Whether ursolic acid is an effective drug in treatment of osteoporosis (OP) and how it exhibit activity effect on OP is unclear. To investigated the potential molecular mechanism of ursolic acid in the treatment of OP and figured out its possible mechanism is necessary. The target genes of ursolic acid were screened by using the database of traditional chinese medicine systems pharmacology, PubMed database and UniProt database. OP-related target genes were searched by GeneCards database, and utilized online mapping tool to obtain common target genes of component-disease. String database was used to construct a protein-protein interaction (PPI) network of component-disease common target genes and perform topological analysis to screen core target genes. DAVID database was performed gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for component-disease shared target genes. Using the core target protein as the receptor and ursolic acid as the ligand, the molecular docking was performed using AutoDockVina 1.1.2 software. A total of 52 ursolic acid-related target genes and 4657 OP-related target genes were excavated, with a total of collective 43 target genes. The above-mentioned PPI network with shared target genes contains 43 nodes and 510 edges, with an average node degree value of 23.32. A total of 24 core target genes were obtained, mainly including tumor protein p53 (TP53), vascular endothelial growth factor A (VEGFA), interleukin-6 (IL6), tumor necrosis factor (TNF), caspase3 (CASP3), matrix metallo protein (MMP9), transcription factor AP-1 (JUN), activator of transcription 3 (STAT3), mitogen-activated protein kinase 8 (MAPK8), and prostaglandin endoperoxidase 2 (PTGS2), respectively. According to KEGG enrichment analysis, there are 126 treatment of OP signaling pathway were enriched. GO enrichment analysis revealed that 313 biological processes were identified. The molecular docking result showed that the binding energies were all lower than −5 kcal/mol, indicating strong binding activity to the protein by the 6 core target gene. The therapeutic effect of ursolic acid on OP may be achieved by regulating TP53, JUN, IL6, VEGFA, CASP3, and MAPK8 genes, respectively. It exhibits possible biological function in the treatment of OP mainly involve positive regulation of apoptotic process, response to drug, incytoplasm, cytosol, protein binding, identical protein binding. Its mechanism may related to multiple therapeutic targets and signaling pathways such as cancer pathway, hepatitis B, and TNF signaling pathway. There are currently 200 million women with OP in the world, and about 30% of the elderly over 50 years old have 1 or more fractures. The situation is very serious. In addition, the disease may also induce other diseases, causing a heavy economic burden to the patient’s family and society. The elderly are the main patient population of OP. At present, most of the clinical drugs for the treatment of OP are chemical drugs that regulate bone metabolism, but they may cause a series of adverse reactions such as kidney damage, which limits their clinical application. Since there are no obvious symptoms in the early stage of the disease, once it is diagnosed, it is relatively late, and even life-threatening, finally resulting in serious and irreversible consequences. OP belongs to the category of “bone atrophy” in the Traditional Chinese Medicine (TCM) which is to invigorate the kidney and replenish the essence, activate blood and remove blood stasis as the basic principle. However, the relevant molecular mechanism of TCM treatment of OP has not been fully elucidated, which restricts the further transformation of related TCM theories to clinical practice. Most of the current new drug screening ideas focus on the targeted therapy and mechanism of drugs on a certain key protein and molecule. Ursolic acid has anti-oxidative, anti-inflammatory, anti-viral, anti-tumor,9 and other effects, and is widely distributed in nature. Relevant studies have shown that ursolic acid may significantly improve kidney damage. The TCM theory believes that kidney deficiency is the main pathogenesis of OP, advocates that the treatment of OP should be paralleled with the protection of renal function. Therefore, according to the theory of “invigorating the kidney and strengthening bones” in TCM, ursolic acid may have a potential OP improvement effect. Network pharmacology is to use the achievements of computer science, molecular biology, pharmacy and other disciplines to carry out multi-gene, multi-target and multi-channel systematic research on the potential targets and pharmacological effects of TCM ingredients, which can provide information and new ideas for the modernization of TCM. Molecular docking is a theoretical simulation method that predicts the binding mode and affinity of the receptor through the interaction between the receptor and the drug molecule. Combining the network pharmacology method and molecular docking technology to preliminarily explore the potential targets and pathways of ursolic acid in the treatment of OP in the study. It may provided a basis for further elucidation of the molecular mechanism of ursolic acid in the treatment of OP.Osteoporosis (OP) is an inflammatory metabolic disease with a complex occurrence and development process, which is closely related to multiple genes and signaling pathways in the body. With the increase of population growth and aging, the incidence of OP is also increasing year by year, which has become a major public health problem in the world.https://old.tcmsp-e.com/tcmsp.php), and the potential target proteins of ursolic acid were retrieved from the PubMed database (https://pubmed.ncbi.nlm.nih.gov) for structural alignment. Through the UniProt database (https://www.uniprot.org), the proteins corresponding to the above targets were converted into human genes, and a database of ursolic acid monomer compounds and their target genes was constructed.The potential target proteins of ursolic acid were retrieved through the database of traditional chinese medicine systems pharmacology (https://www.genecards.org) to obtain the disease target genes of OP.Using “osteoporosis” as the keyword, a human gene search was performed in the GeneCards database  to draw venn diagram for component-disease common target genes.The two groups of targets screened in 2.1 and 2.2 were entered into the online mapping tool (https://string-db.org/cgi/input.pl), set the biological species as “homosapiens (Homo sapiens),” and construct proteins with common target genes -protein interaction relationship (PPI) network, and import the construction results into Cytoscape 3.8.0 software for visual display. In the visual network diagram, the common target gene is represented by a node. The darker the color and the larger the shape, the greater the degree of the corresponding target gene, that is, the more important the target gene is; the edge represents the common target gene. The thicker the line, the greater the combination score between target genes, that is, the more significant the correlation between target genes and target genes. Topological analysis of the PPI network was performed with the help of the “networkanalyzer” tool in the Cystoscape 3.8.0 software. The median value of 3 parameters, including degree value, betweenness centrality, and closeness centrality, is used as the reference index. Among them, betweenness centrality represents the number of shortest paths passing through a node, the greater the betweenness centrality, the greater the influence of the node; the closeness centrality represents the average distance between the node and other nodes in the network, and the closeness to the center, the higher the degree of nodes, the better the information flow; the target gene with a degree value greater than the average degree value is selected as the core target gene, and the degree value, betweenness centrality and closeness centrality are all ranked. The topology analysis results of the top 10 core target genes are displayed.Import the common target genes of component-disease into the String database (https://david.ncifcrf.gov). Among them, GO functional annotation analysis mainly involves biological process (BP), molecular function (MF), cellular component (CC) functions. Set the species to “homosapiens” and the identifier to “official genesymbol ,” retain entries with a corrected P value less than .05, and sort them according to the corrected P value from small to large. Select the top 20 entries and import them into RStudio 3.6.3 software to visualize the enrichment results.GO functional annotation and KEGG pathway enrichment analysis were performed on component-disease shared target genes using the DAVID database (https://www.rcsb.org), and save it in the “protein data bank” format as the protein receptor. Using AutoDockTools 1.5.6 to removes water molecules, separates proteins, adds non-polar hydrogen and other treatments to the protein receptor, calculates the gasteiger charge of its structure to ensure that the atoms follow the atomic type in autodock, and then saves it as a “pdbqt” format file. Download the 2-dimensional structure of ursolic acid from PubChem database (https://pubchem.ncbi.nlm.nih.gov), save it in “mol2” format, and utilized AutoDockTools 1.5.6 after hydrogenation, charging, detecting the central node of ligand molecule and searching and defining the rotatable bond, the software will save it as a “pdbqt” format file as the ligand. The receptor is set to be rigid and the ligand is set to be flexible, then Autodock vina 1.1.2 software was used for molecular docking of protein receptor encoded by core target gene and ursolic acid ligand. The binding ability of ligand and receptor is evaluated by binding energy. If the binding energy is less than 0, it means that ligand and receptor can spontaneously bind, and the smaller the value, the higher the binding activity. Pymol 2.4.0 software was used for visual display of docking results finally.Download the 3-dimensional structure of the protein encoded by the core target gene from the protein data bank database (30H48O3 (PubChem CID:64945) and its molecular weight is 498.737. Ursolic acid is a kind of triterpenoids in natural plants, the 2-dimensional structure of ursolic acid was presented in Figure The molecular formula of ursolic acid is CThe PPI visualization network of component disease common target genes was shown in Figure P value was less than .05, and a total of 313 GO functional items were enriched in BP (284), MF (58), and CC (28) items, respectively. Among them, BP is mainly involved in positive regulation of apoptotic process, response to drug, positive regulation of gene expression, negative regulation of apoptotic process, apoptotic process, positive regulation of transcription from RNA polymerase II promoter, inflammatory response, negative regulation of cell proliferation, positive regulation of cell proliferation, response to lipopolysaccharide etc. CC is mainly involved incytoplasm, cytosol, nucleus, nucleoplasm, plasma membrane, extracellular space, extracellular region, macromolecular complex, extracellular exosome, mitochondrion etc. MF is mainly involved in protein binding, identical protein binding, enzyme binding, protein kinase binding, protein homodimerization activity, macromolecular complex binding, peptidase activity, cytokine activity, ubiquitin protein ligase binding, transcriptional activator activity etc. The bubble plot of the GO functional enrichment analysis  is shown in Figure Screening was conducted according to the criterion that the corrected  in BP 28, MF (58)P value was less than .05, and a total of 126 KEGG signaling pathways were enriched. The visual display results of the top 20 items with the corrected P value are shown in Figure Escherichia coli infection, colorectal cancer, relaxin signaling pathway, prostate cancer, platinum drug resistance and many other pathways.Screening was performed according to the standard that the corrected The binding energies (docking affinity) of ursolic acid to core target proteins tumor protein p53 (TP53), transcription factor AP-1 (JUN), interleukin- 6 (IL6), vascular endothelial growth factor A (VEGFA), CASP3, and mitogen-activated protein kinase (MAPK8) were −10.5, −9.8, −10.6, −10.6, −10.5, −11.5 kcal/mol , respectively , MAPK and intracellular phosphatidylinositol kinase (PI3K), and these 3 factors are closely related to the occurrence and development of OP.–31 It was revealed that TNF-α may not only induce the expression of nuclear factor kappa B (NF-κB) receptor activator ligand (RANKL) in osteoblasts, but also directly act on RANKL to induce the formation of osteoclasts and promote bone resorption. It was found that ursolic acid may protects reduce the levels of cytokines TNF-α and IL-1β that against ulcerative colitis via anti-inflammatory and antioxidant effects in mice. The above researches suggests that IL-6 and TNF-α are potential targets of ursolic acid in the treatment of OP.OP is a systemic bone disease characterized by progressive loss of bone mass, fragility, and susceptibility to fractures. On the other hand, activated MAPK may promote the generation of osteoblasts, thus improving the symptoms of OP. Prostaglandins induce macrophage colony-stimulating factor (M-CSF) and RANKL expression, and also promote osteoclast differentiation and inhibit osteocyte function. Prostaglandin endoperoxidase 2 (PTGS2) is the rate-limiting enzyme in the production of prostaglandins and has a certain inhibitory effect on the above-mentioned activities of prostaglandins. Vascular endothelial growth factor (VEGF) both supports angiogenesis and promotes the function of osteoblasts that may partially regulate the functions of osteoblasts and osteoclasts through autocrine, endocrine and paracrine mechanisms, and promote bone formation and reduction of bone resorption. The caspase family is a key factor in regulating the process of apoptosis, among which caspase 3 is one of the most critical apoptotic effectors in this family and is considered to be the executor of apoptosis. When caspase 3 is activated, apoptosis cannot be reversed. In the present study, through the GO functional annotation analysis of shared targets, it was found that GO biological processes were mainly concentrated in the process of apoptosis, regulation of cell proliferation, and drug response. Through the KEGG pathway enrichment analysis of common target genes, it was revealed that they were mainly enriched in tumor pathway, TNF signaling pathway, hepatitis B, PI3K/Akt signaling pathway, human HTLV-I infection and other pathways. Among them, the PI3K/Akt signaling pathway is closely related to bone tissue metabolism. Activation of the PI3K/Akt signaling pathway may stimulate the proliferation and differentiation of osteoblasts while inhibiting their apoptosis. In addition, PI3K can stimulate the formation of osteoclast actin filaments, regulate cell chemotaxis, attachment and proliferation, and inhibit the PI3K expression reduces bone resorption by mature osteoclasts. Previous studies have been confirmed that ursolic acid can inhibit the conduction of PI3K/Akt signaling pathway, resulting in up-regulation of downstream pro-apoptotic protein expression, activation of endoplasmic reticulum stress pathway, and ultimately promoting cell apoptosis. Relevant studies have confirmed that there is a close relationship between chronic hepatitis B virus infection and OP, and its mechanism of action may be related to the chronic inflammation caused by the virus infection. Hepatitis B virus infection can induce the secretion of inflammatory cytokines such as TNF-α, IL-1, and IL-6, which can increase the expression of RANKL and stimulate bone resorption. The combined effect of the above-mentioned inflammatory cytokines is the main cause of OP caused by chronic hepatitis B virus infection, which can lead to decreased bone formation and increased bone resorption, thereby reducing bone mineral density. In addition, HBV infection inhibits hepatic 25-hydroxyvitamin D production and vitamin D uptake, thereby increasing bone loss and reducing bone formation. Hepatic decompensation caused by long-term hepatitis B virus infection affects collagen synthesis in bone matrix by inhibiting fibroblast growth, and may co-suppress osteoblast function by increasing the expression of carcinoembryonic fibronectin.MAPK8 is a member of the MAPK family and plays an important role in regulating inflammatory and immune responses. On the one hand, MAPK plays an important role in the formation and activation of osteoclasts that affect the development of OP by mediating the NF-κB signaling pathway.In the present study, molecular docking technology was used to verify the molecular docking between ursolic acid and the encoded proteins of core target genes TP53, JUN, IL6, VEGFA, CASP3, and MAPK8. The results showed that ursolic acid has strong docking activity with the above-mentioned core target gene-encoded protein , which further indicates that ursolic acid can interact with the core target gene-encoded protein receptor. Stable binding and play its role in the treatment of OP. The results preliminarily verified the reliability of the above-mentioned network pharmacological prediction results, and laid a foundation for the subsequent research on the mechanism of action of ursolic acid in the treatment of OP.Taken together, the therapeutic effect of ursolic acid on OP may be through the regulation of core target genes such as P53, VEGFA, IL6, TNF, CASP3, MMP9, JUN, STAT3, MAPK8, and PTGS2. It may mainly related to cancer pathways, hepatitis B signaling pathway, TNF signaling pathway and many other key pathways. Its mechanism may involves multiple therapeutic targets and signaling pathways, reflecting the characteristics of TCM for treating diseases with multiple targets and multiple pathways. However, the potential targets and pathways of ursolic acid in the treatment of OP predicted in the present study still need to be further verified by relevant experiments.Shuqing Zhou and Jingxin Mao conceived and designed the research. Jingxin Mao, Bowen Yang, and Qiuwen Zhu carried out the analysis and wrote the paper in the study. Shuqing Zhou and Jingxin Mao make the equally contribution to the manuscript. All authors declare that there have not any commercial or associative interest that represents competing interests in connection with the work submitted.Conceptualization: Jingxin Mao, Shuqing Zhou.Data curation: Bowen Yang, Qiuwen Zhu, Jingxin Mao.Formal analysis: Bowen Yang, Xiaodong Wang.Funding acquisition: Jingxin Mao.Investigation: Bowen Yang.Methodology: Bowen Yang.Resources: Qiuwen Zhu, Jingxin Mao, Shuqing Zhou.Software: Qiuwen Zhu, Xiaodong Wang.Supervision: Qiuwen Zhu, Jingxin Mao, Shuqing Zhou.Validation: Qiuwen Zhu, Xiaodong Wang, Jingxin Mao.Visualization: Qiuwen Zhu, Xiaodong Wang, Shuqing Zhou.Writing – original draft: Bowen Yang.Writing – review & editing: Jingxin Mao.


Based on a Gene Expression Omnibus (GEO) chip analysis combined with network pharmacology and molecular docking technology, in this study we explored the molecular targets and mechanism of the wuyao–ginseng medicine pair in the prevention and treatment of diarrhea-type irritable bowel syndrome (IBS-D). The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to search for the chemical constituents and targets of wuyao and ginseng. The UniProt database was used to search for the target gene name. In the GEO database, IBS was searched to obtain GSE36701 and GSE14841 microarray data. We imported the intersection targets into the STRING database to construct a protein–protein interaction (PPI) network. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (Go) pathway analyses were performed using the Metascape database. A total of 30 active ingredients of wuyao–ginseng, 171 drug targets, 1257 IBS differentially expressed genes, and 20 drug-disease intersection genes were obtained from the GEO data. We screened the results and obtained the core active ingredients beta-sitosterol, DMPEC, Boldine, etc.; the core targets NCOA2, EGFR, VEGFA, etc.; and the key pathways P13K-Akt, MAPK, etc. The wuyao–ginseng medicine pair may be involved in inflammation-related signaling pathways, acting on disease targets such as NCOA2, EGFR, and VEGFA as well as pathways such as P13K-Akt and MAPK, thereby playing a key role in the prevention and treatment of IBS-D. IBS is a dysfunctional gastrointestinal disorder characterized by recurrent abdominal pain or discomfort and changes in bowel habits . The epiThe TCM disease name of IBS is diagnosed as “diarrhea”, “constipation”, “abdominal pain”, etc., according to different clinical symptoms . SimotanPanax ginseng C.A.M. Pey and can stimulate vitality without damaging positivity or the spleen and lung. Research has shown that Panax ginseng has the effects of invigorating vitality, invigorating the spleen, benefiting the lungs, and soothing the mind and eyesight. The active ingredients it contains  are also important to exert its medicinal effects.Ginseng, with its bitter-sweet taste, is the dry root and rhizome of These two medicines, one supplementing and one dispersing, can precisely target the pathogenic mechanism of the IBS intestinal barrier that causes spleen deficiencies and dampness, correcting deficiencies and evil excesses. They have the synergistic effect of supplementing, dispersing, and strengthening the intestine and stopping diarrhea ,9. In thhttp://tcmspw.com), UniProt protein (https://beta.uniprot.org), Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/), STRING (https://cn.string-db.org), and Metascape (https://metascape.org) databases were used. GraphPad Prism 8 (https://www.graphpad.com), Ticket letter (http://www.bioinformatics.com.cn), and Ctyoscape 3.8.2 (https://cytoscape.org) along with the Ctyoscape 3.8.2 software cytoNCA plug-in were also used.The TCMSP Chinese Medicine Pharmacology  value (%) ≥ 30 and druglikeness (DL) value ≥ 0.18 were set to screen out the eligible active ingredients contained in wuyao–ginseng, respectively. The components without corresponding targets were then screened out to obtain the relevant active ingredients . The reviewed active components of wuyao–ginseng were imported into the UniProt protein database. Homo Sapiens was selected as a species, affirmed under the “reviewed” conditions, and the drug targets were converted into a Gene Symbol. The selected drug ingredients and drug targets were inputted into the Ctyoscape 3.8.2 software and then the active ingredient drug target network was constructed. The network was topologically analyzed with the cytoNCA plug-in. The core network between the active ingredients and the drug targets of wuyao–ginseng was then screened according to the median values of betweenness centrality (BC) and degree centrality (DC). p-value < 0.05 of genetic variations were used for the standard analysis to map the volcano and heat maps [Chips GSE36701 and GSE14841 were selected to search for irritable bowel syndrome in the GEO database. GSE36701 contained 40 healthy volunteers (HVS) and 53 IBS-D subjects with diarrhea as the main feature. GSE14841 consisted of 4 healthy volunteers and 5 IBS-D subjects. In the healthy volunteers and patients with IBS-D with multiple genetic differences, an absolute value |logFC| ≧ 0.5 and a eat maps . On the basis of the above, the drug targets and differential genes were introduced into vitalization letters. Intersection targets were generated and a Venn diagram was drawn. The network and type files were created according to the intersection genes and their active components. The files were imported into the Ctyoscape 3.8.2 software to draw the network pharmacological map. Multiple proteins were selected in the STRING database and the intersection genes were imported. Homo Sapiens was selected as the species and the PPI network was constructed. The tsv file of the network was then exported. After importing the tsv file into Ctyoscape 3.8.2, the cytoNCA plug-in was used to screen the core genes according to the median value of the BC and DC. The PPI network was then constructed and a visual analysis conducted. p-value for the analysis and a bubble map was drawn. The GO enrichment analysis results contained three modules: the BP ; the CC (cellular component); and the MF (molecular function). The top 10 items were selected for the analysis according to their p-values and bar charts were drawn. The Metascape database was used to carry out the KEGG and GO enrichment analysis for the differential genes of the drug target. A KEGG enrichment analysis was conducted to analyze the action pathway of the control of EBS-D by wuyao–ginseng. The top 10 items were selected according to the http://zinc.docking.org) database and the PDB (https://www.rcsb.org/) database, respectively; these were then interconnected one by one using Auto Dock 4.2.6. The binding strength and activity of the target and active compounds were evaluated based on the Docking Score. Pymol 2.5.0 software was used for the docking analysis and drawing.The core component interacted with the core target. The 3D structures of the core components and the core targets were searched for in the ZINC  ≥ 30 and DL ≥ 0.18 as the screening conditions, a total of 31 active ingredients of wuyao–ginseng was obtained. Wuyao contained 9 active ingredients and ginseng contained 22 active ingredients. The targets of the active ingredients were mapped to the certified human gene targets in the UniProt database. After the null values were removed, 30 active ingredients  and 171 A total of 30 active ingredients of wuyao–ginseng and their targets were imported into the Ctyoscape 3.8.2 software to construct a visualization diagram of the active ingredient drug targets of wuyao–ginseng. With the help of the cytoNCA plug-in, nodes larger than the BC and larger than the DC median value were screened (a node represented the active ingredients and drug targets of wuyao–ginseng). A new visualization network was extracted and established to construct a core network diagram of the active ingredients and drug targets of wuyao–ginseng . A totalp-value < 0.05 for the standard selection. A total of 160 differentially expressed genes (DEGs) was obtained. The GSE14841 chip had a sample total of 9, including 4 HV samples and 5 IBS-D subjects. A total of 1280 DEGs was obtained by screening under the same condition. According to the differential genes of each chip, the respective heat map and volcano map were drawn  and 53 cases of IBS-D subjects, was used to conduct a variance analysis using a |logFC| ≥ 0.5 and a re drawn . A totalA total of 171 drug targets was obtained from wuyao–ginseng. A total of 1257 differentially expressed genes of IBS-D was obtained from the GSE36701 and GSE14841 chip database. From these, 20 overlapping genes were obtained , which were drawn as a Venn diagram .The active components of wuyao–ginseng and the selected intersection genes were imported into Ctyoscape 3.8.2 software to draw the network pharmacological map. Nodes larger than the BC and larger than the median value of the DC were screened out. A new visualization network was then extracted and established to construct the core network diagram of the active ingredient disease target of wuyao–ginseng, with 26 nodes and 19 edges . AccordiA total of 20 intersection genes was imported into multiple proteins in the STRING database. The species was manually selected to construct the PPI network and its tsv file was exported; this was then imported into the Ctyoscape 3.8.2 software for an analysis with the help of the cytoNCA plug-in , cell component (CC), and molecular function (MF). Through the GO enrichment analysis, 186 BF entries, 17 CC entries, and 23 MF entries were obtained. The top 10 columns were selected, respectively, according to their ar chart . The resAutoDock software was used to rank the top 6 compounds  chromone, quercetin, and Ginsenoside Rh2), The compounds were interlinked with the 7 top core targets  one by one, and the matching degree between the compounds and the core targets was determined from the binding energy (BE) values. In general, a BE ≤ −4.25, a BE ≤ −5.00, and a BE ≤ −7.00 Kcal/mol indicated that a compound had a certain good and strong binding energy with the target. According to the heat map analysis drawn in R4.1.2 , among tAt the same time, Pymol 2.5.0 software was used to analyze the docking mode of the compound and core target combination, respectively. aconitum officinalis mainly include volatile oils, isoquinoline alkaloids, quiterpene furans, and flavonoids [At present, the pathogenesis of IBS-D has not been fully elucidated. Probiotics, antibiotics, immunosuppressants, 5-HT antagonists, and fecal microbiota transplantation therapy have been widely used to treat IBS-D . Howeveravonoids ,14, whicavonoids ,16,17. Gavonoids . Ginsengavonoids . The comavonoids , which aIn this study, a total of 171 drug targets was obtained from the TCMSP database, among which 150 targets were obtained from radix aconitum and 23 targets were obtained from radix aconitum and ginseng. A core network map of the active ingredient drug target of wuyao–ginseng was constructed to explore the active ingredients and targets of wuyao–ginseng. Subsequently, IBS were searched for in GEO database and two chip databases, GSE36701 and GSE14841, were screened. The two chip databases, GSE36701 and GSE14841, were analyzed and 1257 DEGs were obtained. These differentially expressed genes were associated with IBS-D. The intersection of 171 drug targets and 1257 DEGs yielded 20 overlapping gene targets, indicating that these genes may be the targets of the control of IBS-D by wuyao–ginseng. The active constituents and intersection genes of wuyao–ginseng were analyzed to construct an active component disease target network pharmacological map of wuyao–ginseng. The main active ingredients were beta-sitosterol, DMPEC, Boldine, 6,7-dimethoxy-2-(2-phenylethyl) chromone, quercetin, and Ginsenoside Rh2. These results suggested that these components were the main components of the control of IBS-D using wuyao–ginseng, and NCOA2 and other genes were the important targets of the control of IBS-D using wuyao–ginseng. Finally, 20 intersection genes were imported into the STRING database to construct a PPI network map. According to the intersection gene PPI network, EGFR, VEGFA, BCL2L1, CASP9, MMP9, and MAPK14 were important targets. The results of the KEGG pathway enrichment analysis showed that the cancer pathway, P13K-Akt signaling pathway, and MAPK signaling pathway were the main pathways in the prevention and treatment of IBS-D using wuyao–ginseng. The GO functional enrichment analysis showed that biological processes were related to the transmembrane receptor protein tyrosine kinase pathways and enzyme-linked receptor protein signaling pathways as well as the cell components, including the plasma membrane protein complexes. The molecular functions were related to protein domain-specific binding. AutoDock software was used to rank the top 6 compounds  chromone, quercetin, and Ginsenoside Rh2). The compounds were interlinked with 7 top core targets  one by one and the matching degree between the compounds and the core targets was determined from the binding energy (BE) values. The results showed that the active ingredients in wuyao–ginseng were beta-sitosterol, DMPEC, Boldine, 6,7-dimethoxy-2-(2-phenylethyl) chromone, quercetin, and Ginsenoside Rh2, which may play a role in the prevention and treatment of IBS-D by improving low-grade inflammation through the NCOA2, EGFR, VEGFA, BCL2L1, CASP9, MMP9, MAPK14, PI3K/AKT, and MAPK signaling pathways.The occurrence and development of IBS-D involve a large number of pathophysiological processes, including low-grade intestinal mucosal inflammation, increased intestinal barrier permeability, and stress . Low-graBased on the above studies, it was found that the key genes were related to the PI3K/AKT signaling pathway and MAPK signaling pathway. An overexpression of NCOA2 in mouse prostate tumors led to the overactivation of PI3K/AKT and MAPK signals, thus promoting the tumor malignancy . The epiIn this study, by means of a gene analysis, network pharmacology, and molecular docking technology, an association analysis between drugs and diseases was conducted and the drug components, targets, pathways, and potential mechanism of action that play a key role in the prevention and treatment of IBS-D were preliminarily predicted, providing a new idea for the prevention and treatment of IBS-D by the medicine of wuyao-ginseng. First, a total of 171 drug targets was obtained from the TCMSP database, among which 150 targets were obtained from radix aconitum and 23 targets were obtained from radix aconitum and ginseng. A core network map of the active ingredient drug target of wuyao–ginseng was constructed to explore the active ingredients and targets of wuyao–ginseng. Subsequently, IBS was searched for in the GEO database and two chip databases, GSE36701 and GSE14841, were screened. The two chip databases, GSE36701 and GSE14841, were analyzed and 1257 DEGs were obtained. These differentially expressed genes were associated with IBS-D. The intersection of 171 drug targets and 1257 DEGs yielded 20 overlapping gene targets, indicating that these genes may be the targets of the control of IBS-D by wuyao–ginseng. Finally, 20 intersection genes were imported into the STRING database to construct a PPI network map. According to the intersection gene PPI network, EGFR, VEGFA, BCL2L1, CASP9, MMP9, and MAPK14 were important targets. The active ingredients in wuyao–ginseng were beta-sitosterol, DMPEC, Boldine, 6,7-dimethoxy-2-(2-phenylethyl) chromone, quercetin, and Ginsenoside Rh2; these may play a role in the prevention and treatment of IBS-D by improving low-grade inflammation through the NCOA2, EGFR, VEGFA, BCL2L1, CASP9, MMP9, MAPK14, PI3K/AKT, and MAPK signaling pathways.


Objective: To study the mechanisms of the Ligusticum chuanxiong Hort.–Piper longum L. herbal pair (LPHP) in the treatment of migraine using network pharmacology.Methods: The active constituents of LPHP and their targets were searched for and screened using the Chinese Medicine System Pharmacology Database. Genes related to migraine were searched on GeneCards, Online Mendelian Inheritance in Man and other databases. Cytoscape was used to construct and combine active component–target and disease–target networks. The core target was screened by network topology analysis, and the Metascape database was used for gene ontology analysis of key targets and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis to explore the molecular mechanisms in the treatment of migraine.Results: A total of 28 active constituents of LPHP were obtained through database screening and literature review, and 60 cross-linking targets were obtained. The target sites were analysed using a protein–protein interaction network to obtain six target proteins with a greater degree of relevance. These were identified as the main targets for the treatment of hypertension, and these key targets were found to be associated with 20 signalling pathways, including neuroactive ligand–receptor interaction, the calcium signalling pathway, the cGMP–PKG signalling pathway, pathways in cancer and the cyclic adenosine 3′,5′-cyclic monophosphate (cAMP) signalling pathway.Conclusion: This study reveals the molecular mechanism of LPHP in the treatment of migraine from the perspective of network pharmacology and provides a basis for further research and molecular mechanism research. Migraine is a common and frequently occurring chronic neurovascular disease. Its onset is characterised by repeated attacks , seriousLigusticum chuanxiong–Piper longum (Lc–Pl) couplet medicines have been shown to have a good effect on migraine. For example, a previous study by our research group . On this platform, we searched for the chemical components of Ligusticum chuanxiong and Piper longum, using these Latin taxonomic names as keywords.This research was based on the traditional Chinese medicine systems pharmacology database and analysis platform  ≥ 30% and drug-likeness (DL) ≥ 0.18. For the common active compounds that did not meet the screening criteria, the relevant literature was searched for supplements through the China National Knowledge Infrastructure (CNKI) platform using “https://www.uniprot.org) was used to standardise the protein target information of the compounds.After screening, the UniProt database (http://www.disgenet.org), the DrugBank database (http://www.drugbank.ca), the GeneCards database (https://www.genecards.org), the Online Mendelian Inheritance in Man database , and the Therapeutic Target Database .Disease-related targets for migraine were collected through the DisGeNET database (http://www.bioinformatics.com.cn/). The active ingredients of the drug and the screened intersection targets were uploaded to the Cytoscape 3.7.2 software to generate the drug-component–target network diagram, and the key chemical components and targets were obtained through network topology analysis.The intersection of potential drug targets and disease-related targets was screened using the WeChat website (The intersection target data were uploaded to the BisoGenet database to construct the protein–protein interaction (PPI) diagram. We then conducted a topology analysis of the PPI network and calculated the topology parameter values, including the degree of connectivity (Degree), betweenness, closeness, local average connectivity, and neighbourhood connectivity. The core targets with the strongest interactions were determined according to the degree value, which refers to the number of connections between a node and other nodes. A higher degree value thus reflects a wider set of connections between a given node and other nodes in the network.Lc–Pl couplet medicines in the treatment of migraine.We conducted gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on the intersection targets using the Metascape database, and screened the main action pathways of Ligusticum chuanxiong and 104 chemical components of Piper longum were obtained.Using TCMSP, all 189 chemical components of Ligusticum chuanxiong  and 13 active components of Piper longum  were obtained. Then, common ingredients that did not meet the screening criteria were identified according to relevant reports published on CNKI, giving eight Ligusticum chuanxiong supplements (including ligustrazine) and one Piper longum supplement (piplartin).The collected chemical components were screened using the conditions OB ≥ 30% and DL ≥ 0.18, and the active ingredients in the blood were screened out. From this process, six active components of Lc–Pl couplet medicines were summarised, resulting in no duplicate ingredients. A total of 28 chemical ingredients were obtained, which were classed as candidate ingredients for the Lc–Pl couplet medicines , further indicating that there might be a synergistic relationship between these two traditional Chinese medicines. Overall, Piper longum had more disease targets; therefore, the order of contribution in the treatment of migraine was determined as Piper longum > Ligusticum chuanxiong.A total of 60 intersections of potential targets of the  website , indicatLigusticum chuanxiong (CX) and Piper longum (BB) were used to construct an active ingredient–direct acting target network diagram in Cytoscape 3.7.2. The network diagram, as presented in The 28 chemical components, the 60 targets, Lc–Pl couplet medicines involve not only the phenomenon of one compound acting on multiple target proteins but also that of different compounds acting on the same target protein.In the network diagram, the hexagonal nodes of various colours represent the active ingredients of different drugs, and the squares represent targets; the area and colour transparency of each node represents its Degree value, with larger areas and darker shading representing increasing importance. As shown by the results, the The network topology parameters for the treatment of migraine were analysed using the built-in Network Analyzer in CytoScape 3.7.2, and the Degree value of each node was calculated. The top-ranked chemical components for Degree were as follows: piperine (MOL001592), tetrahydro tanshinone I (MOL001601), myricetone (MOL002135), piplartin (MOL002848), sesamin (MOL001558), 4-methoxy-N- benzamide (MOL001586), ferulic acid (MOL000360), senkyunolide E (MOL002145), senkyunolide I (MOL002143), piperlonguminine (MOL001559), and pipernonaline (MOL001560).ß 2-adrenergic receptor (ADRB2), cardiac sodium channel gene (SCN5A), acetylcholine M1 receptor (CHRM1), cyclooxygenase 1 (PTGS1/COX1), γ-aminobutyric acid type A receptor α1 (GABRA1), α1 adrenergic receptor B (ADRA1B), solute carrier family 6 member 3 (SLC6A3), muscarinic acetylcholine receptor M3 (CHRM3), phosphodiesterase 3α (PDE3A), and retinoic acid X receptor a (RXRA). The above might thus be the key chemical components and action targets of Lc–Pl couplet medicines. These findings also reflect the multi-component and multi-target therapeutic characteristics of Lc–Pl couplet medicines on migraine.The top-ranked targets for Degree were cyclooxygenase 2 (PTGS2/COX2), The 60 intersection targets were then uploaded to the BisoGenet plug-in of Cytoscape 3.7.2 to analyse the protein interactions between the targets, obtain their corresponding indirect action targets and construct the PPI network. The resulting network, containing 2,785 nodes and 65,592 edges, is shown in Lc–Pl couplet medicines for migraine, and these six core targets also affect each other. It is of interest to note that the closest relationship is indicated between TARDBP and FUS, which are both related to amyotrophic lateral sclerosis.  with the strongest interaction values were screened under the conditions shown in lerosis. .p-value as the screening criterion, the top 15 and the top 20 entries were selected for analysis, respectively, and the histogram and bubble chart were drawn using tools on the Bioinformatics website. The GO results are shown in The intersection targets were used for GO function enrichment analysis and KEGG pathway enrichment analysis in the Metascape database. Taking As shown in p < 0.01) include the neuroactive ligand–receptor interaction pathway, calcium signalling pathway, cGMP-PKG signalling pathway, pathways in cancer and the adenosine 3′,5′-cyclic monophosphate (cAMP) signalling pathway. On this basis, we infer that Lc–Pl couplet medicines might be the result of a multi-channel synergistic effect in the treatment of migraine.The KEGG pathway enrichment analysis screened 20 signal pathways, as shown in Lc–Pl couplet medicines, as presented in Lc–Pl couplet medicines in the treatment of migraine.The target pathway network for Lc–Pl couplet medicines in the neural activity ligand–receptor interaction pathway, we mapped the relevant targets to this pathway, as shown in To further explore the relationship between Lc–Pl couplet medicines, including ligustrazine, senkyunolide E, piperlonguminine, and piperine, and 11 important ingredients. Some of these, including the calcitonin gene-related peptide receptor antagonist folic acid  and β2-adrenergic receptor (ADRB2) were correlated with the highest numbers of chemical components , indicating that these targets are closely related to Lc–Pl couplet medicines.In terms of the direct action targets of the chemical components of Lc–Pl couplet medicines include myricetone, senkyunolide A, piperine, and piplartin, which bind to targets such as PTGS2 and exert anti-inflammatory effects through multiple pathways, including the tumour necrosis factor, interleukin-17 and nuclear factor kappa B signalling pathways.The existing research on migraine suggests that neurogenic inflammation is the key link in its onset  and thatThe ADRB2 gene has a series of biological effects due to its regulation of the level of the second messenger cAMP in the cell. It is widely expressed in a variety of tissue cells  and playCalcitonin gene-related peptide (CGRP) is a marker of trigeminal microvascular activation and is widely believed to be an important factor in migraine attacks . A previLc–Pl couplet medicines mainly treat migraines via the neural activity ligand–receptor interaction pathway, the calcium signalling pathway, the cGMP-PKG signalling pathway, pathways in cancer and the cAMP signalling pathway. Approximately one-third of the identified targets are involved in the neural activity ligand–receptor interaction pathway, including adrenergic receptors , dopami [ADRA2B ], 5-HT r [ADRA2B ], opioid [ADRA2B  and GABR [ADRA2B ], glutam [ADRA2B ] and cap [ADRA2B ]. These With the development of systems biology, researchers have discovered that disease is a pathogenic factor that invades the biological system, disrupts the balance of the biological network and leads to abnormal functioning of the organism. In this way, the disease can be regarded as a specific interference of the biological network , and druLc–Pl couplet medicines. Our findings are broadly consistent with existing research results, which provide a degree of support for the treatment of migraine with lutongning granules, composed exclusively of Ligusticum chuanxiong and Piper longum. A limitation of this study is that it only discusses predictability based on the network pharmacology approach; therefore, further experimental research is needed to verify the relevant targets and signal pathways.This paper has discussed the mechanisms of migraine treatment by


The aim of this paper was to evaluate the intratubular penetration area of a bioceramic sealer, using continuous wave (CW), vertical condensation (VC) with two different types of gutta-percha  and bioceramic-coated (BG)) and single cone (SC) technique with BG gutta-percha, in different root thirds.A total of 150 mature single-root human teeth  were prepared and randomly divided into five groups (n = 30). Teeth were filled using a bioceramic sealer  and two different types of gutta-percha, with CW and VC techniques, the teeth in the control group were filled with SC technique and BG gutta-percha. The teeth were sectioned and evaluated as one-third portions in each case under a confocal laser microscope. The penetration area measurements were carried out with the Autocad ® programme. Data was analyzed using the one-factor ANOVA test (p < 0.05) and Post Hoc Test (p < 0.05).The ANOVA Test showed significant differences in the penetration areas of the five obturation techniques (P < 0.05). The Post Hoc Test exhibited significant differences in multiple comparisons (P < 0.05). There was more dentinal tubule penetration in the coronal third than in the apical third in all techniques.The intratubular penetration of the bioceramic sealer was influenced by the obturation techniques tested, but not by the different gutta-percha tested. There was more penetration of sealer in the warm obturation techniques than the SC, regardless of the type of gutta-percha used. Bioceramic sealer; Confocal laser scanning microscopy; Tubule penetration; Warm gutta-percha techniques; Single cone. It iBioceramic sealer penetration into dentinal tubules is essential in order to create a mechanical anchorage between the sealer and the dentinal tubules  and chemPreviously, the usual root canal filling practice was to apply a considerable amount of gutta-percha and a small proportion of sealer . Kim et EndoSequence BC Sealer HiFlow® (HiFlow)  is a bioceramic sealer developed to be heat-resistant ; it is sEndoSequence BC Sealer® and EndoSequence BC Sealer HiFlow® have the same composition as TotalFill BC Sealer®   and TotalFill BC Sealer HiFlow® (HiFlow) . The comRecently, the behaviour of other bioceramic sealers, such as BC Sealer or BioRoot RCS®  has been investigated after exposure to heat. The chemophysical properties were investigated during or shortly after heat exposure , 22, 23.In the early 2000s, specialized manufacturers introduced root obturation techniques with the “monoblock” concept in which the gutta-percha, the sealer and the dentin generated a single unit . CurrentThe TotalFill BC filling system consists of TotalFill BC Points® bioceramic-coated (BG) gutta-percha and BC Sealer; the obturation system makes use of the moisture naturally present in the canal to start the setting reaction . BC SealThere is a general lack of information available concerning the capacity of tubule penetration of BC Sealer when used together with gutta-percha tips with different compositions  and different tapers (0.2–0.4-0.6–0.8).The aim of this paper was to evaluate the intratubular penetration area of a calcium silicate-based sealer (HiFlow), using two warm obturation techniques, continuous wave (CW) and vertical condensation (VC) with two different types of gutta-percha  and BG) and also SC technique with BG in different root thirds. The null hypothesis asserts there are no differences between the penetration areas obtained for each of the obturation techniques.2This piece of research was approved by the Research Ethics Committee of UCV, (Registration number: UCV/2019-2020/001.).2.1To carry out the study, 150 human teeth with a single root were selected . The teeth were extracted for periodontal reasons. Roots with acute curvatures, immature apex, resorption, previous endodontic treatment, calcification, fractures or initial apical sizes larger than 15 were rejected. After extraction, the teeth were immersed for one hour in a 5.25% sodium hypochlorite solution (NaOCl) after which the root surfaces were cleaned with a Gracey® 1-2 curette  and then stored in a saline solution.2.2Two preoperative X-rays were taken in two views to check the presence of a single canal. Buccolingual and mesiodistal parallel radiographs were obtained for each tooth. After opening the root canal system with a tapered cone burr  and constant irrigation, the canal was located with a DG16® endodontic probe . The root of the clinical crown was separated at the amelocemental junction with a handpiece diamond disc and water cooling; a size 10 or 15 K file was then introduced into the canal space, the working length (WL) was established 0.5 mm from the apical foramen by visual observation.All canals were prepared with Protaper Gold®  according to the producer’s instructions. The shaping files S1 (250 rpm and 3 Ncm) and S2 (250 rpm and 1 Ncm) were used with circumferential movements and brushing at the working length, while the finishing files F1 (250 rpm and 1.5 Ncm) and F2 (250 rpm and 2.5 Ncm), were used with a pecking motion with the Gold Reciproc™ motor . After each file was used, the canal was flushed out with 5.25% NaOCl solution. The permeability of the canals was checked by inserting a size 10 file through the apical foramen after instrumentation was completed.As the final irrigation protocol, canals were irrigated for 1 min with 5 ml of 5.25% NaOCl, 1 min with 5 ml of 17% EDTA, and 30 s with 5 ml of chitosan-hydroxyapatite precursor, 10 ml of saline solution was used for a final flush out and also used in the established order of different irrigants , 30. The2.30.1% of Rhodamine B™  was added to the bioceramic sealer in relation to the weight for its subsequent observation through the confocal laser microscope, thanks to the fluorescent property of the dye.The samples were then randomly divided into 5 experimental groups (n = 30). The samples were sealed with the different obturation techniques set forth as follows .Table 1G2.4Once all the samples were sealed, they were stored at 37 °C and 100% humidity in a laboratory incubator for 14 days to allow complete sealer setting. The root was divided into three parts at the dental laboratory of Catholic University of Valencia, taking a sample from each third: the coronal, middle and apical third . Horizontal cuts were made using a 0.3 mm diamond disc handpiece with water cooling , 1 mm th2).The measurements of the penetration areas of the sealer were carried out with AutoCad® Software from the images obtained and collected in a data sheet. Fist, each image was scaled to 500 μm in order to obtain a correct measurement of all its elements. The appropriate AutoCad tool function was applied to the area of tubular sealer penetration and the canal area, to obtain the penetration area . The penAll measurements were recorded by one of the authors. In case of doubt on first viewing, the sample was polished, and a new image was then obtained for analysis. All data was recorded, and then analysed.2.5The statistical analysis of the data collected for the present study was carried out using SPSS 23 software using a confidence level of 95% and considering them statistically significant (p < 0.05). As the sample size is sufficiently large, (n = 30), we used parametric methods of comparison. The one-way ANOVA test was used to compare means and to determine the statistical and significant effect of the study variables  . The Pos3In all thirds, the ANOVA test showed significant differences between at least two of the obturation techniques (p < 0.05) .In the coronal third, the Post Hoc test showed there was a statistical difference between the SC and VC with NG (p = 0.001), the penetration average was lower using the SC technique. There was no statistical difference observed compared to the other techniques .In the middle third, the Post Hoc test revealed there was a statistical difference between the SC and CW with NG (p = 0.017), CW with BG (p = 0.035) and VC with NG (p = 0.001). In this third, the penetration average was lower in the SC. There was no statistical difference compared to the other techniques .In the apical third, the Post Hoc test exhibited there was a statistical difference between the SC and CW with NG (p = 0.031), CW with BG (p = 0.045), the penetration average was lower in the SC. There was no statistical difference compared to the other techniques .4During the study, we used the confocal laser microscope, since the conditioning of the specimens for the scanning electron microscope needs to be dried . The samRhodamine B could be suitable with the bioceramic sealers , becauseThe correct evaluation of the samples in the microscope was difficult because the precision cuts and an adequate level of polishing were essential. The principal problem was one of accuracy: the root cuts had to be 1 mm deep, thin and completely straight. The samples of the apical third were particularly difficult to manipulate because of their size.The penetration of a sealer is vitally important and it may be influenced by several factors such as the chemophysical properties of the sealer, the obturation technique and the anatomy of the root canal system . AdditioThe viscosity and flow of endodontic sealers are important in determining the effectiveness that they penetrate in the dentinal tubules . The infThe warm obturation techniques with bioceramic sealers have become a controversial theme. There were significant changes in the properties of the bioceramic sealers after heating during the thermoplasticized obturation techniques . ApatiteChen et al. compared  two biocIt is commonly believed that increasing the pressure in the warm obturation techniques produces a significant level of sealer penetration in the dentin tubules, however the literature demonstrates contradictory results . For theper se [We used one group of SC with BG points as the manufacturers recommended this type of BG points and bioceramic sealer with the SC technique , 47. Theper se . Al-Hiyaper se  used theOur results showed a greater degree of penetration in the warm obturation techniques versus SC in all thirds, the area of penetration for each technique was greater in the coronal third compared to the apical portion. These results are in agreement with the those of previous studies , 15, 26.In the study by Eymirli et al. , the resOsiri et al.  studied Eid et al.  comparedThe clinical relevance of our results may serve as a reference because the high ability of dentinal tubule penetration of the sealer with warm obturation techniques may improve the sealing action in the tubules, and therefore the antibacterial effect.The blended use of HiFlow with the CW technique exhibited a good dentinal tubule penetration, so it is possible to achieve better apical sealing . In addi5In conclusion, within the limitations of this study, the warm obturation techniques (CW and VC) showed more intratubular penetration of the calcium silicates-based sealer than the SC. The different gutta-percha tested in warm obturation techniques (NG and BG) were not influenced in the penetration area. For each type of gutta-percha and technique, dentinal tubule penetration was higher in the coronal section than in the apical section.Alberto Casino Alegre: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Susana Aranda Verdú: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Jose Ignacio Zarzosa López; Jorge Rubio Climent, Dr; Antonio Pallarés Sabater, Dr; Eliseo Plasencia Alcina, Dr: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


In this study, we evaluated the cultivation impacts on four predominant CH4 production pathways, including acetate, carbon dioxide (CO2), methylamines, and methanol, in a wetland and cultivated cropland in northeastern China. The results showed significant suppression of CH4 production potential and the abundance of genes for all four methanogenic pathways in cropland. The consistency between CH4 production and methanogenesis genes indicates the robustness of genomic genes in analyzing methanogenesis. The suppression effects varied across seasons and along soil profiles, most evident in spring and 0 to 30 cm layers. The acetate pathway accounted for 55% in wetland vs. 70% in the cropland of all functional genes for CH4 production; while the other three pathways were stronger in response to cultivation, which presented as stronger suppressions in both abundance of functional genes  and their percentages in four pathways . The structural equation models showed that substrate availability was most correlated with CH4 production potential in the wetland, while the positive correlations of acetate, CO2, and methylamine pathways with CH4 production potential were significant in the cropland. The quantitative responses of four CH4 production pathways to land conversion reported in this study provide benchmark information for validating the CH4 model in simulating CH4 cycling under land use and land cover change.Wetland conversion to cropland substantially suppresses methane (CH To achieve these, we quantify the relative abundance of functional genes involved in four methanogenic pathways based on metagenomes across four seasons and soil profiles. The controls of soil edaphic and micrometeorological characters were also determined. We expect that (1) all four pathways would be inhibited after cultivation but to different extents; (2) cultivation would enhance the contribution of the acetate pathway rather than the CO2 in area  Merr.] was cultivated in cropland from May to October every year without any fertilization and stand litter removal  in Tongjiang City, northeastern China. Sanjiang Plain is the largest freshwater wetland in China, with ~10,560 kmSoil cores were collected from natural wetland and cultivated cropland in autumn (10–12 October 2019), winter (27–29 December 2019), spring (13–15 May 2020), and summer (19–21 July 2020), respectively. Three replications in each ecosystem, and we totally have 24 soil cores. Every soil core was of depth 100 cm and divided into 10 sections . A total of 240 samples were packed in polyethylene bags immediately and brought back to the laboratory with ice packs within 24 h for further analysis.4 production rates were determined by lab incubation. About 20 g of dry weight soil and 100 ml ddH2O were put into a bottle. Then, the bottle was flushed with nitrogen gas for 5 min to keep the anaerobic condition. Gas was extracted immediately when the bottle was sealed using injectors with a three-way valve on the lid. Every soil sample has three replications. Then, all the bottles were incubated at 30°C for 5 h. After the incubations, gas was extracted again using injectors with three-way valves. The CH4 concentration was measured by chromatography . The changes in CH4 concentration, with normalized to dry weight soil, represent CH4 production potential. Local CH4 flux includes measurement by the chamber in our experimental sites  AND (Reclamation OR Culti* OR Land-use-change* OR conversion* OR transition*) AND (methane* OR CH* OR mcrA OR pmoA).” A total of 876 papers matched the keywords. The criteria for selecting proper studies were as follows: (1) both pristine wetland and conversed farmland were included in the same study area within the same measurement time; and (2) the variables refer to CH4 flux. A total of 438 pairs of observed variables taken from 23 publications were regarded as global CH4 flux.Potential CHtes Hao, . The gloSoil edaphic characters, including soil total carbon (T.C.), total nitrogen (T.N.), total phosphorus (T.P.), and total sulfur (T.S.), were measured using high-temperature combustion and heating digestion methods, respectively . We extracted 214 DNA samples, and 26 soil samples failed to extract DNA. Metagenomic libraries were constructed using NovaSeq Reagent Kits from all samples with ~400 bp insert sizes. Sequencing 150 bp reads were performed on Illumina NovaSeq 6000  platform at Majorbio Bio-Pharm Technology Co., Ltd. . Totally 1.6 billion paired-end reads were produced, resulting in an average of 50 million reads per sample.DNA was extracted according to the protocol using fastDNAhttps://github.com/OpenGene/fastp, version 0.20.0)   . The CD-HIT  was used to construct a non-redundant gene catalog. Then mapped high-quality reads to the non-redundant gene catalog with 95% identity using SOAPaligner   for taxonomic annotations. The genes annotations were conducted using Diamond  with an e-value cutoff of 1e−5.After removing adaptor sequences and trimming, high-quality reads were obtained by removing low-quality reads, including reads with N bases, length < 50 bp, and quality < 20 using fastp (2/CO2 pathway (M00567) (shorted as CO2 pathway in the following text), methanol pathway (M00356), and methylamines pathway (M00563), according to KEGG database . Four pathway modules of methanogenesis were identified: acetate pathway (M00357), Hdatabase . The abudatabase . All dat4 fluxes were added 10 before log-transformation and calculation to avoid negative values. Differences between natural wetland and cultivated cropland were determined with the Kruskal–Wallis test. Dunn's test was used for multiple comparisons and the P-value adjustments method was “Bonferroni.” The linear regression analysis between percentages of four pathways and micrometeorological and edaphic characters was performed using a package of lme4 in R version 4.0.2.The CH4 production potential. Before SEM analysis, the bivariate relationships between all variables with simple linear regressions were checked to ensure the appropriateness of the linear models. The adequacy of the model was determined by the χ2 test, root mean square errors of approximation (RMSEA), and Goodness-of-fitness index (GFI). All data used in SEM were normalized. Biomass is referred MBC, MBN, MBP, MBS, and MBC/MBN ratio. The SEM was performed by a package of “lavaan” in R version 4.0.2.Structural equation modeling (SEM) analysis was employed to identify the direct and indirect influence of micrometeorological and edaphic characteristics, biomass, and functional genes on CH4 flux on global and local scales and production potential were estimated. Our results showed that CH4 flux and production potential were suppressed in cultivated cropland  decreased after cultivation from CH4 emission (6.75 mg C m2 h−1) to slight uptake (-0.023 mg C m2 h−1)  . CH4 procropland . Moreovecropland .p < 0.05, 2 (M00567), methanol (M00356), and methylamines pathways (M00563) decreased by about 19, 52, 68, and 62% in cropland, respectively . Community structures of methanogens were significantly distinguished in wetland and cropland , across seasons  and along layers .The effects of cultivation on methanogenic composition and community structures were also significant . Eight o4 production, the percentages of four pathways were calculated and found a transition of CO2, methanol, and methylamines pathways to the acetate pathway after cultivation . The dominant path was acetate in the wetland, which accounted for 55% of total methanogenic gene abundance . The CO2 pathway accounted for 20%, the methanol pathway accounted for 8%, and the methylamines pathway accounted 15%. In cropland, the percentage of acetate pathway increased to 70%, while CO2, methanol, and methylamines pathways decreased to 15, 6, and 9%, respectively . Besides, the promotion of acetate percentage and the inhabitation of other three percentages in cultivated cropland were more remarkable in spring and summer than that in autumn and winter , with the highest in spring and lowest in autumn (2 pathway varied insignificantly across seasons in cropland (p = 0.60). Abundances of acetate, methanol, and methylamines pathways significantly changed across four seasons in cropland (p < 0.05), with the highest in winter and lowest in autumn for acetate pathway, while highest in autumn and lowest in spring for methanol and methylamines pathways . At the same time, the fluctuations in the abundance of CO2 and methanol pathways along soil profiles were insignificant . In cropland, acetate and CO2 paths were remarkably different along with soil profiles (p < 0.05), with the highest at 0–10 cm and 40–50 cm, while the lowest at 50–60 and 0–10 cm, respectively . The micrometeorological character indirectly affected CH4 production potential through a strong positive effect on biomass and edaphic characters . Biomass, edaphic, and micrometeorological characters related to four pathways without altered CH4 production potential directly , while acetate and methylamines pathways positively correlated with CH4 production potential . In non-summer seasons, CH4 production potential was significantly related with biomass  and indirectly related with meteorological and edaphic characters in the wetland  and CO2 pathway genes , as well as negatively correlated with edaphic character  (SEMs showed that different characters correlated with CHn-summer . In the directly . In the  wetland . However < 0.05) .2 = 0.25 in total and R2 = 0.10 in wetland, p < 0.05), while decreased with edaphic characteristics . However, the proportions of methanol and methylamines pathways increased with edaphic factors  but decreased with meteorological characteristics . The proportion of CO2 pathway declined with both edaphic  and meteorological characteristics .The regression of four pathway percentages with edaphic and meteorological characteristics showed that the relation of acetate and the other three pathway percentages were opposite . The ace4 flux and production potential  can be found at: NCBI—PRJNA853804.CS and XX conceived the project. XZ, YZ, and CG carried out soil sampling. XZ carried out lab experiments. NW carried out data analysis and interpretation with all other authors. NW and XX wrote the manuscript with assistance from other co-authors. All authors contributed to the article and approved the submitted version.This study was partially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA28020502), the Top Notch program in China, the National Key R&D Program (2016YFA0602303), the National Natural Science Foundation  of China, Ecology Innovation Team (2020CXTD02) in Minzu University of China, and Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. This study was partially supported by the National Natural Science Foundation of China (No. 32171873).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Panax notoginseng saponins (PNS) have been reported to have good anti-inflammatory effects. However, the anti-inflammatory effect mechanism in rheumatoid arthritis (RA) remains unknown. The focus of this research was to investigate the molecular mechanism of PNS in the treatment of RA. The primary active components of PNS were tested utilizing the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and Analysis Platform based on oral bioavailability and drug-likeness. The target databases for knee osteoarthritis were created using GeneCards and Online Mendelian Inheritance in Man (OMIM). The visual interactive network structure ‘active component – action target – illness’ was created using Cytoscape software. A protein interaction network was built, and associated protein interactions were analyzed using the STRING database. The key targets were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) biological process enrichment analyses. The effects of PNS on cell growth were studied in human umbilical vein endothelial cells (HUVECs) treated with various doses of PNS, and the optimum concentration of PNS was identified. PNS was studied for its implication on angiogenesis and migration. The active components of PNS had 114 common targets, including cell metabolism and apoptosis, according to the network analysis. The therapeutic effects of the PNS components were suggested to be mediated through apoptotic and cytokine signaling pathways. In vitro, PNS therapy boosted HUVEC proliferation. Wound healing, Boyden chamber and tube formation tests suggested that PNS may increase HUVEC activity and capillary-like tube branching. This study clarified that for the treatment of RA, PNS has multisystem, multicomponent, and multitargeted properties.  PNS has multisystem, multicomponent, and multitargeted properties in the treatment of RA.PNS might increase the angiogenic impact of HUVECs to treat RA.PNS might activate apoptotic and cytokine signaling pathways to treat RA.1.Rheumatoid arthritis (RA) is an inflammatory illness that causes persistent synovitis as well as cartilage and bone deterioration over time. This persistent inflammation ultimately results in synovial hyperplasia, joint deformity, and functional impairment in the joints . The worPanax notoginseng reduces blood stasis, prevents bleeding, improves blood circulation, and relieves pain  × 100% [Cells were seeded at a density of 1.0 × 10] × 100% .2.7.52 for 30 minutes to allow the Matrigel to solidify. Then, 1.5 × 104 cells were introduced to the incubator for another 8 hours. To examine tube development, cells were photographed using an inverted microscope (100×). At least 5 different micrographs were obtained at various locations for each sample. The number of network circles in each image was used to evaluate the ability of the HUVECs to create tube-like structures [Matrigel  was melted into a liquid at 4°C overnight the day before the test. A 96-well plate was covered with 40 μl of Matrigel the next morning at 4°C for 30 minutes. The plate was then heated at 37°C in a humidified incubator with 5% COructures .2.8.GraphPad Prism 8.0 software was utilized for statistical analysis, and all data are displayed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and nonparametric tests were used to compare data across groups, followed by the Shapiro–Wilk normality test. Statistical significance was defined as a P value of less than 0.05.3.The purpose of this study was to explore the pharmacological mechanisms of PNS acting on RA using pharmacology and experimental validation. This approach consisted of three parts. The effective active components and prospective targets of PNS were first investigated. Moreover, appropriate RA targets were found using GeneCards and the OMIM databases. Second, the target genes of PNS therapy for RA were assessed, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Finally, experimental evidence revealed that PNS increased the angiogenic impact of HUVECs, including proliferation, migration, and differentiation into capillary-like structures. This technique may provide data to support the clinical use of PNS for the treatment of RA, as well as aid in PNS drug screening and serve as a good reference for the investigation of the mechanisms of other traditional Chinese medicine therapeutics.3.1.After screening the TCMSP database, we found 119 active PNS components, but only 8 of them were shown to be effective. However, one of the active ingredients, ginsenoside f2, had no related targets, so there were only 7 active ingredients. We deleted 111 unrelated active compounds from the list (Supplementary Text 1).3.2.There were 716 PNS targets in the TCMSP database. We collected the gene names of the targets from the UniProt database, deleted the invalid and duplicate targets, and ultimately identified 167 active substances. A total of 2859 RA-related gene targets were discovered after searching and combining the major databases (Supplementary Text 2). We found 114 common target genes by mapping the 167 chemical targets to 2859 RA-related target genes .Figure 3.3.As shown in 3.4.We submitted our information to the STRING database to determine how the overlapping genes interact and then built a PPI network with 114 nodes and 351 edges . In Figu3.5.ClusterProfiler was used to find the enrichment of the GO terms BP, MF, and CC. We used a p < 0.05 criterion to select the top 10 GO analysis findings . In the ClusterProfiler was utilized to conduct KEGG pathway enrichment analysis with pvalueCutoff = 0.05 to understand the functions of the PNS therapy targets in the signaling pathways. 3.6.3.6.1Different PNS concentrations increased the proliferation of HUVECs relative to the control group at every time point. Specifically, the 6.25 mg/L dose was shown to be the most effective for promoting cell growth . As a re3.6.2The ability of endothelial cells to migrate is crucial for angiogenesis. Boyden chamber and scratch wound experiments were used to determine the effects of PNS on HUVEC mobility . When co3.6.3The tube formation assay was used to assess the impact of PNS on HUVEC vessel-forming capacities. HUVECs were seeded onto Matrigel and treated with either 6.25 mg/L PNS or DMEM. Compared to DMEM-treated control cells, PNS-treated cells developed more capillary-like networks . Quantit4.In this research, a network pharmacology method was used in conjunction with experimental validation to determine the underlying mechanism of action of PNS against RA. We discovered 114 target genes and 8 active chemicals in this study by combining the active ingredient targets of PNS with the goal of RA therapy. Quercetin has been found to have an anti-inflammatory actions in these 8 active chemicals. Quercetin reduces inflammation in RA mice by reducing neutrophil activity, resulting in a therapeutic impact on the disease . Other PThe component–target gene network, on the other hand, demonstrates that these active substances have a similar target, indicating that they may have synergistic benefits in the treatment of RA. PNS can act on PTGS2 as one of the hypothesized targets for RA therapy. There are six active chemicals in PNS that can act on PTGS2. It has been shown that PNS may selectively inhibit COX-2 and hence aid in the treatment of RA. This indicates that numerous active compounds in TCM may work together to cure illnesses by acting on a single target . In termFor these target genes, we next performed GO and KEGG enrichment analyses. The apoptotic signaling route, cytokine-mediated signaling pathway, and other pathways closely linked to the incidence and progression of RA were discovered to be the most important signaling pathways associated with RA. This suggests that PNS may include many active components that work together to prevent and cure RA via different mechanisms. For example, several apoptotic signaling pathways are involved during the course of RA. Following the US FDA approval of tofacitinib, a JAK3-selective small molecule inhibitor (SMI), for the medical treatment of RA, the critical role of JAK/STAT pathway activation in RA was further confirmed. The Janus family of kinases (JAKs), which includes JAK1, JAK2, JAK3, and TYK2, are nonreceptor protein tyrosine kinases. The production of aberrant hematopoietic stem cell development, hematological malignancies, autoimmunity, and some immunodeficiency disorders has been demonstrated to require abnormal activation of JAK/STAT signaling by JAK mutations or constitutive TYK2 signaling . InhibitPNS was shown to increase angiogenesis in vitro in the current investigation. PNS improved various aspects of angiogenesis, including endothelial cell proliferation, migration, and differentiation into capillary-like structures. In the CCK8 assay, 6.25 mg/L PNS was most beneficial for cell proliferation at 48 h, indicating that PNS had no toxic effect on HUVECs, and 6.25 mg/L PNS also performed better in all three subsequent experiments compared to the control group. A previous study reported that PNS and its main constituents Rg1 and R1 significantly enhanced the migration of endothelial cells and angiogenesis in response to myocardial infarction injury . AnotherThere were several limitations to this investigation. First, several additional validated trials must be carried out. Furthermore, there are no in vivo experimental data in this work. Therapeutic research and development need a thorough knowledge of the primary gene targets of PNS and RA.5.A network pharmacology approach was used in conjunction with experimental validation in this research to elucidate the underlying mechanism of action of PNS in the treatment of rheumatoid arthritis. These results suggest that the primary active chemicals of PNS, their fundamental targets, and pathways may provide fresh insights into the development of natural herb-related therapies for rheumatoid arthritis. Additionally, we experimentally demonstrated that PNS alleviates rheumatoid arthritis by boosting the angiogenic impact. The current study establishes evidence for the therapeutic application of PNS in the treatment of rheumatoid arthritis.Click here for additional data file.


However, the potential mechanisms of its anticataract effects have not been fully explored. In this study, network pharmacology was used to investigate the potential mechanism underlying the actions of CS against cataracts, and molecular docking was performed to analyze the binding activity of proteins and compounds. qPCR was performed to detect the mRNA level of genes, and the cell apoptotic rate was measured using flow cytometry. We identified 13 active compounds from CS and 105 targets, as well as 238 cataract-related targets. PPI networks were constructed, and fifty key targets were obtained. These key targets were enriched in the regulation of transcription, apoptotic process, and signal transduction pathways. Molecular docking demonstrated that the compounds of CS exhibited good affinity to some critical targets. Furthermore, CS prevented the apoptosis of human lens epithelial cells induced by UVB lights by decreasing the gene expression of CASP3, ESR1, and TP53 and increasing the CRYAB gene expression. The present study attempted to explain the mechanisms for the effects of CS in the prevention and treatment of cataracts and provided an effective strategy to investigate active ingredients from natural medicines. Further studies are required to verify these findings via  Detailed information is presented in p < 0.05) were identified, and the top 20 pathways mainly contained cancer-related pathways, signal transduction pathways, and virus-related pathways (The compound-target network consisted of 117 nodes (12 active compounds and 105 targets) and 152 edges, as shown in pathways .p < 0.05), with 281 in the biological process, 43 in the cellular component, and 49 in the molecular function. In addition, a total of 67 pathways (p < 0.05) were affected by cataracts, and the top 20 enriched pathways are shown in A total of 238 target genes related to cataracts were identified from the OMIM (48), MalaCards (8), and GeneCards (232), after removing the duplicates. The PPI network (removing nodes without any connection) of these targets was constructed , which iIn addition, module analysis obtained a cluster of 6 targets with score = 5.60 from the PPI network of cataract target genes . EnrichmWe generated the PPI network of potential anticataract targets of CS, as shown in α-bisabolol, SB203580, and HDM201 were selected as inhibitors of ESR1, AKR1B1, CASP3, MAPK14, and TP53, respectively. The top 5 cavity sizes and Vina scores of each compound-target or inhibitor-target docking were obtained from CB-Dock. It is generally believed that the lower Vina score indicates a more stable binding state between a protein and a compound. In addition, if a cavity size is close to or bigger than the ligand, the accuracy of docking tends to increase [Five important targets  from the network pharmacology analysis were selected to perform molecular docking analysis with their target compounds. Fulvestrant, (-)-kusunokinin, increase . MoleculExcessive apoptosis of lens epithelial cells is implicated in the pathogenesis of several types of cataract formation. Herein, we detected the effect of CS on the mRNA expression of several hub genes located in the central network and the apoptosis of HLEC. The results demonstrated that UVB induced the upregulation of CASP3, TP53, CRYAB, and ESR1  and HLECCataracts, the major cause of blindness, are characterized by blurry vision. It has been reported to be associated with various risk factors, including smoking, hypertension, steroid consumption, diabetes, and ionizing radiation , 34. CS In this study, we identified 13 active compounds in CS and 105 potential targets of these active compounds in total, and 238 cataract-related targets were also obtained from the three public databases. Four genes, including ESR1, MAPK14, CASP3, and AKR1B1, were shared between CS compound targets and cataracts' targets, indicating their possible anticataract action. Central network analysis obtained a central network with 50 key targets, which significantly enriched the pathways correlated with cataracts, such as the thyroid hormone signaling pathway, PI3K-Akt signaling pathway, and MAPK signaling pathway. The potential mechanisms of CS against cataracts were for the first time comprehensively investigated in the present study, which laid a theoretical foundation for the clinical application of CS in the treatment of cataracts and further research.α/VEGF signaling pathway and has the potential to be developed for the prevention and treatment of diabetic retinopathy [Among the active compounds in CS, the top three active ingredients with the most targets were aloe emodin, stigmasterol, and rhein, indicating their potential role in the treatment of cataracts. Aloe emodin is an anthraquinone derivative, which possesses the antiangiogenic effect on laser-induced choroidal neovascularization by inhibiting the HIF-1inopathy . In addiinopathy . Stigmasinopathy . It was inopathy , 38. Rheinopathy –41. Oxidinopathy , 43, andα overexpression has previously been reported in lens epithelial cells [αB-crystallin (CRYAB) may play an important role in the treatment of cataracts. It is a chaperone that maintains protein stability and preserves lens transparency [Network analysis suggested that four shared targets may play crucial roles in the treatment of cataracts, including aldose reductase (AKR1B1), caspase-3 (CASP3), mitogen-activated protein kinase 14 (MAPK14), and estrogen receptor (ESR1). AKR1B1, an NADPH-dependent aldo-keto reductase, is involved in diabetic cataracts and retinopathy . A previal cells , indicatal cells . Module sparency , 52 by psparency . The docAs demonstrated in network pharmacology analysis, the hub genes were enriched in the apoptosis process. Meanwhile, the apoptosis of lens epithelial cells contributes to cataract development. Therefore, we investigated the impacts of CS on the apoptosis of human lens epithelial cells. As expected, CS treatment could reduce the UVB-induced elevated apoptosis rate of HLECs. Several apoptosis-related genes were also regulated by CS, including CASP-3, TP53, ESR1, and CRYAB, indicating that CS may prevent HLEC apoptosis via regulating these hub genes. Although the gene expression of MAPK14 and AKR1B1 was not affected by CS treatment, the activities of these proteins required further validation to identify their roles in the treatment of cataracts by CS.In addition, the PPI data of compound targets and cataract-related targets were obtained to construct the PPI network. Enrichment analysis of these two sets of targets revealed a series of shared pathways, such as the PI3K-Akt signaling pathway, MAPK signaling pathway, and FoxO signal pathway. To obtain the central network of CS anticataract targets, we merged the PPI network of compound target and cataract-related targets. KEGG pathway enrichment analysis showed that the key targets of CS against cataracts were mainly enriched in the thyroid hormone signaling pathway, MAPK signaling pathway, and PI3K-Akt signaling pathway, indicating the involvement of these pathways in the treatment of cataracts.de novo synthesis of GSH. However, Kosano et al. demonstrated that thyroxine treatment accelerated the GSH-GSSG cycle rather than de novo synthesis of GSH to maintain a certain level of hepatic GSH necessary for reducing elevated LPO [The thyroid hormone signaling pathway participates in the regulation of growth, development, and glucose metabolism. The modulation of glycolysis and carbon flux reprogramming can increase the glutathione (GSH) syntheses and activate the antioxidant enzymes , which aated LPO .γ-Klotho gene [The MAPK signaling pathway is another enriched pathway for CS in the treatment of cataracts, which involves various cellular functions, including cell proliferation, differentiation, and migration. Hashida et al. found the association of cataract formation with the upregulation of MAPK cascade protein . In additho gene . Androgrtho gene . Peng ettho gene . TherefoNotably, the PI3K-Akt signaling pathway might be associated with the ingredients of CS and anticataract activity. It has been demonstrated that the PI3K-Akt signaling pathway is involved in the pathogenesis of cataracts , 63. MeaIn conclusion, this study used a network pharmacology approach to explore the potential mechanisms of CS acting on cataracts. Key targets and pathways involved in the treatment of cataracts using CS were identified, which provided evidence for the clinical application of CS in cataract treatment and further studies. CS treatment regulated the gene expression of several hub genes in HLEC and prevented the apoptosis of HLEC, which may contribute to the cataract treatment. However, from a critical point of view, further experiments are required to validate other findings. This study also provided clues to evaluate the synergy of herbs in the treatment of other complex diseases.