel informative cues from the perspectives of bioinformatics analysis and systems immunology, which warrant further experimental validation in mice and may serve as models for humans.The original contributions presented in the study are included in the article/HA and YA designed the project HA designed and implemented the PathwayKO platform, conducted computations and analyzed data. HA, FM and YA interpreted results, wrote manuscript and approved the final manuscript. All authors contributed to the article and approved the submitted version.