Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular behaviors, including the response to stress and cell differentiation, and are highly conserved across eukaryotes. MAPK pathways can be activated by the interaction between the small GTPase Cdc42p and the p21-activated kinase (Ste20p in yeast). By studying MAPK pathway regulation in yeast, we recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is regulated in a similar manner and is turned over by the 26S proteasome. This turnover did not occur when Ste20p was bound to Cdc42p, which presumably stabilized the protein to sustain MAPK pathway signaling. Although Ste20p is a major component of the fMAPK pathway, genetic approaches here identified a Ste20p-independent branch of signaling. Ste20p-independent signaling partially required the fMAPK pathway scaffold and Cdc42p-interacting protein, Bem4p, while Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p. Interestingly, Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p, Rga1p, which unexpectedly dampened basal but not active fMAPK pathway activity. These new regulatory features of the Rho GTPase and p21-activated kinase module may extend to related pathways in other systems. Mitogen-activated protein kinase (MAPK) pathways are evolutionary conserved modules that regulate a multiplicity of cellular responses including the response to stress (like osmotic stress), as well as cell proliferation, cell differentiation, and survival , 2. In rSaccharomyces cerevisiae. Three of the five yeast MAPK pathways in yeast are regulated by the Rho GTPase Cdc42p and PAK kinase Ste20p . This tQ61l+TD A in diffreported , Cdc42pQreporter A. Surpric42pQ61L , inducedc42pQ61L A, ste20ΔFRE-lacZ , showed r Ste20p B, ste20Δimilarly B. Cdc42pn Ste50p , 80, 81 n Ste50p  were als. Cells lacking Rga1p and Ste20p showed similar levels of P∼Kss1p than cells lacking Rga1p alone . The rga1Δ ste20Δ double mutant also rescued the signaling defect of the ste20Δ mutant based on the FUS1-HIS3 reporter . Collectively, these experiments support the idea that Cdc42p regulates a Ste20p-independent branch of the fMAPK pathway. Further support comes from the fact that induction of the fMAPK pathway in WT cells by newly identified stimuli can induce signaling in cells lacking Ste20p , FRE-LacZ (B), or P∼Kss1p analysis (C). As previously reported . FUS1-HIS3 reporter activity in the rga1Δ ste20Δ double mutant was also somewhat reduced by the deletion of BEM4 . However, there was some signaling in the triple mutant, which indicates that at least one other protein is involved in Ste20p-independent signaling in some contexts. Therefore, Cdc42p induces Ste20p-independent signaling at least partly through Bem4p.We next sought to identify factors that mediate Ste20p-independent signaling. A good candidate was the adaptor protein Bem4p, which interacts with Cdc42p and Ste11p , 29. CdcUS1-LacZ A, FRE-LaFRE-LacZ B, or P∼Kanalysis C. As prereported , Cdc42pQe mutant , A–C. FUP120L, (FUS1-lacZ (G) and FUS1-HIS3 analysis (D). As expected, a plasmid expressing the hyperactive allele of the MAPKKK STE11-4 did bypass the need for Ste20p. Therefore, Msb2p and Sho1p may selectively regulate the Ste20p branch of the fMAPK pathway. To summarize, the fMAPK pathway is composed of Ste20p-dependent and Ste20p-independent branches that are regulated by different combinations of proteins.We next tested whether fMAPK pathway components act preferentially in the Ste20-dependent branch. Hyperactive versions of fMAPK pathway sensors Msb2p  and ShoP120L, ] were teUS1-lacZ G and FUSanalysis D. As expA), we next tested whether Cdc42pQ61L+TD bypassed the requirement for Ste20p in the HOG and mating pathways. Cdc42pQ61L+TD did not bypass the requirement for Ste20p in the HOG  or mating pathways . Therefore, Cdc42pQ61L+TD may preferentially stimulate the Ste20p-independent branch of the fMAPK pathway.Because Ste20p is a common component shared between three Cdc42p-dependent MAPK pathways . Overexpression of these genes also induced a growth defect, which did not require an intact fMAPK pathway . Similarly, deletion of the BMH1 or BMH2 genes caused a decrease in FUS1-HIS3 activity (A). We were unsuccessful in disrupting both genes, which is lethal in some strain backgrounds , and overexpression of BMH1 induced an elongated cell morphology by microscopy . Overexpression of BMH1 and BMH2 did not induce the FUS1-HIS3 reporter in cells lacking Ste20p or Ste11p . Similarly, the filamentous cell morphology induced by BMH1 was dependent on Ste20p and Ste11p . These results indicate that the Bmh proteins function in the Ste20p branch of the fMAPK pathway.Bmh1p and Bmh2p are members of the 14-3-3 family of proteins  that weractivity . To conf pathway A. Overex pathway A, ste11Δactivity A. We werkgrounds , 89 and kgrounds . Overexpe growth B, WashedBMH1 or BMH2 did not influence the growth of cells in media supplemented with salt  or impact mating, based on sensitivity to pheromone . These results indicate that the Bmh1p and Bmh2p proteins do not play a major role in regulating the HOG or mating pathways. The phosphorylation of the MAP kinases for each of the Cdc42p-dependent MAP kinase pathways was also examined. To examine basal and activated conditions, cells were grown in media containing galactose over a time-course experiment, which induces phosphorylation of the fMAPK Kss1p P∼Kss1p, , 87, theE). Overexpression of BMH1 did not impact P∼Hog1p levels at any time point tested . In fact, the level of P∼Hog1p was lower for several time points tested including no-salt environments (t = 0). Therefore, overexpression of Bmh1p might result in a slight reduction in HOG pathway activity.A similar approach was used to evaluate the role of Bmh1p in regulating the HOG pathway. Phosphorylation of the MAPK Hog1p (P∼Hog1p) was assessed by growth of cells in salt, which caused a rapid increase in P∼Hog1p levels E. OverexG, Control, compare 0 min to 5 min). Overexpression of BMH1 did not cause an additional increase phosphorylation of Fus3p above the control  and did not affect the mating phenotype of shmoo formation (B). Overexpression of BMH1 did show one difference from the control at 0 min, which was higher levels of P∼Fus3p . These results suggest that Bmh1p and Bmh2p do not regulate the mating pathway, except possibly for basal levels. Overall, these results show that Bmh1p and Bmh2p mainly regulate the Ste20p branch of the fMAPK pathway.In response to the mating pheromone α-factor after 5 min), WT cells show ∼7-fold increase in P∼Fus3p . To further explore this phenotype, the fMAPK pathway was examined under basal (glucose replete) and pathway-activated  conditions. In glucose-replete conditions, the rga1Δ mutant showed elevated levels of P∼Kss1p . By comparison, Rga1p did not dampen fMAPK pathway activity under pathway-activating conditions . Cells lacking Rga1p also showed a similar pattern of invasive growth as WT . These results were surprising because Rga1p is expected to function under conditions when Cdc42p is active in the fMAPK pathway. Therefore, Rga1p regulates the fMAPK pathway under basal conditions but not in conditions when the pathway is active and cells are undergoing invasive growth.Rho GTPases are inactivated by GAPs that stimulate their intrinsic GTPase activity. Rga1p is the main GAP for Cdc42p in the fMAPK pathway , and as Fig. 2C) but had a modest effect when cells were induced by osmotic stress . Similarly, Rga1p did not impact osmotolerance, as seen in cells lacking the redundant Sln1p branch of the HOG pathway . Cells lacking Rga1p also showed higher basal mating pathway activity (D) but did not impact P∼Fus3p levels after treatment with α-factor (D). Similarly, the growth arrest in response to α-factor was not impacted in cells lacking Rga1p (E). Therefore, Rga1p negatively regulates Cdc42p-dependent MAPK pathways mainly under basal conditions.Rga1p also dampened basal HOG pathway activity C but hadc stress C, KCl. S pathway B, ssk1Δ.activity D but didα-factor D. Similang Rga1p E. TherefRDI1 had subtle phenotypes, the activity of MAPK pathways was examined in cells overexpressing RDI1 by a galactose-inducible promoter. Overexpression of RDI1 would be expected to promote Cdc42p extraction from membranes and cause a decrease in MAPK pathway activity. As expected, overexpression of RDI1 caused a reduction in fMAPK pathway activity under basal  and activating conditions  (A). RDI1 overexpression also caused a reduction in invasive growth . Overexpression of RDI1 also dampened the HOG  and mating  pathways but to a lesser degree than fMAPK . The fMAPK pathway was more affected than the HOG and mating pathways by functional tests (B and E). The interaction between Bem4p and Cdc42p is required for fMAPK pathway activation . These results indicate that Cdc42p levels at the plasma membrane are critical for MAPK pathway signaling. Moreover, the activity of the fMAPK pathway is highly sensitive to Cdc42p levels at the plasma membrane.Rho GTPases are also regulated by Rho GDP-dissociation inhibitors which control their subcellular localization by sequestering GTPases from membranes , 93. Rdins  A. RDI1 oe growth B, washedtivation . Overexpcdc24-4 mutant, which is defective for fMAPK pathway activity (A). All of the phosphosite mutants tested  rescued the viability defect of the cdc24::NAT mutant, based on growth on 5-floroorotic acid, and retained function in the fMAPK pathway based on FUS1-HIS3 (B) and P∼Kss1p analysis (C). Therefore, the phosphorylation of Cdc24p is not critical for regulation of the fMAPK pathway. In Candida albicans, expression of the CDC24 gene is induced during hyphal growth and is a transcriptional target of the homologous Cek MAPK pathway  such that it is only activated in the presence of pheromone . These rNAT, HYG, and KanMX6 using PCR-based approaches using published templates (PH-A (PC3624), Cdc24Linker-A (PC3625), Cdc24PH-A, Linker-A (PC3623), and Cdc24T35A (PC3626) were generously shared by Rong Li . WT cells and control conditions were set to a value of one and adjusted for other samples accordingly.Band intensities quantitation of P∼Fus3p, P∼Kss1p, Cdc42p and GFP-Cdc42p, and ubiquitin were detected under nonsaturated conditions and normalized to the housekeeping protein Pgk1p using the Image Lab Software (FUS1-HIS3 growth reporter in cells lacking STE4 (ste4Δ), show basal activity of the fMAPK pathway by this reporter  were grown in SD-URA media to maintain plasmid selection  and media lacking histidine (SD-URA-HIS) or supplemented with 3-amino-1,2,4-triazole to evaluate fMAPK activity.The fMAPK pathway activity was evaluated by assessing the ing STE4 , 127. Cereporter . WT cellFRE-lacZ (FUS1-LacZ (ste4 background) (ssk1Δ) were examined, which is redundant with the Ste11p branch (The FRE-lacZ  and FUS1kground)  reporterkground)  to evalukground) . Cells lp branch . The matp branch . For halhttps://www.zeiss.com/microscopy/en/service-support/support/discontinued-products.html; Zeiss). Actin stanning was performed as described (https://imagej.nih.gov/ij/download.html). Fluorescence images were converted to Grayscale and inverted using ImageJ.Differential interference contrast, fluorescence microscopy using FITC, and rhodamine filter sets were used in an Axioplan 2 fluorescence microscope (Zeiss) with a Plan-Apochromat 100x/1.4 (oil) objective with the Axiocam MRm camera (Zeiss). Images were analyzed using Axiovision 4.4 software (escribed  using Phhttps://www.graphpad.com/scientific-software/prism). Data were analyzed by one-way ANOVA test followed by a Tukey’s multiple comparison test to generate p-values.All statistical tests were performed with Prism 7 (GraphPad; pjcullen@buffalo.edu.All the data are included in the manuscript and supplemental files and are available upon request at This article contains


Background: Ganoderma lucidum (G. lucidum) is a popular traditional remedy medicine used in Asia to promote health and longevity, which has also been highlighted for anti-cancer effects. This study investigated the molecular pharmacological mechanism of G. lucidum triterpenes in influencing intestinal flora imbalance in non-hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) based on 16S rRNA sequencing technology and network pharmacology analysis.Methods: 16S rRNA sequencing data of fecal samples from normal controls and HCC patients were obtained from the SRA database. G. lucidum triterpenes and HCC-related targets were screened by BATMAN-TCM, ETCM, and GeneCards databases. The TCGA-LIHC dataset was downloaded through the TCGA database to analyze the differential expression of key genes. NHBV-related HCC-related transcriptome RNA sequencing dataset was downloaded via the GEO database.Results: Abundance of intestinal flora in the HBV-related HCC and NHBV-related samples was higher than that of control samples. The intestinal flora of NHBV samples was mainly enriched in apoptosis and p53 pathways. Totally, 465 G. lucidum triterpenes-related targets were intersected with 4186 HCC-related targets, yielding 176 intersected targets. Among them, apoptosis and p53 pathway factors were located at the core of the protein-protein interactions network. Ganosporelactone B, the active component of G. lucidum triterpenes, had the lowest binding free energy to CASP3. CASP3 expression were upregulated in HCC tissue samples, and had higher predictive value in NHBV-related HCC patients.Conclusion: Therefore, Ganosporelactone B, the active ingredient of G. lucidum triterpenes, improves the imbalance of intestinal flora and ultimately curtails development of NHBV-related HCC. Liver cancer is now the sixth most common cancer and the fourth leading cause of cancer death worldwide . In 2020Currently, surgery remains the treatment of choice for HCC. However, as most patients with HCC already have cirrhosis or have reached an intermediate to advanced stage at the time of presentation, only 10%–20% of patients meet the criteria for complete surgical resection . In addiGanoderma lucidum  is a medicinal mushroom historically used in Asian countries to treat a wide variety of diseases and prolong life . It has There is a functional link between the liver and the intestine, with the liver being the first barrier organ against bacteria of the intestinal origin or their metabolites . DysreguThis study aimed to investigate the key mechanism of G. lucidum triterpenes in treating HCC by improving the imbalance of intestinal flora based on 16S rRNA sequencing, network pharmacology and network pharmacology, and bioinformatics technologies. This study is expected to provide new evidence for the prognosis of HCC patients treated with G. lucidum triterpenes and new ideas for the future precision treatment of NHBV-related HCC patients.https://www.ebi.ac.uk/ena/browser/search) (BioProject number: PRJNA428932). 16S rRNA sequencing data for the project samples were further downloaded via the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra/), including 33 healthy control fecal samples, 22 fecal samples from non-HBV-related HCC patients and 35 fecal samples from HBV-related HCC patients. All data used in the study were obtained from publicly available databases and therefore did not require ethics committee approval.Phenotype information for all samples from the HCC-related project was retrieved through the EMBL-EBI database (https://github.com/biobakery/biobakery/wiki/kneaddata). multiQC was used for sequence quality control, and kneaddate was used to remove host and contaminating sequences. Relative abundance of microbial taxa was obtained by mapping microbial species trees and annotating differences by GraPhlAn (https://github.com/biobakery/graphlan.git). To assess the diversity of species complexity in the samples, Alpha diversity analysis was performed by Richness index and Chao1 index, and Beta diversity was analyzed by Constrained Principal Coordinates Analysis (CPCoA) (http://huttenhower.sph.harvard.edu/lefse/) analysis, with a linear divergence analysis (LDA) score threshold set at 2.0. LDA scores indicate the degree of effect of significantly different species between groups, with higher scores indicating higher scores indicate greater differences in characteristics between the two groups.Samples were assessed by applying multiQC and kneaddata ( (CPCoA) . WilcoxoQIIME data were transformed using R package Compositions followed by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States States (PICRUSt) was used to predict metagenome pathways for each primer set using the Kyoto Encyclopedia of Genes and Genomes (KEGG) . The unshttps://tcmsp-e.com/tcmsp.php). The targets corresponding to the active ingredients of G. lucidum triterpenes were searched individually through the BATMAN-TCM database (http://bionet.ncpsb.org.cn/batman-tcm/). The targets corresponding to the active ingredients of G. lucidum triterpenes were also retrieved individually through the ETCM database (http://www.tcmip.cn/ETCM/index.php/Home/Index/). The screening results of BATMAN-TCM and ETCM databases were merged, and duplicate genes were removed, which were the G. lucidum triterpenes-related targets.The active ingredients of G. lucidum triterpenes were obtained through the TCMSP database (https://www.genecards.org/) was searched for “hepatocellular carcinoma,” and the corresponding targets were downloaded. Differentially expressed genes in the HCC dataset (TCGA-LIHC) were screened by the GEPIA database (http://gepia2.cancer-pku.cn/#index). The GeneCards and GEPIA database screening results, were merged, and duplicate genes were removed, resulting in HCC-related targets.The GeneCards database (http://jvenn.toulouse.inra.fr/app/index.html), the G. lucidum triterpenes-related targets were intersected with the HCC-related targets to obtain the intersected targets. KEGG enrichment analysis of the intersecting targets was performed using the R package clusterProfiler, and bar graphs were plotted. In addition, we obtained maps of apoptosis pathway regulatory mechanisms (https://www.kegg.jp/pathway/hsa04210) and p53 pathway regulatory mechanisms through the KEGG database (https://www.kegg.jp/kegg/).Using the jvenn tool (https://string-db.org) with the species restriction “Homo sapiens” to obtain the protein interaction network. The regulatory network was further imported into Cytoscape (v3.8.2) software to visualize the network relationship map and to tag Apoptosis and p53 pathway-related factors.The intersection targets of G. lucidum triterpenes and HCC were imported into the STRING database  software to further construct the “G. lucidum triterpenes component-target-pathway” network.https://pubchem.ncbi.nlm.nih.gov/), followed by conversion of the 2D structures into 3D structures using ChemBio3D (v14.0) software, while the 3D structures were optimized using the MM2 algorithm with minimum free energy. The mol2 format file was exported. The small molecule ligands were further saved as “pdbqt” files using AutoDockTools (v1.5.7)  .https://www.rcsb.org) were downloaded, specifically: NFKBIA (PDB ID: 1ikn), NFKB1 (PDB ID: 6ttu), BAX (PDB ID: 5w61), CASP3 (PDB ID: 1rhq), BAK1 (PDB ID: 2jcn), BCL2 (PDB ID: 2w3l), TNF (PDB ID: 1a8m), AKT1 (PDB ID: 1h10), CCND1 (PDB ID: 2w9z), CCNE1 (PDB ID: 1w98), IGF1 (PDB ID: 1b9g), PTEN (PDB ID: 7juk), CDKN1A (PDB ID: 2zvv). These 13 target protein receptors were processed using PyMOL (v2.5.4) software for dehydration and organic matter removal to derive protein molecules. The receptor protein molecules were hydrogenated, and the charge was calculated using AutoDockTools (v1.5.7) and saved as “pdbqt” files to determine the active pocket of the receptor protein  software to calculate the Binding Affinity and derive a molecular docking schematic .https://portal.gdc.cancer.gov/) to download data related to the TCGA-LIHC dataset, which contains 50 normal liver tissue samples and 3 RNA sequencing data from 74 cancer tissue samples from HCC patients (HTSeq-FPKM) and survival information from 371 HCC patients. Patients were divided into high and low-expression groups based on median values of key gene expression. Kaplan-Meier survival analysis was performed to compare the difference in overall survival (OS) between the two groups, with p < 0.05 selected as the cut-off value.Download the TCGA-LIHC dataset through The Cancer Genome Atlas (TCGA) database (https://www.ncbi.nlm.nih.gov/geo/). It contained 307 normal liver tissue samples, 61 samples from NHBV patients, and 19 HBV patient liver tissue samples. The difference in CASP3 expression between the three groups was analyzed using an unpaired t-test, with p < 0.05 indicating a statistically significant difference. Patients were divided into high and low-expression groups according to the median value of CASP3 expression. Kaplan-Meier survival analysis was performed to compare the difference in overall survival (OS) between the two groups, with p < 0.05 selected as the cut-off value. ROC analysis was performed using the R package, and ROC curves were plotted to assess the predictive value of CASP3. ROC curves and the area under the ROC curve  were used to assess the sensitivity and specificity of the survival analysis model.The HBV-related HCC-related dataset GSE10143 was downloaded via the Gene Expression Omnibus (GEO) database , where HCC patients were divided into HBV-related HCC group (HBV group) and NHBV-related HCC group (NHBV group).Alpha diversity is a method to calculate the species composition within a sample, including both quantitative and abundance information. We found the alpha diversity indices. Richness index and Chao1 index significantly differed between HCC patients (NHBV and HBV groups) and normal control (HC group) fecal samples. In addition, there were also significant differences between the NHBV and HBV groups . The resBeta diversity is the study of differences in species composition between communities. We calculated the Beta diversity distance matrix using three common similarity/distance indices  and conducted Constrained Principal Coordinates Analysis (CPCoA) based on bray_curtis distances. Analysis (CPCoA) based on the bray_curtis distance found a significant separation between the three sample groups .Further species composition analysis was performed, and the R package edgeR was used to calculate differences in intestinal flora abundance between the three groups, and volcano, heat, and Manhattan plots were plotted, showing that at the phylum level, there were significant differences in operational taxonomic units (OTUs) between the NHBV and HBV groups compared to the HC group, with NHBV There were 119 significantly Depleted OTUs and 75 significantly Enriched OTUs in the NHBV group and 87 significantly Depleted OTUs and 27 significantly Enriched OTUs in the HBV group .In addition, our preliminary analysis of species composition at the phylum (Phylum) level showed that the species composition of the gut flora of HCC patients (NHBV group and HBV group) and normal controls (HC group) differed significantly at the phylum (Phylum) level, with Bacteroidetes being more abundant in the HBV group and Proteobacteria being more abundant in the NHBV group were more abundant, which is consistent with evidence from the literature  Figure . A TenarAcinetobacter, Moraxellaceae, Pseudomonadales, Streptococcus, Streptococcaceae, Lactobacillus, and Lactobacillaceae were significantly more abundant in fecal samples from patients in the NHBV group than in the HC group, while the relative abundance of Alloprevotella, Fusobacterium, Fusobacteriaceae, Fusobacteriales, Fusobacteria, Fusobacteria, Ruminococcus2, Clostridium_ XIVa, Megamonas, Selenomonadales, Negativicutes, Veillonellaceae were significantly more abundant in relative terms than the NHBV group  level and performed LEfSe analysis. BV group . In addiBV group .It has been shown that Proteobacteria increased and Ruminococcus decreased in the intestinal flora of HCC patients in the presence of a vascular invasion group compared to the absence of a vascular invasion group , which iHelicobacter pylori infection, and Phosphonate and phosphinate metabolism functional pathways  abundance data by PICRUSt2 software. Further, we visualized the functional composition of the intestinal flora by STAMP software. Our analysis showed that the differential intestinal flora between the NHBV and HC groups were mainly enriched in Apoptosis, Cardiac muscle contraction, Parkinson’s disease, Carotenoid biosynthesis, Vasopressin-regulated water reabsorption, p53 pathway, Flavonoid biosynthesis, Influenza A, Toxoplasmosis, Colorectal cancer, Small cell lung cancer and other functional pathways . The difpathways . Phosphipathways .It has been well documented that Apoptosis and p53 pathways play an important role in tumors such as HCC . As showPrevious evidence shows that G. lucidum can improve intestinal flora dysbiosis and reduce AOM/DSS-induced colitis and tumorigenesis by inhibiting TLR4/MyD88/NF-κB pathway . As the Firstly, we obtained 28 active ingredients of G. lucidum triterpenes through the TCMSP database. Further, we searched the BATMAN-TCM database for each of the 28 active ingredients and obtained 415 targets with a threshold of Score >5.0. The ETCM database was also searched for targets for each of the 28 active ingredients, using a threshold of Score >0.8 (the default threshold), and 108 targets were obtained. The screening results of BATMAN-TCM and ETCM databases were merged, and duplicate genes were removed to obtain 465 G. lucidum triterpenes-related targets.2 FC| > 1 and p < 0.05, and 3202 genes were screened. The GeneCards and GEPIA database screening results, were taken and merged, and duplicate genes were removed to obtain 4186 HCC-related targets. We then intersected the 465 G. lucidum triterpenes-related targets with the 4186 HCC-related targets to obtain 176 intersected targets  with a threshold of |Log targets . The KEG targets .We imported 176 intersecting targets into the String database, limiting the species to humans to obtain protein interaction relationships. We found that Apoptosis and p53 pathway factors (dark green nodes) were located at the core of the protein interaction network . The speBased on the “component-target” relationships in the BATMAN-TCM and ETCM databases, we obtained 13 Apoptosis and p53 pathway-related targets  corresponding to the 10 G. lucidum triterpenes components , and further constructed the “G. lucidum triterpenes Component-Target-Pathway” network relationship . The speThe activity of many drugs and other biomolecules is expressed through interactions with receptor macromolecules. Evaluating the Binding Affinity between receptors and ligands is a central issue in structure-based computer-aided drug molecular design . We thenAs shown in To further investigate the key targets of G. lucidum triterpenes affecting the occurrence and development of NHBV-related HCC. We downloaded the TCGA-LIHC dataset through the TCGA database. We performed differential analysis on CASP3, PTEN, NFKBIA, BCL2, CDKN1A, and TNF expression. The results showed that compared with the Normal group, CASP3, PTEN, and CDKN1A were significantly upregulated in the Tunor group. In contrast, the expression of NFKBIA, BCL2, and TNF was not significantly expressed . SurvivaThe previous results of functional enrichment of the gut flora showed that the gut flora of NHBV patients is closely related to apoptosis and p53 pathways. Therefore, we explored the prognostic value of the apoptosis and p53 pathway factors CASP3 in NHBV-related HCC patients. The GSE10143 dataset was retrieved from the GEO database, which contained 307 normal liver tissue samples, 61 liver tissue samples from NHBV-related HCC patients, and 19 liver tissue samples from HBV-related HCC patients. We found that CASP3 expression was significantly upregulated in the NHBV group compared to the Normal group, while there was no significant difference in CASP3 expression in the HBV group . The resThe above results suggest that CASP3 has a good prognostic value in patients with NHBV-related HCC and that the Apoptosis and p53 pathway factor CASP3 is a key gene mediating the imbalance of intestinal flora for the development and progression of NHBV-related HCC.In this study, based on the analysis of 16S rRNA sequencing data, we found that the abundance of intestinal flora was significantly higher in both the HBV-related HCC group (HBV group) and the NHBV-related HCC group (NHBV group) than in the HC group (HC group), and there were significant differences in species composition between groups at the phylum and genus levels, with the NHBV-related HCC was more significantly different than HBV-related HCC. Based on genus abundance data, KEGG enrichment analysis of the gut flora revealed that the NHBV group gut flora was mainly enriched in functional pathways such as apoptosis and p53 signalling.The intestinal tract is the immune organ of the human body, and the intestinal flora is known as the “second human genome”. The intestinal flora regulates the immune response of the host. It is involved in important physiological functions of various systems in the human body, and dysbiosis of the intestinal flora affects the mechanisms of disease development . NumerouAs the 16S rRNA gene is the most commonly used molecular marker for systematic taxonomic studies of prokaryotic microorganisms, allowing precise quantification of all species in the gut microbiome, 16S rRNA sequencing has become one of the most commonly used high-throughput sequencing-dependent histological technologies . Chung MIt is worth mentioning that Proteobacteria are increased at the portal level in patients with chronic liver disease and HCC compared to healthy individuals . In addiClostridium butyricum (a butyrate-producing probiotic) inhibits the development of intestinal tumors by regulating Wnt signalling and intestinal flora  gene is a cysteine-aspartate protease that plays a central role in the execution phase of apoptosis  Supplem. Yu N etDihydroartemisinin has been shown to inhibit gastric carcinogenesis and invasion by regulating STAT1/KDR/MMP9 and p53/BCL2L1/CASP3/7 pathways . AlthougTo further investigate the prognostic value of CASP3 in HCC patients, we performed bioinformatics analysis based on the TCGA-LIHC dataset in the TCGA database and the GSE10143 dataset in the GEO database. The results showed that the mRNA and protein expression of CASP3 was significantly upregulated in cancer tissue samples from HCC patients compared to normal liver tissue samples. CASP3 predicted the survival rate of NHBV-related HCC patients at 1, 3, and 5 years better. The above results will be useful for the precise treatment of NHBV-related HCC patients in the future. It will be valuable for guiding the clinical use of G. lucidum triterpenes.In summary, we can tentatively conclude that Ganosporelactone B, the active ingredient of G. lucidum triterpenes, can improve the imbalance of intestinal flora by targeting the activation of apoptosis and p53 pathways through the inhibition of CASP3 and ultimately prevent the occurrence and development of NHBV-related HCC . The pre


We demonstrate the utility of the package by providing examples of its application in single-cell, bulk transcriptome, and microbiome analyses. ggkegg may empower researchers to analyze complex biological networks and present their results effectively.The Kyoto Encyclopedia of Genes and Genomes (KEGG) database serves as a valuable systems biology resource and is widely utilized in diverse research fields. However, existing software does not allow flexible visualization and network analyses of the vast and complex KEGG data. We developed https://github.com/noriakis/ggkegg.The package and user documentation are available at:  In omics analysis, researchers obtain data on various biological entities, such as genes, enzymes, and compounds, with the primary objective of investigating their functional significance and behavior in living organisms. To aid in this analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) provides a comprehensive set of tools and databases specifically designed to facilitate the comprehension of systems biology . KEGG isgraphite acquires pathways from sources such as KEGG and Reactome, stores them in the graphNEL format, and provides an interface for topological analyses  and image files from KEGG, enabling the output of images that reflect various user-defined values on the graph. Employing graphviz or native KEGG images, it generates high-quality visualizations that are widely used in diverse research , no library has implemented functionality for programmatically visualizing these characteristics in detail, which is deemed crucial for analyses such as functional analysis of many orthologs identified in microbiome analysis. Moreover, integration with other packages for omics analyses is considered important and seamlessly integrating analysis results, such as inferred gene regulatory networks and multiple enrichment analysis results, and combining customized KEGG plots with other plots generated in the R environment prove challenging.ggkegg to extend these packages. ggkegg retrieves information such as the KEGG PATHWAY and MODULE, formats them into a structure that is easy to analyze, and offers a series of functions for further analyses and visualization. ggkegg can also be viewed as an extension of ggplot2, an R package that composes plots using the grammar of graphics . KEGG information can be manipulated and analyzed using a grammar of data manipulation, dplyr, and directly visualized using the graph visualization library, ggraph (https://github.com/thomasp85/ggraph). All the information in KGML is accessible in one tbl_graph object by taking additional steps such as parsing of group nodes like compounds by adding additional identifiers representing the groups, along with components belonging to these groups and edges indicating their relationships. Building on this, the calculation functions of various edge and node importance measures, such as the centrality degree found in igraph and tidygraph  based on detailed criteria using the KEGG API.tbl_graph object is supported. This simplifies graph-based analyses such as calculating edge betweenness using KEGG information and these values.For convenient integration with various packages for omics analyses in the R and Bioconductor environments, we prepared several functions. As a basic function, the integration of various numerical values for nodes and edges, such as expression matrices obtained from omics analysis or statistical values, into the DESeq2 is a package widely used for differential expression or abundance analyses in transcriptomic and microbiome studies. The package allows direct incorporation of result tables, such as log2 fold change and P-values, into plots, thereby facilitating a more straightforward and profound comprehension of the analysis outcomes . The genes enriched in both datasets are colored in gold. Walktrap clustering was performed on the overall network, and the results are presented as rectangles covering nodes. The differences in gene expression between multiple datasets, relationships among genes and clusters identified in network analysis across multiple pathways are comprehensible in one plot. Also, a specific cluster, including genes such as CDK1, CDC25A, and CDC25B was upregulated in both cell lines, suggesting this cluster is globally disrupted in BKPyV infection.Seurat, and the markers of each cluster were identified using the Seurat function FindAllMarkers at a specified threshold (enrichKEGG function in clusterProfiler. These markers within the pathway Antigen Processing and Presentation, as well as their average log2 fold changes, were plotted on the raw pathway image with the same color as the reduced dimension plot (The next example demonstrates the interpretation of marker genes identified by comparing cell clusters in single-cell analyses and elucidating their functional significance in KEGG PATHWAY. We utilized a dataset of peripheral blood mononuclear cells available from 10x Genomics. The data was processed using ion plot . Clusterfastp, and MIDAS was employed to assess the coverage of microbial genomes. The pan-genome information for each interesting species was obtained, and centroid gene annotations for enzyme commission numbers were converted to KO identifiers (ComplexHeatmap and simplifyEnrichment packages (We next performed a metabolic functional analysis of microbial species. We first show the analysis of functional module completeness for bacterial species genomes. We obtained mouse gut metagenomic data from a study that investigated colorectal cancer and cigarette smoking (packages . They inWe finally present an example of analyzing publicly available KO information identified in a study that investigated the gut microbiome of Crohn’s disease patients using global metabolic map . We addiggkegg and three examples of its application in analyzing transcriptomic and microbiome datasets. All these analyses can be performed exclusively within the R environment. KEGG is one of the most widely used biological pathway databases, and ggkegg can connect to network analysis and visualization libraries in the R environment such as ggplot2, ggraph, and tidygraph. This maximizes the benefits of the grammar of graphics, thereby facilitating efficient and comprehensible visualization of complex biological pathway information in KEGG.We presented an R package ggplot2, ggkegg may enable convenient importing and plotting, providing valuable assistance for future package development. In addition, it can be easily combined with plots from other R packages, further assisting in the creation of publication figures with customizable elements harnessing the power of ggplot2 in the R environment. The documentation is linked from the GitHub repository, detailing use cases accompanied by codes.One limitation of the package is that KEGG MODULE, NETWORK, and PATHWAY raw files sometimes have irregular formatting, which may result in incorrect parsing. In such cases, the package provides warnings. This package is expected to become an essential tool for enhancing the understanding of analyses conducted in R and Bioconductor. It can greatly contribute to various analyses, including single-cell, bulk RNA-Seq, and microbiome analyses, by integrating network analysis and visualization. Moreover, while developing other packages using btad622_Supplementary_DataClick here for additional data file.


To explore the mechanism of Tripterygium wilfordii polyglycoside (TWP) in the treatment of membranous nephropathy (MN) by network pharmacology. TCMSP and DrugBank databases were used to screen the main targets of the main active components of Tripterygium glycosides, and OMIM and Gene Cards databases were used to search the gene targets of MN. UniProt database was used to normalize all the targets to get the intersection targets of TGs and MNs. Synergistic genes were uploaded to the STRING platform to construct a protein-protein interaction network and screen related core targets. Gene Ontology and Kyoto Genome Encyclopedia analyses of core targets were performed using the DAVID database. AutoDockTools software was used to verify the molecular docking between the active components of TGs and the synergistic genes. We identified 126 potential targets for the active component of Tripterygium glycosides, 584 MN-associated disease targets, and 28 co-acting genes. It mainly involves AGE-RAGE signaling pathway, lipid and atherosclerosis, IL-17 signaling pathway, fluid shear stress and atherosclerosis, NF-kappa B signaling pathway and other pathways and biological pathways in diabetic complications. The active component of that Tripterygium glycosides and the active site of the synergistic core target can the bond energy is less than −5kJ/mol. Tripterygium glycosides can regulate the release of inflammatory factors to treat MN through multiple active components, multiple disease targets, multiple biological pathways and multiple pathways, which provides a basis for broadening the clinical use of traditional Chinese medicine in the treatment of MN. Most membranous nephropathy (MN) is an autoimmune disease, which is caused by the deposition of immune complexes in renal blood vessels leading to the thickening of glomerular capillary wall. According to the etiological classification, about 4 fifths of patients have no definite etiology and are called idiopathic membranous nephropathy. The other 20% of MN are often secondary to other diseases. Such as systemic lupus erythematosus, vasculitis, rheumatoid arthritis, viral hepatitis B and so on, called secondary membranous nephropathy. In addition, drugs, poisons, tumors or environmental factors can also cause secondary membranous nephropathy.In the history of traditional Chinese medicine (TCM), there is no unified diagnosis of membranous nephropathy, but according to its clinical symptoms, it can be classified as “edema,” “turbid urine,” “consumptive disease” and other diseases. Modern Chinese medicine practitioners believe that the pathogenesis of MN is deficiency in origin and excess in superficiality, which is closely related to the 4 viscera of lung, spleen, liver and kidney. The pathogenesis of MN is based on the deficiency of spleen and kidney, and different pathological factors such as dampness, heat, phlegm and blood stasis may occur in different pathological stages. Therefore, attention should be paid to the spleen and kidney in the treatment, while taking into account the clearing of heat, eliminating dampness, promoting blood circulation and dredging collaterals.Membranous nephropathy (MN) is a kind of glomerular disease that can occur at any age with a variety of autoantigens to activate the immune response, damage the podocytes and induce proteinuria. Moreover, it is the most common cause of nephrotic syndrome in adults and one of the major risk factors for end-stage renal disease (ESRD).,5Therefore, TCM has prominent advantages in the treatment of MN, such as improving serum albumin, reducing urinary protein, alleviating edema and fatigue, and has few adverse reactions, which has attracted more and more attention from physicians. Studies have found that Tripterygium glycosides have significant clinical efficacy and good safety in the treatment of MN, and significantly improve the quality of life of patients.Tripterygium wilfordii is the root, leaf and flower of Celastraceae plant Tripterygium wilfordii. It was first recorded in Shen Nong Herbal Classic that it is pungent, warm in nature, bitter in taste, poisonous, and enters the liver and kidney meridians. It is recorded in Chinese Materia Medica that it has the effects of expelling wind and removing dampness, promoting blood circulation and dredging collaterals, detumescence and relieving pain, killing insects and detoxifying.Modern clinical studies have confirmed that Tripterygium wilfordii has anti-tumor, anti-inflammatory, immunosuppressive and other effects, mainly for the treatment of rheumatoid arthritis, systemic lupus erythematosus, nephrotic syndrome, allergic purpura, psoriasis and other refractory diseases, with significant clinical efficacy. Modern pharmacological studies have found that it has very obvious anti-inflammatory and immunosuppressive effects, and can be widely used in the treatment of inflammatory and immune diseases; a meta-analysis reported that Tripterygium wilfordii has a beneficial effect on the remission of idiopathic refractory nephrotic syndrome.The Tripterygium glycosides (TGs) are extracted from the root of Tripterygium wilfordii, and their physiological activities are produced synergistically by a variety of components, including triptolide and celastrol. TGs not only retain the immunosuppressive effects of Tripterygium wilfordii crude drugs, but also remove many toxic components. Has immunosuppressive and anti-inflammatory effect, it has been proved that it can be used for the treatment of inflammatory and immune diseases. The MN rat model treated with TGs showed that it could significantly reduce the pathological renal injury of MN rat model, reduce the production of inflammatory cytokines, and improve oxidative stress, thereby significantly reducing the accumulation of less inflammatory injury and oxidative lesions.The pathogenesis of MN is complex, which can be caused by a variety of endogenous and exogenous reasons, and there are many targets of TGs. Therefore, the specific mechanism and molecular biology of TGs in the treatment of MN are not clear.KDIGO IMN clinical practice guidelines recommend that the main treatment of MN is hormone combined with cyclophosphamide or calcineurin inhibitors, but this therapy has low effective rate, high recurrence rate, and is prone to complications such as infection and renal function damage.Therefore, depending on the method of network pharmacology to mine the targets of TGs in the treatment of MN, and to verify the molecular docking, it will provide theoretical basis and experimental direction for the subsequent cellular and molecular experiments of TGs in the treatment of MN, and promote the clinical application of TGs for MN.TCM network pharmacology, as a new mode of modern TCM research and development, contains the holistic concept of TCM, including TCM unique diagnosis and treatment ideas and rich clinical experience, and constitutes a research mode characterized by “network and “system.”TCMSP, GeneCards, OMIM, Uniprot, Venny 2.1, Cytoscape v3.9.1, String Platform, DAVID database, RCSB PDB database, etc.Targets of TGs were searched in TCMSP and DrugBank databases with the search terms “triptolide, tripterine,” and the species was limited to “Homo sapiens.” Through GeneCards, OMIM and other databases, the disease targets of MN were searched with the keyword “membranous nephropathy.”UniProt KB search function in UniProt database was selected, the database selection was limited to “Reviewed (Swiss-Prot)” and the species was “Human,” the targets were queried, and the above targets were normalized to obtain TGs and MN target sets.The active components of TGs and potential disease targets of MN were introduced into the database of functional protein association networks (STRING), and the species was defined as Homo sapiens by Multiple proteins tool. Acquire a protein interaction, setting an interaction threshold to be a highest confidence “(>0.9), hide that free protein in the result, at the same time, save the PPI network as a TSV format file. The data were imported into Cytoscape _ v3.9.1 software for topological analysis of the network, and the protein-protein interaction network was drawn to screen the core proteins and obtain the core targets of the interaction.DAVID database was used for gene ontology analysis and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of core targets. Biological Process, Cell Component and Molecular Function modules were selected for GO enrichment analysis. Pathway analysis was carried out through KEGG to obtain the pathways that were related to the pathogenic mechanism of MN and enriched with most of the key genes, and the data results were visualized.TGs were used as ligands, and the potential core targets of TGs for MN were used as receptors for molecular docking. The 3-dimensional structure of TGs was downloaded from the Pubchem database, and the core target structure was downloaded from the RCSB PDB database. AutoDockTools 1. 5. 7 software was used to remove water molecules and ligands from the above files. At the same time, hydrogenation, electron addition and other operations were carried out, and the semi-flexible docking mode with default docking parameters was selected. The binding energy of the ligand molecule is <0 kJ/mol, indicating that the ligand molecule can spontaneously bind to the receptor protein. The binding energy is less than −5.0 kJ/mol, indicating that the binding energy is better, and the smaller the binding energy, the better the docking.126 target genes of active components of TGs, 584 potential target genes of MN and 28 crossed target gene spots were screened through relevant databases  value from large to small; the length of the bar graph represents the number of genes enriched in the pathway, the horizontal axis represents the proportion of genes in the pathway, and the vertical axis represents the top 20 functional pathways enriched by genes. The results are shown in Figure The core target has a high degree of active sites and can form more than 2 hydrogen bonds, and the binding energy is less than −5 kJ/mol, so the core target has good binding activity. The binding energy of triptolide with PTGS2 was the lowest (−10.5 kJ/mol), and the binding activity was the strongest; while the binding energy of celastrol with MMP2 was the lowest (−9.9 kJ/mol), and the binding activity was better. TGs can form a good binding activity with the core target, which proves that TGs have outstanding performance in the treatment of MN. See Table  In clinical practice, the most commonly used drugs for TCM treatment of MN include Tripterygium wilfordii Chinese patent medicines, Astragalus membranaceus Chinese patent medicines, Rehmannia glutinosa Chinese patent medicine, fermented Cordyceps sinensis powder Chinese patent medicine, and Salvia miltiorrhiza Chinese patent drugs, with significant efficacy and few adverse reactions.A large number of clinical studies have shown that TCM is effective in the treatment of MN, with less side effects and high safety, and has achieved good clinical results. We summarized the randomized controlled trials (RCTs) of Tripterygium glycosides (LGT) in the treatment of MN, and found that LGT could improve the clinical efficacy of MN patients receiving basic treatment and significantly reduce the urinary protein content of patients. This study provides sufficient evidence-based medical evidence for TCM in the treatment of MN, and also lays the foundation for the promotion and application of TGs. See Table In recent years, great progress has been made in the study of TCM syndrome differentiation of large samples of MN. By analyzing the TCM prescription and clinical symptoms of adult MN, many medical scientists concluded that the TCM syndrome differentiation of adult MN was mainly based on “deficiency syndrome and excess syndrome.” Deficiency syndrome included deficiency of both qi and yin, deficiency of lung-spleen qi, deficiency of spleen-kidney Yang, and deficiency of liver-kidney yin; the syndromes of excess syndrome include blood stasis, rheumatism, retention of water and dampness, and retention of dampness and heat.Chinese scholars have found that the incidence of MN in different provinces in China is quite different. In Northeast China, MN has exceeded IgA, occupying the first place in primary glomerular diseases; the incidence in other areas of China is also increasing year by year.Membranous nephropathy (MN) is one of the common causes of renal failure. Its clinical manifestation is nephrotic syndrome, with the highest incidence among people aged 30–50. Modern medical studies have confirmed that about 60–70% of cases of primary membranous nephropathy have autoantibodies against phospholipase A2 receptor (PLA2R), and MN is defined as an autoimmune disease. At the same time, Jilin Chen et al conducted a retrospective cohort study and found that CXCL8 was highly correlated with the prognosis of MN;MMP9, MMP2 and TGFB1 are significantly expressed in patients with MN, which can be used as diagnostic markers of MN. MMP9 is significantly correlated with proteinuria, and MMP9 is significantly increased in patients with proteinuria in nephropathy,27; Studies have found that glomerular inflammation in MN lesions has different signaling pathways, and PTGS2 and monocytes/macrophages are involved in the inflammatory process of nephritis;Tumor necrosis factor (TNF), a pleiotropic cytokine with proinflammatory and immunomodulatory properties, has an important role in renal disease, which predicts renal progression in patients with MN;Immunohistochemistry confirmed the high expression of VEGFA in MN renal biopsy specimens. VEGFA plays an important role in the pathogenesis of IMN through PI3K/AKT signaling pathway, providing a new target for the treatment of IMN;Researchers found that CXCL12 in serum and urine of MN patients was significantly increased, and podocyte proliferation was inhibited in vitro induced podocyte injury model, CXCL12/CXCR4 and phosphorylated STAT3 (p-STAT3) were increased, and CXCL12/CXCR4 and p-STAT3 promoted cell proliferation. It can be used as a therapeutic target for MN patients; upregulation of cell tumor antigen p53 (TP53) expression is closely related to renal pathological changes in MN; CASP3 can reduce PLA2R activation and PI3K/AKT/mTOR pathway inhibition in PLA2R-activated podocytes, helping to protect podocytes from apoptosis.In this study, PPI network analysis screened out the core targets of TGs in the treatment of MN according to the value >10, and now the top 10 targets are listed, including CXCL8, MMP9, PTGS2, TNF, VEGFA, STAT3, MMP2, TP53, TGFB1, and CASP3. Jingyuan Xie et al Conducted a 9-cohort study involving 12820 individuals in East Asian and European populations. Genome-wide association studies of 4 East Asian cohorts with 4 841 individuals (1 632 primary MN cases and 3 209 controls) and 5 European cohorts with 7 979 individuals (2 150 primary MN cases and 5 829 controls) identified gene targets that were highly associated with MN. These include anti-phospholipase A2 receptor (PLA2R), nuclear factor NF-kappa-B p105 subunit (NFKB1), interferon regulatory factor 4 (IRF4), and human leukocyte antigen (HLA).The core gene targets of this study were enriched in 102 KEGG pathways, and the results showed that the treatment of MN by TGs was highly related to the following pathways: diabetes-related, immune-related and cell survival-related pathways.In the podocytes of the kidney, the AGE-RAGE signaling pathway stimulates vascular endothelial growth factor, which causes proteinuria by increasing vascular permeability, and promotes the release of transforming growth factor-β1, leading to the production of glomerular extracellular matrix and tubular epithelial stroma,36;The AGE-RAGE signaling pathway activates nicotinamide adenine dinucleotide phosphate oxidase, which in turn activates the signaling pathways mediated by mitogen-activated protein kinase, extracellular regulated protein kinase, extracellular signal-regulated kinase ERK1/2, and P38 through reactive oxygen species, and activates the NF-κB pathway through phosphorylation.,38TGs can alleviate renal damage and reduce urinary protein levels in MN patients through AGE-RAGE signaling pathway. Immune-related pathways include Interleukin 17 (IL-17) signaling pathway, NF-κB signaling pathway, Th1, Th2 and Th17 cell differentiation, Toll-like receptor signaling pathway, T cell receptor signaling pathway and HIF-1 signaling pathway. IL-17 signaling pathway can activate NF-κB and induce NF-κB-dependent cytokines to up-regulate the expression of inflammatory genes;IL-17 has a pathogenic role in immune-mediated glomerular diseases. The activation of NF-κB signaling pathway can combine with other pathways, leading to renal inflammation and fibrosis. The HIF-1 signaling pathway is clearly upregulated when renal function is impaired by fibrosis.TGs can treat MN by inhibiting renal local inflammation and hyperimmunity through IL-17 signaling pathway, Th1 and Th2 cell differentiation, toll-like receptor signaling pathway, NF-κB signaling pathway and HIF-1 signaling pathway. Cell survival related signaling pathways include TNF signaling pathway, apoptosis, NF-κB signaling pathway and IL-17 signaling pathway. The TNF signaling pathway induces podocyte apoptosis by inducing podocyte retinoic acid receptor responder 1 overexpression. While mediating renal inflammation and fibrosis, NF-κB signaling pathway also aggravates renal injury and proteinuria by inducing apoptosis through oxidative stress.,43TGs can reduce renal cell apoptosis and autophagy, alleviate renal injury and reduce proteinuria by inhibiting the activation of signaling pathways such as TNF and NF-κB.The diabetes-related pathway is the AGE-RAGE signaling pathway in diabetic complications and insulin resistance, which activates the NF-κB pathway and promotes the expression and release of IL6 and TNF, leading to cell activation and tissue damage.At present, there are few reports on the construction of drug-disease molecular networks in MN. Network pharmacology is helpful to further reveal the mechanism of TGs in the treatment of MN, to provide a direction for further study of the pharmacological and pharmacodynamic material basis of TGs in the treatment of MN, to provide a direction for the next target therapy, and to lay the foundation for the clinical promotion of traditional Chinese medicine. In this study, network pharmacology and molecular docking were used to predict the mechanism of action of TGs in the treatment of MN, and it was found that the mechanism was mainly related to the reduction of urinary protein and clinical symptoms by alleviating inflammatory response, immunosuppression and glomerulosclerosis.However, in this study, the treatment of MN by TGs was theoretically verified, and a large number of clinical trials and related mechanism target studies were lacking. Therefore, further animal experiments are needed to verify its targets, which will lay a theoretical foundation for the next large sample, multi-center, randomized, double-blind and controlled clinical trials.Conceptualization: Pengyu Xu, Guangchu Fu.Data curation: Haishen Zhao, Kejun Shi.Formal analysis: Haishen Zhao, Kejun Shi.Funding acquisition: Pengyu Xu.Investigation: Pengyu Xu.Methodology: Pengyu Xu, Manya Wang.Project administration: Pengyu Xu, Manya Wang, Hong Ye, Xubo Su.Resources: Manya Wang, Pin Zang.Software: Pengyu Xu, Hong Ye, Pin Zang.Supervision: Pengyu Xu, Hong Ye, Xubo Su.Validation: Kejun Shi, Pin Zang.Visualization: Xubo Su.Writing – original draft: Pengyu Xu, Guangchu Fu.Writing – review & editing: Pengyu Xu, Guangchu Fu.


Cucumis sativus L.) are a global popular vegetable and are widely planted worldwide. However, cucumbers are susceptible to various infectious diseases such as Fusarium and Verticillium wilt, downy and powdery mildew, and bacterial soft rot, which results in substantial economic losses. Grafting is an effective approach widely used to control these diseases. The present study investigated the role of wax and the phenylpropanoid biosynthesis pathway in black-seed pumpkin rootstock-grafted cucumbers. Our results showed that grafted cucumbers had a significantly higher cuticular wax contents on the fruit surface than that of self-rooted cucumbers at all stages observed. A total of 1132 differently expressed genes (DEGs) were detected in grafted cucumbers compared with self-rooted cucumbers. Pathway enrichment analysis revealed that phenylpropanoid biosynthesis, phenylalanine metabolism, plant circadian rhythm, zeatin biosynthesis, and diterpenoid biosynthesis were significantly enriched. In this study, 1 and 13 genes involved in wax biosynthesis and the phenylpropanoid biosynthesis pathway, respectively, were up-regulated in grafted cucumbers. Our data indicated that the up-regulated genes in the wax and phenylpropanoid biosynthesis pathways may contribute to disease resistance in rootstock-grafted cucumbers, which provides promising targets for enhancing disease resistance in cucumbers by genetic manipulation.Cucumbers ( Cucumis sativus L., 2n = 2x = 14), a member of the cucurbit family [Cucumbers  were detected in BG cucumbers compared to those in SR cucumbers . Gene OnDEGs in the biological process category, polysaccharide metabolic process (GO:0005976), cellular glucan metabolic process (GO:0006073), glucan metabolic process (GO:0044042), cellular polysaccharide metabolic process (GO:0044264), and cellular carbohydrate metabolic process (GO:0044262) were upregulated in BG cucumber. Among the cellular component categories, GO terms related to cell wall, cell periphery, extracellular region, apoplast, and external encapsulating structure  were up-regulated in BG cucumber. Interestingly, the GO terms chromatin, DNA packaging complex, DNA-directed RNA polymerase II, holoenzyme, protein-DNA complex, and mitochondrial part were all down-regulated in BG cucumber. In the molecular function category, the majority of xyloglucan: xyloglucosyl transferase activity, DNA-binding transcription factor activity, transcription regulator activity, and glucosyltransferase activity-related DEGs were up-regulated in BG cucumber.Pathway enrichment analysis was performed to elucidate the biological functions of the identified DEGs using KEGG analysis. Using transcriptome sequencing, 1132 DEGs were assigned to 86 KEGG pathways in the BG cucumbers compared with those in the SR cucumbers . The topCsaV3_3G010290 was significantly upregulated in BG cucumbers compared to SR cucumbers; however, CsaV3_7G034800 did not show any difference in gene expression between BG and SR cucumbers. In BG cucumbers, phenylpropanoid biosynthesis-related genes, the expression of, for example, CsaV3_4G002300, CsaV3_4G002320, CsaV3_4G002330, CsaV3_6G039690, and CsaV3_6G039710, were significantly up-regulated, whereas the expression of CsaV3_4G023630, CsaV3_7G005720, and CsaV3_7G031610 were significantly down-regulated.DEGs involved in wax and phenylpropanoid biosynthesis with more than two-fold differences between BG and SR cucumbers were selected for further qRT-PCR analysis. The primers used for quantitative real-time PCR (qRT-PCR) are listed in MdMYB30-ectopic-expression in Arabidopsis. Moreover, MdMYB30 contributes to increased resistance against Pst DC3000 in Arabidopsis, thus indicating that epicuticular wax content and composition might enhance disease resistance against the bacterial pathogen Pst DC3000 [CsWAX2 overexpression showed a significant improvement in resistance to pathogens [Recent studies have investigated the relationship between the cucumber cuticular wax layer and disease resistance, in addition to the impact of grafting. The cuticular wax layer functions as a physical barrier that limits the entry of pathogens and acts as a reservoir of signals that trigger plant defense responses . In cucut DC3000 . Cuticlet DC3000 . For exaathogens . This stThe resistance to diseases in cucumber plants is an interesting area of study in plant physiology. It has been found that this resistance is mediated by the phenylpropanoid biosynthesis pathway, a vital biochemical pathway in plants that is responsible for the production of secondary metabolites such as flavonoids and phenylpropanoids . These sFusarium oxysporum infection, as reported by Dong [Recently, transcriptomics has become an essential tool for deciphering the molecular mechanisms underlying disease resistance in cucumber. Wu  used tra by Dong .In this study, high-quality and reliable RNA-seq was performed on self-rooted and grafted cucumbers with black seed pumpkin rootstocks. Functional annotation of DEGs and KEGG analysis identified 13 and 9 genes that were up-regulated and down-regulated, respectively, in grafted cucumbers in the phenylpropanoid biosynthesis pathway. Recently, the phenylpropanoid biosynthesis pathway has been shown to play an important role in the mediation of cucumber disease resistance. Studies have reported that this pathway produces a range of secondary metabolites, such as lignin and flavonoids, which contribute to plant defense responses. For instance, Zhang  identifiCsMYB60, a MYB transcription factor that regulates the expression of phenylpropanoid pathway genes, enhanced cucumber resistance to disease. The phenylpropanoid biosynthesis pathway in cucumber plants is a crucial component of their disease resistance strategy. Understanding this pathway can provide valuable insights into improving disease resistance in other crop plants as well, potentially leading to improved crop yields and food security. Future research in this area could focus on ways to enhance the activity of this pathway, thereby boosting the plant’s natural defenses against pathogens.Further investigations provided more detailed insights into the roles of specific genes related to the phenylpropanoid pathway in cucumber disease resistance. For example, Li  found thThe cucumber variety ‘Zhongnong 18′ was used as the grafted scion, and a blackseed pumpkin variety was used as the rootstock. The rootstock blackseed pumpkin seeds were sown 5 d earlier than those of the ‘Zhongnong 18′ variety. When the scion cotyledons were unfolded, grafting was carried out using the insertion method. Both rootstock and grafting scions were grown in a 32-well tray and maintained in a greenhouse at the Shenyang Agricultural University in August 2021. Cucumber self-rooted seedlings were used as the control and black seed pumpkin rootstock grafting seedlings were used as the experimental treatment. In total, there were 20 plants per treatment with 3 replicates that were randomly arranged. Protective rows were set up around for daily management. The abbreviations ‘BG’ and ‘SR’ represent grafted and self-rooted populations.The wax content of fruit skin from self-rooted and grafted cucumbers were measured using an HP-200 portable tri-stimulus colorimeter . The colorimeter was standardized using rectification plates before measurement. The ΔL value was used to represent the wax content on the fruit surface . Three rFruit skin from the self-rooted and grafted cucumbers (12 d after blooming) were used for RNA extraction. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system , and the RNA was sent to Beijing Novogene Bioinformatics Technology Company  for sequencing and bioinformatics analysis. The Illumina NEBNext UltraTM RNA Library Prep Kit  was used for library construction. To select cDNA fragments 370–420 bp in length, the library fragments were purified using the AMPure XP system . PCR was performed using the Phusion High-Fidelity DNA polymerase, Universal PCR primers, and an Index (X) Primer. After PCR amplification and purification of the PCR products, the library quality was assessed using an Agilent Bioanalyzer 2100. After cluster generation using the TruSeq PE Cluster Kit version 4-cBot-HS , the library preparations were sequenced on an Illumina Novaseq platform, and 150 bp paired-end reads were generated.Clean data (clean reads) were obtained by removing reads containing adapters, poly-N, and low-quality reads from raw data. All downstream analyses were based on clean, high-quality data. Feature Counts (version 1.5.0-p3) was used to count the number of reads mapped to each gene. Subsequently, the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of each gene was calculated based on the length of the gene and the read count mapped to the gene. Differential expression analysis was performed in R using the DESeq2R package (version 1.20.0). All DEGs were functionally annotated using the KEGG, GO, Karyotic Orthologous Groups, PfAM, Swiss-Prot, TrEMBL, and NR databases.−∆∆Ct was calculated [First-strand cDNA was prepared using SuperScript III Reverse Transcriptase (Invitrogen). qRT-PCR was performed as described previously  using SYlculated . Gene-spp = 0.05) was used to separate the means.This experiment was plotted using Excel, Origin 2022 software, NovoMagic (accessed on 20 August 2022) cloud platform, and SPSS 19.0 software for difference significance analysis. To test whether the data differences were significant, Duncan’s least significant range test (The phenylpropanoid biosynthesis pathway is a rich source of plant metabolites, necessary for lignin biosynthesis, and also the starting point for the production of many other important compounds. The phenylpropanoid biosynthesis pathway also plays a critical role in the response of cucumber to various pathogens. The identification of specific genes involved in this pathway provides promising targets for genetic manipulation to enhance disease resistance in cucumber. However, further studies are required to elucidate the intricate molecular mechanisms involved in the phenylpropanoid pathway and explore additional potential targets for manipulating cucumber disease resistance.


Polygonum cuspidatum  is one of the commonly used traditional Chinese medicines for treating gouty arthritis (GA), but the specific mechanism is not clear. This study employed network pharmacology and molecular docking techniques to examine the molecular mechanisms underlying the therapeutic effects of HZ on GA. The network pharmacology approach, including active ingredient and target screening, drug-compound-target-disease network construction, protein-protein interaction (PPI) networks, enrichment analysis, and molecular docking, was used to explore the mechanism of HZ against GA. Ten active ingredients of HZ were predicted to interact with 191 targets, 14 of which interact with GA targets. Network pharmacology showed that quercetin, physovenine, luteolin, and beta-sitosterol are the core components of HZ, and IL (interleukin)-1β, IL-6, and tumor necrosis factor (TNF) are the core therapeutic targets. The mechanism of HZ in GA treatment was shown to be related to the IL-17 signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway, and is involved in the inflammatory response, positive regulation of gene expression, cellular response to lipopolysaccharide, and other biological processes. Molecular docking showed that all four core compounds had good binding properties to IL-1β, with luteolin and beta-sitosterol showing better docking results than anakinra, suggesting that they could be used as natural IL-1β inhibitors in further experimental studies. The mechanism of action of HZ against GA has multi-target and multi-pathway characteristics, which provides an important theoretical basis for the study of the active ingredients of HZ as natural IL-1β inhibitors. According to relevant reports, the global prevalence of GA is 0.1% to 10%. Along with the development of the condition, GA may also be combined with other medical diseases, such as nephropathy,,4 complicating treatment and seriously affecting the patients’ quality of survival and prognosis. If not treated aggressively, there is risk of teratogenicity and disability. Clinically commonly used drugs for GA, such as colchicine, nonsteroidal anti-inflammatory drugs, glucocorticoids, and febuxostat, are effective but can have adverse effects such as liver function impairment, renal function impairment, and gastrointestinal reactions.,7 It is also prone to relapse after drug discontinuation, and patient compliance is poor.Gouty arthritis (GA) is a disease characterized by disruption of purine metabolism and deposition of monosodium urate (MSU) crystals around various joints, which can lead to an inflammatory response in the affected joints. Under the guidance of TCM theory, HZ is also used to treat GA with significant efficacy and fewer side effects. However, the mechanism of action of HZ medicinal substances against GA is unclear.Traditional Chinese medicine (TCM) has characteristics such as excellent efficacy, affordability price, and few side effects in the prevention and treatment of GA. Unlike Western medicine, traditional Chinese medicine has the advantages of “multi-component, multi-target, and multi-pathway.” Therefore, the use of TCM to prevent and treat GA is a key research topic. Polygonum cuspidatum  is a perennial shrubby herb that is the rhizome of Polygonum cuspidatum, a plant belonging to the Polygonaceae family. It has the effects of clearing heat and detoxifying, promoting dampness and eliminating jaundice, dispersing blood stasis and relieving pain, resolving phlegm and relieving cough, and is often used to treat arthritis, hepatitis, jaundice, hyperlipidemia, and cough. and when the upstream pathway of IL-1β production is blocked, the level of IL-1β can be effectively lowered, alleviating gout attacks and reducing pain in patients. IL-1β is a critical cytokines in the acute inflammatory response to gout and can cause an inflammatory response. More importantly, it can simultaneously induce the expression of various pro-inflammatory cytokines such as interleukin-6, interleukin-8, and tumor necrosis factor-alpha, as well as adhesion molecules and chemokines, exacerbating the inflammatory response. Therefore, blocking or antagonizing IL-1β could be a new target for gout treatment. Clinically relevant therapeutic agents targeting gout-producing IL-1β have also been undertaken. Anakinra is a recombinant human IL-1 receptor antagonist approved for marketing by the US Food and Drug Administration. Although anakinra is not an Food and Drug Administration-approved indication for gout treatment, the 2012 American College of Rheumatology guidelines for treating gout still recommend anakinra for treating refractory gout.–14 In summary, due to the enormous potential of HZ, we hope to bring more and safer clinical effects to treat patients with GA by exploring its mechanisms of action.In vitro and in vivo studies have confirmed that interleukin-1-beta (IL-1β) plays an essential role in the formation and destruction of gout, In this study, we first used the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database to identify HZ’s effective ingredients and targets of HZ. Second, the GA genes were obtained using the Online Mendelian Inheritance in Man (OMIM) and Disgenet databases. Potential treatment targets were identified by the intersection of the 2 groups. Functionally enriched analysis at bioinformatics.com.cn. Cytospace software and the STRING database were used to produce the drug-component-target-disease network and protein-protein interaction (PPI) network. Finally, we used molecular coupling to verify the ability of the HZ active ingredient to bind to the target and compared it with anakinra. We used network pharmacology to reveal the therapeutic value and potential mechanisms of HZ in GA, hoping to provide more advice and guidance for the treatment of GA.Network pharmacology is a method that integrates pharmacology, molecular biology, medicine, bioinformatics, and statistics and is suitable for studies related to herbal medicines with complex compositions.https://tcmsp-e.com/tcmsp.php) database was used to screen HZ compounds. Furthermore, oral bioavailability ≥ 30% and drug-likeness ≥ 0.18 were used as screening conditions to identify the active compounds and related targets of HZ. The UniProt (https://www.uniprot.org/) database was used to translate the acquired targets into gene names. Complexes corresponding to nonhuman genes and nonhuman genes were deleted.The TCMSP (https://www.disgenet.org/) database and the OMIM (https://omim.org/) database, and the same genes were deleted from both databases to obtain the final gout-related genes.“Gout” was used as a keyword to search the DisGeNET https://bioinfogp.cnb.csic.es/tools/venny/index.html), and the intersected targets were potential targets for HZ intervention in GA.Drug targets were intersected with disease targets using Venny 2.1.0 (https://string-db.org/). Set the species as “Homo sapiens” and set a confidence level greater than 0.4. The free targets in the network were then hidden, and the protein-protein interaction (PPI) network was constructed. The obtained network diagram was then used to build a PPI network using Network Analysis in the Cytoscape software. Where lines represent relationships and nodes represent related targets. In this network, the lines indicate the existence of relationships between them, and the nodes indicate relevant targets.The intersecting targets obtained were imported into the STRING 11.5 database (https://david.ncifcrf.gov/) was used for enrichment analysis of the intersecting genes, and P values processed the data and visualized the results obtained using bioinformatics.com.cn.The DAVID Bioinformatics Resources (https://pubchem.ncbi.nlm.nih.gov/) database, and then the 2D structure was converted into a 3D structure using Chem3D software. In addition, the IL-1β (PDB DOI: 10.2210/pdb5R8Q/pdb) protein molecular structures were downloaded from the PDB database (https://www.rcsb.org/), and the receptor protein was routinely pretreated with PyMol (version 2.2.0) software for dehydration, hydrogenation, and removal of irrelevant ligands. Finally, molecular docking of the ligands to the target receptor was performed using the AutoDockTools software. To evaluate the docking results, we downloaded the IL-1 receptor antagonist anakinra (CID: 90470007) from the PubChem database as a ligand for molecular docking. Finally, the lowest binding energy results were selected separately and visualized using PyMol software.The 2D structure of the active components of HZ was downloaded from the PubChem , followed by luteolin (54 genes), beta-sitosterol (34 genes), and physovenine (34 genes). The results of compound screening are shown in Table The OMIM and DisGeNet databases were used to identify GA-related disease targets. The 2 databases were pooled and duplicate values were removed to obtain a total of 213 disease targets, which were intersected with drug targets to obtain 14 potential therapeutic targets. Venny 2.1.0 was used to import the GA disease targets and HZ targets to create a Venny diagram. The interaction between HZ and GA targets is shown in Figure The OMIM and DisGeNet databases were used to identify GA-related disease targets. Moreover, the 2 databases were pooled, and duplicate values were removed to obtain 213 disease targets, which were intersected with drug targets to obtain 14 potential therapeutic targets. Finally, we used Venny 2.1.0 to import the GA disease and HZ targets to create a Venny diagram. The interaction between HZ and GA targets is shown in Figure Potential targets and active compounds were imported into the Cytoscape software to produce an HZ-component-target-GA network map. A total of 203 nodes and 319 edges were obtained from the network diagram. All nodes were sorted by degree value using the Network Analyzer function in the software tool, with the most significant compound being quercetin (degree: 145), followed by luteolin (degree: 55), physovenine (degree: 35), and beta-sitosterol (degree: 35). Therefore, they are potentially vital compounds for the treatment of HZ with GA. The details of the HZ-compound-target-GA network are shown in Figure P values. We then imported the results into bioinformatics.com.cn to produce an advanced bubble chart. The results show that the biological processes of the potential core targets were mainly involved in the inflammatory response, positive regulation of gene expression, cellular response to lipopolysaccharide, positive regulation of DNA-templated transcription, lipopolysaccharide-mediated signaling pathway, protein kinase B signaling, cellular response to organic cyclic compounds, positive regulation of mononuclear cell migration, humoral immune response, vascular endothelial growth factor production, etc. In addition, the pathway enrichment results mainly included the AGE-RAGE signaling pathway in diabetic complications, Malaria, Rheumatoid arthritis, interleukin-17 (IL-17) signaling pathway, Amoebiasis, Chagas disease, lipid and atherosclerosis, cytokine-cytokine receptor interaction, nonalcoholic fatty liver disease, Inflammatory bowel disease, etc. Biological process enrichment is shown in Figure We imported potential targets into DAVID Bioinformatics Resources for analysis to investigate the biological mechanism of GA treatment with HZ. We then filtered the results to the top 20 biological process and pathway enrichments based on  In addition, we downloaded the two-dimensional structure of the IL-1 receptor antagonist anakinra from Pub Chem for molecular docking to compare it with the 4 core components of HZ. The results showed that the binding energies of all 5 compounds were greater than 7.0 kcal-mol-1, indicating a strong affinity for IL-1β. Among them, the lowest binding energy of Luteolin and Beta-sitosterol was significantly lower than that of anakinra. The compound binding energies are presented in Table Molecular docking of quercetin, luteolin, physovenine, and beta-sitosterol with IL-1β. The lower the docking binding energy of the compound to the target molecule, the more tightly the 2 molecules are bound. Binding energies < −4.25 kcal-mol-1 indicate some binding function between the active ingredient and the protein, good binding function at binding energies < −5.0 kcal-mol-1, and powerful binding function at binding energies < −7.0 kcal-mol-1. The pathogenesis of GA is complex, as MSU can be deposited in cartilage, synovium, and surrounding tissues when the concentration of uric acid is too high, exceeding the solubility of uric acid and becoming supersaturated. The deposited MSU irritates the synovial membrane of the joint, resulting in pathological reactions such as dilatation of synovial blood vessels, increased permeability, and leukocyte exudation, resulting in acute inflammatory symptoms of GA such as joint redness, swelling, and pain. IL-1β plays an essential role in GA as a critical cytokine in the inflammatory response. Related studies,29 have pointed out that when MSU crystals stimulate a GA episode, they cause a massive release of IL-1β and upregulate the levels of neutrophils, which are associated inflammatory mediators. Excess neutrophils in the blood are selectively adsorbed to the surface of endothelial cells and enter the joint space through blood circulation, triggering an intense inflammatory response. It follows that IL-1β is an essential factor in GA attacks. In the present study, we revealed that HZ’s potential targets for GA treatment interact with each other through a network pharmacological approach. In other words, the richness of active ingredients in HZ makes it possible to achieve a multi-target synergistic effect in anti-GA. Furthermore, the PPI network map , Toll-like receptor 2 (TRL2), nuclear factor-kappa B dependent, and NOD-like receptor protein 3 inflammasome, and regulating these 2 pathways or targets can improve the inflammatory response in GA.,38–40 This evidence suggests that these model pathways play an important role in the development of GA and that regulation of the relevant courses or targets can effectively control disease progression. Therefore, HZ treats GA through multiple biological functions and pathways.The biological process and pathway enrichment analysis of core targets revealed that HZ treatment of GA is multi-target and multi-pathway in nature and involves multiple biological processes Fig. . Among tFinally, we validated the core components of HZ with IL-1β using a docking technique. The results showed that quercetin, physovenine, luteolin, and beta-sitosterol showed good binding activity to the target protein Fig. . We alsoNetwork pharmacology plays an important role in the elucidation of the mechanisms of drug action. However, the present study had some limitations. On the one hand, this study screened the core targets under certain conditions, so potential genes outside the states may have been missed. Furthermore, these genes may also have some value. On the other hand, the results obtained in this study need to be further confirmed by experiments.Overall, we validated the core components of HZ with IL-1β using a molecular docking technique, and the results showed that quercetin, physovenine, luteolin, and beta-sitosterol showed good binding activity to the target protein. We also compared these with anakinra and found that luteolin and beta-sitosterol were better. This suggests that these 2 compounds are promising natural IL-1β inhibitors, but they need to be investigated further experimentally.Conceptualization: Xiao Ge, Jiaojiao Zhao, Jiyong Huang.Data curation: Xiao Ge, Jiaojiao Zhao.Formal analysis: Xiao Ge, Yan Zhang.Investigation: Xiao Ge, Rulu Fang.Methodology: Xiao Ge, Yan Zhang.Writing – original draft: Xiao Ge, Rulu Fang.Writing – review & editing: Jiyong Huang.