The problems caused by antibiotic abuse have swept the world, and the Chinese government has responded to calls for a comprehensive ban on antibiotics. However, not using antibiotics also challenges China’s existing livestock industry. Based on this, we carried out a nontargeted metabolomics analysis of the jejunum, ileum, and cecum of diarrhea rabbits and normal rabbits fed with antibiotic-free diets, respectively, to find out the mechanism of action of each intestinal segment group and between different intestinal segments. The screened differential metabolites were mostly related to intestinal barrier, intestinal inflammation, and autophagy after a KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. In this paper, we analyzed the metabolic pathways that were significantly different between different intestinal segments and illustrated the mechanism and potential connections of the screened differential metabolites in different intestinal segments in the form of charts.For many years, antibiotics in feed have been an effective and economical means to promote growth and disease resistance in livestock production. However, the rampant abuse of antibiotics has also brought very serious harm to human health and the environment. Therefore, the Chinese government promulgated laws and regulations on 1 July 2020, to prohibit the use of antibiotics in feed. To improve the effect of antibiotic-free feeding on China’s existing rabbit industry, we used the nontargeted metabolomics method to detect the differences between diarrhea rabbits (Dia) and normal rabbits (Con) on an antibiotic-free diet. A total of 1902 different metabolites were identified. A KEGG analysis showed that in the cecum, metabolites were mainly concentrated in bile secretion, antifolate resistance, aldosterone synthesis, and secretion pathways. The ileal metabolites were mainly concentrated in tyrosine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, steroid hormone biosynthesis, alanine, aspartate, and glutamate metabolism. The metabolites in the jejunum were mainly rich in panquinone and other terpenoid compound quinone biosynthesis, AMPK (adenosine 5′-monophosphate (AMP)-activated protein kinase) signal, inositol phosphate metabolism, and pentose phosphate pathway. After a deep excavation of the discovered differential metabolites and metabolic pathways with large differences between groups, it was found that these metabolic pathways mainly involved intestinal inflammation, intestinal barrier, and autophagy. The results showed that panquinone and other terpenoids could increase AMPK activity to promote cell metabolism and autophagy, thus trying to prevent inflammation and alleviate intestinal disease symptoms. In addition, we discussed the possible reasons for the changes in the levels of seven intestinal endogenous metabolites in rabbits in the diarrhea group. The possibility of improving diarrhea by adding amino acids to feed was discussed. In addition, the intermediate products produced by the pentose phosphate pathway and coenzyme Q had a positive effect on steroid hormone biosynthesis to combat intestinal inflammation. Animal husbandry is so dependent on antibiotics because antibiotics can prevent animal diseases ,2, promoIn this study, we used metabolomics to determine the type and quantity of different metabolites in the intestine of diarrhea rabbits and normal rabbits. The differential metabolites were analyzed using the KEGG and the metabolic pathways with large differences between groups were screened out. The aim was to clarify the metabolites and metabolic pathways associated with intestinal inflammation and intestinal barrier function by analyzing and comparing the relationship between metabolic pathways in the same and different intestinal segments. It provides new ideas and a theoretical basis for the prevention and treatment of gastrointestinal diseases in rabbits in production practice.This study was approved and implemented according to the ethical standards of the Animal Feeding and Use Committee of the School of Animal Science and Technology, Sichuan Agricultural University, Sichuan Province, 611130.This experiment took the Tianfu black rabbit as the experimental object. All the Tianfu black rabbits were raised on the Leibo rabbit farm. There were 105 breeding male rabbits and 768 breeding female rabbits at the Leibo rabbit farm. According to the conventional feeding and management methods at the rabbit farm, each rabbit was reared in a clean rabbit cage (600 × 600 × 500 mm) and was free to drink water. In January 2021, the rabbit farm began to use an antibiotic-free diet to feed rabbits in response to the ban. Subsequently, typical diarrhea symptoms such as deformity, watery stool, and sepsis occurred.Six rabbits with typical diarrhea symptoms and six without diarrhea symptoms and similar body weight (845 g ± 20) were selected from 200 35-day-old female rabbits. The detailed procedure for collecting rabbit intestinal contents followed previous report by Tao et al. . The jejParaffin sections were dewaxed to water, hematoxylin staining nucleus, eosin staining cytoplasm, and dehydration sealing. The histopathological changes of the whole tissue biopsy were examined under a microscope , and the normal microscopic imaging system area and obvious pathological changes were recorded.We used a Vanquish ultrahigh-pressure liquid chromatography (UHPLC) system  and Novogene Co., Ltd.  Orbitrap Q Exactive TM HF mass spectrometer  for the UHPLC-MS/MS analysis. Gut samples’ injection was done using a Hypersil hypostyle column , a linear gradient of 17 min, and a flow rate of 0.2 mL/min . Orbitrahttps://www.mzcloud.org/ (accessed on 9 November 2020)), mzVault, and MassList databases in order to obtain relatively accurate qualitative and quantitative results. We used statistical software tools R (R version rule 3.4.3) and Python (Python version 2.7.6) on CentOS (CentOS version 6.6). When chromatographic data did not have a normal distribution, the area normalization method was used to convert them to a normal distribution.We used compound found 3.1  of the UHPLC–MS/MS system for processing the raw data files, for each metabolite in peak alignment, peak picking, and quantitatively. The main parameters were as follows: 0.2 m retention time tolerance; actual quality of tolerance, 5 PPM ; signal The staining of intestinal tissues showed a coagulative necrosis of the jejunum mucosa in the Dia group and neat cells in the Con group. The number of lymphocytes in ileal lymph tissue in the Dia group decreased, and the number of lymphocytes in the Con group was normal. We observed a full-thickness coagulative necrosis of the cecum wall. On the contrary, the small intestine of the Con group was intact without pathological changes .The correlation analysis of quality control samples showed that the correlation coefficient between the intestinal tissue metabolites data of quality control samples was close to 1. These results showed that the PLS-DA anion and cation model had a good prediction ability, and there was no overfitting .p values < 0.05. In total, 652 different kinds of metabolites were found in the appendix (485 kinds for the positive ion mode and 167 for the negative ion mode), 619 species in the ileum (399 in the positive ion mode and 220 in the negative ion mode), 631 species in the jejunum .The metabolic analysis system  determinDiarrhea is one of the clinical manifestations of intestinal diseases in rabbits . In thisThere were significant differences between groups in the jejunum anion mode, inositol phosphate metabolism, and pentose phosphate pathway. D-erythrose 4 phosphate, D-sedoheptulose 7 phosphate and glyceraldehyde 3 phosphate were detected in the pentose phosphate pathway, which were all upregulated compared with the control group. It indicated that the activity of the pentose phosphate pathway in diarrhea rabbits increased. One possible reason is that inflammatory mediators mediate metabolic disorders . During In the jejunum cation mode, the AMPK signaling pathway and biosynthesis of panquinone and other terpenoid compounds such as quinone were significantly different between groups, and there was a regulatory role between them. AMPK is a key protein involved in multiple signaling pathways. Terpene compounds have the effect of activating the AMPK signaling pathway ,25. OnceIn the ileum anion mode, compared with the control group, steroid hormone biosynthesis in diarrhea rabbits increased. We tried to find the potential relationship between the metabolites screened in this experiment and the increase of steroid hormone biosynthesis. We found that it may be affected by the pentose phosphate pathway  and coenWhether coenzyme Q can also regulate steroid hormones except testosterone remains to be studied. On the study of the inhibitory effect of steroid hormone synthesis on inflammation, Merk et al. reviewed the role of epithelial barrier as an alternative pathway for local and systemic synthesis . Their rListeria monocytogenes infection. This provides new insights and ideas for amino acids involved in immune response. In recent years, studies on the effects of amino acids on intestinal health and the function of mammals have explored the effects of glutamine, arginine, glutamic acid, tryptophan, methionine, and lysine [The repair of intestinal epithelial barrier damage caused by intestinal inflammation requires a lot of amino acid decomposition and synthesis . A totald lysine ,52,53. HIt was found that compared with the control group, the expression levels of the inositol phosphonic acid metabolism, phosphopentose pathway, steroid hormone synthesis, panquinone and other terpene quinone biosynthesis, and AMPK signaling pathway in the diarrhea group were increased. Intestinal inflammation led to a redistribution of energy. The metabolic level of damaged parts increased, indicating that the expression levels of the inositol phosphate metabolism and phosphopentose pathway were significantly upregulated. A large amount of NADPH generated by the enhancement of the pentose phosphate pathway provided a reducing agent for the synthesis of steroid hormones. Under the combined action of panquinone and NADPH, the synthesis of steroid hormones increased, thereby exercising its function of inhibiting inflammation. In addition, the difference between groups for the AMPK signaling pathway was large, and the detected levels of the three metabolites were upregulated. In this study, the synthesis of terpene compounds increased, thereby activating the AMPK signaling pathway. This study also found that the levels of NAD + in the three intestines decreased, and an excessive downregulation of NAD + could lead to the imbalance of autophagy. This may be due to the dysfunction of autophagy caused by the combined action of the AMPK and mTOR signaling pathways, thereby causing intestinal inflammation.The metabolic levels of 14 amino acids were affected. In addition to confirming their relationship with intestinal health in other species, tyrosine and histidine were found in diarrhea rabbits for the first time. Whether these two will have an impact on the intestinal health of rabbits and the specific mechanism of action are our next focus of research.


The aim of this study is to explore pathological mechanisms of bone fragility in type 2 diabetes mellitus (T2DM) patients.  Identifying common genes for T2DM and osteoporosis by taking the intersection is shared by the Comparative Toxicogenomics Database (CTD), DISEASES, and GeneCards databases. The differentially expressed genes (DEGs) and the differentially expressed miRNAs (DEMs) were identified by analyzing the Gene Expression Omnibus (GEO) datasets . FunRich and miRNet were applied to predict potential upstream transcription factors and downstream target genes of candidate DEMs, respectively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore potential mechanisms using Metascape. Eventually, a miRNA-gene network was constructed by Cytoscape software.  271 common targets and 35 common DEGs between T2DM and osteoporosis were screened out in the above databases, and a total of ten DEMs were obtained in the GSE70318. SP1 was predicted to potentially regulate most of the DEMs. Enrichment analysis showed the PI3K-Akt signaling pathway and AGE-RAGE signaling pathway in diabetic complications may play an important role in diabetic skeletal fragility. Two genes (NAMPT and IGFBP5) were considered as key genes involving in the development of diabetic osteoporosis. Through the construction of the miRNA-gene network, most of the hub genes were found to be potentially modulated by miR-96-5p and miR-7-5p.  The study uncovered several important genes, miRNAs, and pathological mechanisms involved in diabetic skeletal fragility, among which the PI3K-Akt signaling pathway and AGE-RAGE signaling pathway in diabetic complications may play important roles. Type 2 diabetes mellitus (T2DM) is a common endocrine metabolic disease in humans and accounts for more than 90% of all patients with diabetes . Accumulβ-catenin pathway has been shown to negatively regulate bone formation in T2DM patients [β-catenin pathway. Studies showed that patients with T2DM have higher serum levels of sclerostin, which can bind to Wnt coreceptors, inhibiting osteoblastogenesis and bone formation [Several factors including obesity, hyperinsulinemia, hyperglycemia, accumulation of advanced glycosylation end products (AGEs), and presence of microvascular disease were considered being involving the pathogenesis of diabetic skeletal fragility . Obesitypatients . Sclerosormation , 17. TheIn this study, we explored the common pathogenesis between T2DM and osteoporosis by combining the multisource T2DM and osteoporosis-related data. Simultaneously, we constructed a miRNA-gene network to identify some potential miRNAs and genes involved in T2DM osteoporotic fracture. Our study offered a new approach to identify pathological mechanisms and potential targets for T2DM with osteoporotic fracture. The research workflow is shown in https://ctdbase.org/), GeneCards (/http://www.genecards.org/), and DISEASES (https://diseases.jensenlab.org/) that were downloaded in October 2021.We selected the “type 2 diabetes mellitus” and “osteoporosis” as the keywords.Data in this study were obtained from public databases and gene expression databases. The Comparative Toxicogenomics Database (CTD) is a publicly available database which provides information about interactions between environmental chemicals and gene products and their effect on human diseases . The Genhttps://www.bioinformatics.com.cn), a free online platform for data analysis and visualization. All these genes were considered to play an important role in the common pathogenesis between T2DM and osteoporosis and were extracted for further analysis.The common disease-related genes between T2DM and osteoporosis from these three databases were obtained using Venn diagram, which was drawn by the website (https://metascape.org/), an excellent integrated analytics platform that combines functional enrichment, interactome analysis, and gene annotation within one integrated porta, was used to perform the functional analysis. The reviewed items include the Kyoto Encyclopedia of Genes and Genomes (KEGG), GO cellular component, GO biological process, and GO molecular function. Min overlap = 3 and Min enrichment = 1.5 were the screening conditions. P < 0.01 was considered statistically significant.The Metascape  (https:/https://string-db.org). An interaction with a corresponding combined score was selected and used to construct a PPI network with Cytoscape software. Cytoscape (version 3.7.2) is an open-source software that creates and surveys the molecular interaction network [The protein-protein interaction (PPI) network was analyzed using the String database ( network .http://www.ncbi.nlm.nih.gov/geo/) by searching the “type 2 diabetes mellitus” and “osteoporosis” terms. Eventually, the three microarray datasets (GSE35958 [n = 19) and T2DM with osteoporotic fractures was selected as the experimental group (n = 19). The platforms of the GSE35958, GSE43950, and GSE70318 were GPL570, GPL10379, and GPL20631, respectively.Gene expression profiles were collected from the Gene Expression Omnibus  were doP value < 0.05 were considered to indicate statistical significance. To show the differential expression of DEMs and DEGs in different samples, the plot and heat map packages in the R studio were applied to draw the volcano map and heat map.GEO2R, an interactive web tool, was used to screen out differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between the experimental group and control group. |logFC| > 1 and the adjusted https://www.mirnet.ca/), a comprehensive atlas of miRNA-target interactions that contains information about miRNA-target interactions resulting from the existing miRNA-target prediction programs  and displays the association in a visual network, was applied to predict the potential target genes of candidate DEMs.To identify the regulatory mechanisms and functions of miRNAs, the miRNet (http://www.funrich.org/), an independent software tool mainly for functional enrichment and interaction network analysis of genes and proteins, was used to predict the potential upstream transcription factors. The P value < 0.05 was considered statistically significant.FunRich   was usedReceiver operating characteristic (ROC) curve analysis, a commonly used method to determine the performance of diagnostic biomarkers, is widely used in biostatistics. Herein, ROC curves were constructed to discriminate T2DM with the osteoporotic fracture group from the control T2DM group for the serum miRNAs, and the areas under the ROC curves (AUCs) were analyzed to measure the diagnostic accuracy of each identified miRNA in the miRNA-gene interaction network. The ROC plot was calculated and visualized using GraphPad Prism v5.0, and the AUC value was used to evaluate the ROC curve, with values between 0 (lowest) and 1 (highest) performance.To integrate the disease-related biological data, T2DM-related genes and osteoporosis-related genes available in the GeneCards, DISEASES, and CTD databases were combined. Finally, 271 genes were determined to be shared among the three resources and the detailed information is listed in Supplemental Table Then, the GO and KEGG analyses on the 271common targets were performed using the Metascape database. P value < 0.05 and |logFC| > 1  were identified by limiting the adjusted  Figures . Among t Figures . In term Figures .P value < 0.05 and |logFC| > 1, ten DEMs were identified in the GSE70318, and they are all downregulated DEMs including miR-550a-5p, miR-500a-5p, miR-181c-3p, miR-96-5p, miR-323a-3p, miR-203a-3p, miR-32-3p, miR-942-3p, miR-7-5p, and miR-16-2-3p. The detailed information about DEMs is shown in Supplemental Table After screening with the threshold of an adjusted Moreover, a total of 1543 genes targeted by these candidate DEMs were predicted by the miRNet database. For better visualization, DEMs with their target genes are displayed in a DEM-target gene network in In the present study, the upstream transcription factors of candidate DEMs were predicted using FunRich software. Eventually, eight upstream transcription factors were considered statistically significant, including SP1, SP4, YY1, EGR1, E2F1, MEF2A, NFYA, and MYC. This result is presented in In order to find the hub genes, a Venn diagram of intersection between the targets of DEMs, 271 common genes, and 35 common DEGs was constructed . It is nTo better investigate the molecular mechanisms of these DEMs in T2DM with osteoporotic fracture, the miRNA-gene network was constructed by Cytoscape software and a total of nine miRNAs and 34 genes were obtained . The miRTo investigate the efficacy of DEMs in the miRNA-gene network as potential biomarkers of osteoporotic fractures with T2DM, we performed ROC curve analysis of these miRNAs. The expression levels of the DEMs were significantly different between experimental and control individuals . AUC valAvailable evidence suggests that osteoporosis and T2DM, two common chronic diseases, may coexist and progressively increase in prevalence and are boosted by aging . The risIn this study, data from the three public databases were extracted to identify the common gene targets between osteoporosis and T2DM. A total of 271 common gene targets were identified to be further analyzed. The result of GO functional analysis showed that the common genes were closely associated with the blood vessel development, response to growth factor, and cellular response to lipid. A previous study reported that chronic hyperglycemia, the main features of diabetic, caused severe impairment in lipid metabolism . VLDL anβ, IL-6, IL-1β, and IL-21, are associated with bone remodeling in diabetes [α (IRE1α), C-Jun n-terminal kinase, etc. These findings have demonstrated that these pathways were associated with bone metabolism in T2DM, which can provide potential directions for the study of the molecular mechanism of T2DM complicated by osteoporosis.The results of KEGG pathway enrichment analysis showed that the common genes were mainly enriched in cytokine-cytokine receptor interaction, T cell differentiation-related pathways, the PI3K-Akt signaling pathway, and the AGE-RAGE signaling pathway in diabetic complications. Emerging evidence has showed that numerous cytokines, including TGFdiabetes , 31. Higdiabetes . In the diabetes  showed tdiabetes . High-gldiabetes . Hypergldiabetes . Moreovediabetes . Higher diabetes . Suzuki diabetes  found thNext, 35 common DEGs between T2DM and osteoporosis were identified by analyzing the GSE35958and GSE43950 datasets. Enrichment analysis results of these DEGs highlighted the role of immune and inflammatory response, which were broadly consistent with the above results. It is noteworthy that the AGE-RAGE signaling pathway in diabetic complications was enriched again, suggesting its importance in diabetic osteoporosis. What is more, a core gene (NAMPT) was found to be shared by multi-datasets. In T2DM, the NAMPT gene codes the protein visfatin, which is critical for beta cell function via mediation of nicotinamide adenine dinucleotide biosynthesis . The sin2H2-type zinc-finger transcription factor [Then, the analysis of the GSE70318 dataset showed that the expression of various miRNAs has experienced some extent of alteration in T2DM patients with osteoporotic fracture compared to T2DM patients. As reported in recent studies, the expression of miRNA can be modulated by transcription factors , 45, andn factor , is the n factor . Followin factor  suggesten factor . These eMoreover, by constructing DEM-gene network, we found that most of hub genes could be potentially targeted by miR-96-5p and miR-7-5p. Prior research demonstrated that miR-96-5p indirectly regulated glutathione levels, a key antioxidant responsible for eliminating the damaging oxidative stress-related reactive oxygen species . HoweverProper recognition of populations at increased fracture risk is indispensable, but the risk of fracture in T2DM individuals tends to be underestimated. At present, researchers generally believe that the pathophysiology of osteoporosis ultimately causing fractures had a heterogeneous etiology with different miRNA expression patterns. Therefore, we performed ROC curve analysis of nine DEMs in the DEM-gene network. Results show that the miR-550a-5p has the highest score of AUC, and it has been verified to inhibit osteogenic differentiation in vitro . AdditioIGFBP5 (insulin-like growth factor binding protein 5) is selected as the key genes in the miRNA-gene network. Insulin-like growth factors (IGFs) have regulatory effects in bone cells, such as regulating cell proliferation, differentiation, and apoptosis, and are controlled by their cognate receptors, IGF-binding proteins (IGFBPs), and IGFBP proteases . It has To our knowledge, few studies have explored the common molecular mechanism between osteoporosis and T2DM by advanced bioinformatics methods. Due to the high rate of osteoporotic fracture in T2DM, we explored the pathological mechanisms of bone fragility in T2DM based on the joint analysis of multi-databases and multi-datasets for the first time, which helped to develop dual-purpose prevention methods. However, there are several limitations to our present study. First of all, the study was performed based only on the serum samples not bone tissue, so studies aimed at bone tissue in T2DM are needed. In addition, the miRNA-gene interactions were only based on predictions from public databases, and further studies with cellular and animal experimental validations are still needed to validate our analysis.In this study, we explored the common pathomechanism between T2DM and osteoporosis and found that the PI3K/AKT signaling pathway and the AGE-RAGE signaling pathway in diabetic complications might play crucial roles in the common pathomechanism. Simultaneously, a miRNA-gene network was constructed and identified some potential miRNAs and genes, which may participate in the pathogenesis of bone fragility in T2DM. We hope that these findings can contribute to deepen our knowledge on the pathologic mechanism of bone fragility in T2DM.


This study systematically explored the mechanism of Rhizoma Coptidis-Eupatorium fortunei in treating type 2 diabetes mellitus (T2DM) by using network pharmacology and molecular docking methods.  The TCMSP database was used to screen out the active ingredients and related targets of Rhizoma Coptidis-Eupatorium fortunei (R-E) drug pair. GeneCards, OMIM, DrugBank, and other databases were used to screen the related targets of T2DM, and then, the UniProt database was used to standardize the relevant targets of T2DM. Then, the Venn analysis was performed on the active ingredient-related targets and disease-related targets of R-E drugs to find the intersection targets. Using the STRING database and Cytoscape software, the PPI network and “drug-active ingredient-target-disease” network are constructed by intersecting targets and corresponding active ingredients. Through the cluster profiler package in the R software, GO function enrichment analysis and KEGG pathway enrichment analysis were carried out on the intersection targets and the screened core targets, and the prediction results were verified by molecular docking.  Taking OB ≥ 30% and DL ≥ 0.18 as the standard, a total of 25 effective active ingredients of R-E drug pairs were screened, including berberine, palmatine, coptisine, and so on. After corresponding, 19 effective chemical components and 284 targets of the R-E drug pair were obtained. After searching multiple disease databases, 1289 T2DM-related targets were screened. After the summary, 159 common targets were obtained in this study. Finally, in the bioinformatics analysis, this study concluded that quercetin, luteolin, berberine, palmatine, and coptisine are the main chemical components of the R-E drug pair. ESR1, MAPK1, AKT1, TP53, IL6, and JUN are the important core targets. GO and KEGG enrichment analyses showed that Rhizoma Coptidis-Eupatorium fortunei could improve T2DM by regulating multiple biological processes and pathways. Molecular docking results showed that berberine, palmatine, and coptisine had higher binding to the core target, and MAPK1, AKT1, and IL6 could stably bind to the active ingredients of Rhizoma Coptidis-Eupatorium fortunei.  Rhizoma Coptidis-Eupatorium fortunei may have therapeutic effects on T2DM such as anti-inflammatory and regulating glucose and lipid metabolism through multiple components, multiple targets, and multiple signaling pathways, which provides a scientific basis for further research on the hypoglycemic effect of Rhizoma Coptidis-Eupatorium fortunei drug pair. The maifunction . T2DM sefunction .Traditional Chinese medicine has prominent advantages in the treatment of T2DM, and the curative effect is remarkable. Rhizoma Coptidis-Eupatorium fortunei medicine pair is a classic medicine pair from the Jianpixiaoke recipe founded by Professor Huang Yanqin, a famous Chinese medicine doctor . RhizomaNetwork pharmacology is an emerging discipline integrating molecular biology, pharmacology, and bioinformatics. It analyzes the network relationship of the “drug-target-disease-pathway” to elucidate the mechanism of action of traditional Chinese medicine in treating diseases . In modehttps://old.tcmsp-e.com/tcmsp.php) [Using “Huanglian” and “Peilan” as the keywords, respectively, the data of R-E drug pairs were obtained through the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) (msp.php) . Active https://pubchem.ncbi.nlm.nih.gov/), this study also developed two similarity-based target prediction networks. Targets, namely, Swiss Target Prediction (http://www.swisstargetprediction.ch/) and STITCH (http://stitch.embl.de/), were retrieved on the server to complement the targets of active ingredients in R-E drug pairs. Finally, through the UniProt (https://www.uniprot.org/) database, the research species is limited to “human,” and the screened targets are converted into corresponding standard genes.The targets corresponding to the active ingredients in the R-E drug pair were collected in TCMSP. Since the targets provided by TCMSP may be incomplete, by using the structural information of the active components obtained from PubChem (https://www.genecards.org/), OMIM database (https://www.omim.org/), DrugBank database (https://go.drugbank.com/), and PharmGKB database (https://www.pharmgkb.org/) to select potential targets of T2DM. The higher the score in the GeneCards database, the stronger the correlation between the target and disease. Therefore, the target with a score ≥ 30 in the GeneCards database is set as the T2DM target. After merging 4 disease database targets, the duplicate values are deleted as disease candidate targets, and the resulting targets are merged. Query the gene name of the target in the UniProt database, and standardize the candidate target.Take “type 2 diabetes mellitus” as the search keyword in the GeneCards database  to construct a common target protein-protein interaction (PPI) network. The biological species were set as “Homo sapiens,” the minimum confidence is 0.900, and the rest of the parameters are default settings to build a PPI network. And import the constructed PPI graph into Cytoscape 3.9.0 for topology analysis. The topological properties of nodes were evaluated using 6 parameters in App-Cyto NCA: Degree Centrality (DC), Betweenness Centrality (BC), Closeness Centrality (CC), Eigenvector Centrality (EC), Network Centrality (NC), and Local Average Connectivity (LAC). The higher the DC, BC, CC, EC, NC, and LAC values of each node, the more important it is in the network. Based on the topological analysis results of PPI, the target above the median was selected as the core target and screened twice, and the screened target was the core target of R-E in the treatment of T2DM.The intersection target of R-E drug pair-T2DM obtained in 2.4 was imported into the STRING database . The obtained macromolecular receptor file was imported into the AutoDockTools software for dehydration and hydrogenation and saved in pdbqt format. Finally, essential targets and active ingredients were imported into the AutoDockTools software for molecular docking, and binding energy was used as the docking evaluation index. Three representative groups of ligands and receptors were selected for data visualization processing using Pymol software.Molecular docking of important targets with key chemical components. The small molecule ligand file was downloaded from the PubChem database, imported into the Chem3D software for optimization, and then processed by the AutoDockTools software to save the small molecule in pdbqt format. The gene ID of the important target was retrieved through the UniProt database, and the three-dimensional structure of the protein receptor was obtained in the PDB database -canadine), palmatine, and stigmasterol are the top 6 chemical components with degree values of 126, 58, 25, 18, 17, and 17. These chemical components may play a key role in the treatment of T2DM with R-E drugs. The network of “Traditional Chinese medicine-active ingredient-target-disease” is shown in Import the intersection target of the R-E drug pair and T2DM into the STRING database for PPI analysis, limit the species to “Homo sapiens,” set the minimum confidence to 0.900, hide the free points, and construct the PPI network. The results show that the network contains 159 nodes and 589 edges, and the average node degree is 7.41. The PPI network is shown in Download the TSV file, and import it into the Cytoscape 3.9.0 software. Apply the cyto NCA plug-in to calculate the BC, CC, DC, EC, LAC, and NC values of each node, and conduct a topological analysis of the results. Finally, the top 14 targets with the largest correlation are obtained, which are JUN, MAPK1, SRC, AKT1, PIK3R1, TP53, TNF, FOS, STAT1, ESR1, IL6, CDKN1A, EGFR, and MYC. These targets may be the core targets of the R-E drug pair for the treatment of T2DM. The core network screening process is shown in The screened core targets, important chemical components, Chinese herbal medicine, and disease are introduced into Cytoscape 3.9.0 to obtain the “Traditional Chinese medicine-active component-core target-disease” network, so as to better screen the core targets and important chemical components of R-E drugs for the treatment of T2DM. The network consists of 25 nodes and 53 sides. The results show that the core targets are ESR1, MAPK1, AKT1, TP53, IL6, and JUN in turn. The important chemical components are HL1 (quercetin), PL1 (luteolin), HL4 (berberine), HL3 , and HL5 (coptisine). The network of “Traditional Chinese medicine-active ingredient-core target-disease” is shown in p < 0.05). Since the p value represents the significance of enrichment, the first 10 items with the lowest p value among BP, CC, and MF were selected, and the bar graph and bubble graph of GO biological function enrichment were made by using the R 4.1.2 software. The GO analysis results are shown in In GO enrichment analysis, the results obtained 2960 GO entries, including 2660 biological process (BP) entries, 95 cell composition (CC) entries, and 205 molecular function (MF) entries . Due to the enrichment significance of the p value table, the first 30 signal channels with the lowest p value are selected, and the KEGG signal channel enrichment bar graph and bubble graph are made by using the R 4.1.2 software. KEGG analysis results are shown in Using KEGG pathway enrichment analysis, 182 KEGG signal pathways were obtained (This study screened the number of core targets enriched in the key KEGG pathway and further analyzed the enriched pathways. It was found that hepatitis B, Kaposi's sarcoma-associated herpesvirus infection, endocrine resistance, proteoglycans in cancer, human cytomegalovirus infection, and breast cancer pathway contained more than 10 core targets, which showed that the relationship between cancer, inflammation, and T2DM was inseparable, Whether there is a close relationship within its mechanism needs further study. The specific enrichment pathways of core targets are shown in This study also conducted GO and KEGG analyses on the core targets of R-E drugs for the treatment of T2DM. A total of 1670 entries were obtained from the enrichment results of GO, including 1531 entries for BP, 41 entries for CC, and 98 entries for MF. GO analysis of core target is shown in The first 6 core targets and 5 important chemical components selected were used as receptors and ligands for molecular docking. The higher the absolute value of binding energy, the more stable the binding between receptor and ligand. The binding energy of -5.0 kcal/mol is set as the threshold for whether the receptor and ligand bind well. Berberine, palmatine, and coptisine have high binding to core targets. MAPK1, AKT1, and IL6 interact well with these key components. This shows that some effective components in R-E drug pairs have good binding properties with protein targets. After comprehensive consideration, the representative molecular docking results are visualized, see β cells, and their distribution in islets, mediate endoplasmic reticulum stress, promote autophagy, inhibit apoptosis, improve basal and glucose stimulated insulin secretion, and protect islets β Cell number and function [The previous research of our research group found that Jianpixiaoke recipe can significantly improve the number of rat islets function . RhizomaThe pathogenesis of T2DM is complex, and there are many important compound components, targets, and pathways, but the mechanism of Rhizoma Coptidis and Eupatorium fortunei in the treatment of T2DM has not been fully clarified. In order to further explore the material basis and possible mechanism of its improvement in patients with T2DM, this study systematically predicted the potential chemical components, key targets, molecular mechanisms, and related pathways of Rhizoma Coptidis and Eupatorium fortunei in the treatment of T2DM through the research methods of network pharmacology and molecular docking. After searching multiple databases, a total of 19 chemical components and 284 targets of R-E drug pair, 1289 T2DM-related targets, 159 common targets of chemical components of drug pair and disease, and 182 potential signal pathways were screened. These results reflect the potential anti-inflammatory mechanism of Rhizoma Coptidis and Eupatorium fortunei in the treatment of T2DM.β cell damage, promotes insulin secretion, and regulates blood glucose balance [β, and β-catenin protein can reduce the level of oxidative stress in renal tissue and protect the renal function of diabetic rats [α-glucosidase [β cellular damage. Coptisine can reduce blood glucose by promoting the transport and absorption of glucose [Quercetin reduces the effect on pancreatic islets through anti-inflammatory and antioxidant effect  balance . Quercettic rats . Luteolitic rats . Other scosidase , significosidase . Berbericosidase , 18. Palcosidase . Li et acosidase  found th glucose .β apoptosis are closely related, and it is also one of the biological indicators for clinical diagnosis of gestational diabetes in early pregnancy [β cells by regulating MAPK/ERK cascade reaction [This study also revealed that ESR1, MAPK1, AKT1, TP53, IL6, and JUN may be the potential key targets of R-E in the treatment of T2DM. ESR1 and islet regnancy . MAPK1 creaction . AKT1 isreaction . As a tureaction . IL6 is reaction , while breaction . JUK is reaction .β apoptosis [κB pathway, upregulate TNF-α expression, and induce insulin resistance [ΚB expression, leading to HIF-1α increased expression [α. It can increase the expression level of p53, improve the hypoxia state of islet cells, and increase the sensitivity of surrounding cells to insulin. As a classic pathway of inflammatory response, TNF signaling pathway is closely related to the occurrence of DM [α is an important inflammatory factor, and its elevated level can interfere with insulin signal transduction. At the same time, TNF-α binding with cell surface receptors can affect the phosphorylation of insulin receptors and disorder blood glucose metabolism [The results of GO functional enrichment analysis suggest that R-E can improve T2DM by regulating a variety of BP and MF, which may interfere with T2DM through biological processes such as glucose homeostasis and lipopolysaccharide reaction. These targets are mainly located in the plasma membrane and its components. In the KEGG enrichment analysis results of this study, the key targets are mainly enriched in AGE-RAGE, endocrine resistance, PI3K-Akt, IL-17, HIF-1, TNF, and other signal pathways. Among them, the pathways involved in glucose and lipid metabolism and insulin resistance include AGE-RAGE, PI3K-Akt, and endocrine resistance. As an important link in the generation and development of microvascular complications of diabetes, the AGE-RAGE signaling pathway regulates the process of impaired islet cell function . Berberipoptosis . Studiessistance . HIF-1 spression . HIF-1 spression . Quercetce of DM . TNF-α itabolism .α and IL6, thereby increasing insulin [In this study, through GO and KEGG analyses of the target and core target of R-E in the treatment of T2DM, it is found that most of the enriched pathways are related to cancer and inflammation. Now, epidemiological studies show that diabetes can induce the incidence and mortality of pancreatic cancer, colorectal cancer, and other tumors . Althoug insulin . MAPK si insulin .Molecular docking results showed that berberine, palmatine, and coptisine had high binding activity to the core target. Among them, MAPK1, AKT1, and IL6 have better docking results with these effective active ingredients. This suggests that berberine, palmatine, and coptisine may be the main active components of Rhizoma Coptidis-Eupatorium fortunei against T2DM.Through the network pharmacology and molecular docking technology, this study confirmed that Rhizoma Coptidis-Eupatorium fortunei may treat T2DM through anti-inflammatory and regulating glucose and lipid metabolism. Network pharmacology and molecular docking have revealed the mechanism of traditional Chinese medicine in the treatment of T2DM at the molecular level, which provides an effective basis for Rhizoma Coptidis-Eupatorium fortunei in the clinical treatment of T2DM. However, at present, network pharmacology is still in the development stage, the computing software and related database layers are different, each has its own characteristics, and the data about various drugs, targets, and diseases are not comprehensive. Therefore, further experimental verification and more rigorous research strategies should be carried out in the future. The research team will continue to carry out further experimental validation and clinical application of Rhizoma Coptidis-Eupatorium fortunei and continue to explore the potential of traditional Chinese medicine in the treatment of T2DM.


To explore the related mechanism of acupuncture affecting obesity by regulating inflammation using bioinformatics methods.  The genes related to obesity, inflammation, and acupuncture and inflammation were mined using GenCLiP 3, and the intersecting genes were extracted using Venn diagram. The DAVID database was employed for pathway enrichment analysis and functional annotation of coexpressed genes. Then, the protein-protein interaction (PPI) network was constructed with the STRING database and visualized by the Cytoscape software and screened out important hub genes. Finally, the Boxplot and Survival Analysis of the hub genes in various cancers were performed by GEPIA.  755 genes related to obesity and inflammation and 38 genes related to acupuncture and inflammation were identified, and 24 coexpressed genes related to obesity, inflammation, and acupuncture were extracted from the Venn diagram. Eight hub genes including interleukin-6 (IL-6), interleukin-10 (IL-10), Toll-like receptor 4 (TLR4), signal transduction and transcriptional activation factor 3 (STAT3), C-X-C motif chemokine 10 (CXCL10), interleukin-17A (IL-17A), prostaglandin peroxide synthesis-2 (PTGS2), signal transistors, and transcriptional activation factor 6 (STAT6) were identified by gene ontology (GO), Kyoto Encyclopedia of Genes (KEGG), and PPI network analysis. Among them, IL-6 is suggested to play an essential role in the treatment of obesity and inflammation by acupuncture, and IL-6 was significant in both Boxplot and Survival Analysis of pancreatic cancer (PAAD). Therefore, in this study, the core gene, IL-6 was used as the breakthrough point to explore the possible mechanism of acupuncture in treating obesity and pancreatic cancer by regulating IL-6. κB (NF-κB) pathway, thereby alleviating inflammation, which can be used as a potential strategy for the treatment of obesity. (2) IL-6/STAT3 is closely related to the occurrence, development, and metastasis of pancreatic cancer. Acupuncture affecting pancreatic cancer through TLR4/NF-κB/IL-6/STAT3 pathway may be a potential method for the treatment of pancreatic cancer. (1) Acupuncture can regulate the expression of IL-6 through the TLR4/nuclear factor- With th (TNF-α) , obesityAt present, the common treatments for obesity include medication, surgery, Chinese medicine, acupuncture, and massage . CompareThe amount of biomedical literature shows an exponential growth, which requires a large quantity of time for researchers to fully read the existing relevant literature. In addition, the diversity of target genes in traditional Chinese medicine makes it inefficient to blindly verify the pathways one by one through experimental methods. Text mining technology is a branch of data mining technology, but compared with other data mining methods, text mining increases the sensitivity of a search by identifying more words, word forms, or phrases and thus broadens the range of studies including related records . The goaIn this study, we used GenCLiP 3 to deeply mine the key genes and pathways related to the occurrence and development of obesity and further screen through gene ontology analysis, pathway enrichment annotation, and protein-protein interaction analysis, in an attempt to find out the key targets and related pathways of acupuncture therapy for obesity, providing a certain reference for subsequent related research and the exploration of therapeutic methods.http://ci.smu.edu.cn/genclip3/analysis.php) is a web-based text mining server that, like other text mining tools, focuses on how genes relate to various topics, such as various biological processes and diseases, by extracting gene-related information from the PubMed literature. The difference is that GenCLiP 3 has established a new model, which integrates Sphinx and MySQL to establish the index of literature and its corresponding genes, and identifies the gene-keyword correlation by comprehensively considering the cooccurrence frequency of genes and keywords to be retrieved in the sentences and abstracts of the literature [GenCLiP 3 , the coexpressed genes related to obesity, inflammation, and acupuncture were obtained for subsequent analysis.In this study, phenotypic and protein (gene) retrieval was set up on the website of GenCLiP 3, with “(obesity' OR ‘adiposis' OR ‘corpulency') AND (Inflammation)” and “(‘acupuncture' OR ‘needling' OR ‘prodding') AND (Inflammation)” as search terms, respectively, to obtain the information of two groups of genes. Then, by intersecting the two groups of genes using a Venn diagram . The first 15 results were independently extracted, and the bubble diagram was drawn with the ggplot2 software package in R language. To further explore the potential relationship between coexpressed genes, we first imported the coexpressed genes into the “Multiple proteins” option of the STRING online website (version 11.0b) and constructed the initial PPI network. Subsequently, we imported the initial PPI network into the Cytoscape software to construct a visual network model for further analysis.Hub genes are often important action targets and focus in the research of biological processes. Next, we screened hub genes according to the network topology through the PPI network. The important clustering functional modules were screened in Cytoscape software using the MCODE plug-in so that we could observe the subnetworks where the coexpressed genes interacted most closely in the dimension of protein. Since the PPI network was constructed through Cytoscape, more nodes and edges tended to mask the true hub genes and affected the screening process. Therefore, we used the CytoHubba plug-in to predict and explore key nodes and subnetworks in a given network. In the Hubba node option of the CytoHubba plug-in, two network topology algorithms, Degree and The Maximum Clique Centre (MCC), were selected to calculate and analyze the PPI network structure and the connection weights between nodes, and then, the respective top ten genes were sequenced. In order to synthesize the important nodes in the biological network, we selected the intersection of the top ten genes screened out by the two algorithms as the final hub genes.http://gepia.cancer-pku.cn) using the published gene sequencing information. GEPIA is an interactive web server that integrates information from gene expression datasets from TCGA and GTEx common databases to help us perform comprehensive and complex data mining tasks [In view of the close relationship between obesity and cancer, we next observed the correlations between hub genes and various types of cancer on the GEPIA website  of most proteins related to obesity, inflammation, and acupuncture were mainly located in intracellular and extracellular compartments. The intracellular compartments included cytoplasm, cytosol, caveola, and mitochondrial intermembrane space, while the extracellular compartments included extracellular region, extracellular space, and external side of plasma membrane, as shown in To elucidate the pathways of coexpressed genes involved in obesity, inflammation, and acupuncture, we performed enrichment analysis of the KEGG pathway, where these genes are mainly enriched in four subcategories: signal transduction pathway, parasitic infection-related pathway, bacterial infection-related pathway, and viral infection-related pathway. Signal transduction pathways include Jak-STAT signaling pathway, Toll-like receptor signaling pathway, HIF-1 signaling pathway, tumor necrosis factor signaling pathway, and cytokine-cytokine receptor interaction. The parasitic infection-related pathways include toxoplasmosis, chagas disease (American trypanosomiasis), and malaria. The bacterial infection-related pathways include inflammatory bowel disease, tuberculosis, leishmaniasis, and pertussis. The viral infection-related pathways include measles, as shown in In order to clarify the tissue-specific interaction between coexpressed genes, a specific protein-protein interaction network was established. The results of the initial PPI network established on the STRING site are shown in Max Depth = 100. The top 10 hub genes screened by MCC method in the CytoHubba plug-in were as follows: IL-6, IL-10, TLR4, STAT3, CXCL10, IL-17A, PTGS2, IL-13, MYD88, and STAT6, as shown in To study the specific hub gene related to obesity, inflammation, and acupuncture, we imported the initial PPI network into the Cytoscape software for visual analysis. The important clustering functional modules obtained by the MCODE plug-in filtering include 14 nodes and 84 edges, as shown in P value < 0.05) and with significance in prognosis analysis, in various cancers, were as follows: AKT1, BCL2, CCK, CCL11, CXCL10, IL1RL1, IL6, JAK2, MAPK14, MYD88, PTGS2, SOD1, STAT3, STAT6, TLR4, and TXNIP. The expression level of IL-6 in PAAD was significantly higher than in normal pancreatic tissues, as shown in P value of 0.048, as shown in After screening by the Boxplot tool on the GEPIA website, in the list of 24 coexpressed genes, genes which showed significantly differential expression  [γ, thereby activating part of the pathway that inhibits lipogenesis. IL-6-deficient mice will be obese due to reduced energy consumption, suggesting that IL-6 can enhance energy metabolism to prevent obesity and insulin resistance [γ. Meta-analysis suggests that the IL-6 pathway is involved in body weight regulation and that regulation of IL-6 signaling may be a potential therapeutic pathway [As an inflammatory factor associated with obesity, the level of IL-6 is positively correlated with obesity , 14. In yte size , 16. In yte size . At the yte size . In obesyte size . There iyte size . The recyte size , 22. Theyte size . Intraveyte size . The rolyte size . On the size [γ) . When ILsistance . Althoug pathway . TherefoκB signaling pathway, such as reducing the secretion of inflammatory factors such as TNF-α, interleukin-1 (IL-1), and IL-6 and effectively reducing pneumonia [κB pathway may also have a significant impact on acupuncture treatment of obesity through IL-6 [κB is an important pathway that regulates IL-6. In the mechanistic study of the drug, modified Zhujing formula [κB. In atherosclerosis (AS) clinical drug trials, SQMXTJN blocked TLR4/NF-κB signaling pathway and downregulated the expression of TLR4, NF-κB, IL-6, TNF-α, and other proteins in the serum of AS patients, thereby reducing inflammation, acting as an antiatherosclerotic agent and slowing down the progression of AS [κB signaling pathway [2+ inward flow, leading to inactivation of TLR4/NF-κB signaling and decreased the expression of IL-6 [κB/IL-6 pathway. It has also been conclusively shown that acupuncture can reduce the activity of NF-κB p65 by modulating intestinal TLR4 and then inhibiting its interaction with NF-κB p65 [κB signaling pathway and downregulating IL-6 expression. The downregulation of IL-6 and other inflammation-related factors by acupuncture is important mechanism for the therapeutic effects of acupuncture on systemic inflammation and various related diseases. As mentioned above, IL-6 is a very promising therapeutic target for obesity, and acupuncture is likely to modulate inflammation and thus affect obesity through the TLR4/NF-κB/IL-6 signaling pathway.Acupuncture plays a therapeutic role in systemic inflammation and various related diseases. Electroacupuncture (EA) regulates downstream effectors of the TLR4/NF-neumonia  and braineumonia , and it neumonia . The TLRugh IL-6 . TLR4/NF formula , Elephan formula  decreaseon of AS . In addion of AS , such as pathway . EA trea of IL-6 ; EA down of IL-6 . Thus, aF-κB p65 . AcupuncP value 0.048). Therefore, we speculated whether acupuncture can be used to play a certain auxiliary role in the treatment of PAAD by targeting IL-6. PAAD is one of the most aggressive and lethal cancer types, and patients have extremely poor prognosis and low survival rate due to no obvious symptoms in the early stage, rapid disease progression, and low efficiency of chemotherapy, while the incidence of pancreatic cancer is increasing year by year, which seriously threatens people's life and health [Numerous studies have shown that obesity is closely related to PAAD . We anald health . Therefod health , 42. Thed health , 44, whid health  and epigd health , 47. In d health , small-cd health , prostatd health , and othd health . Relatedd health . Regenerd health , 55. Reld health . In addid health . This thd health . TherefoIn this study, making full use of the intelligent NLP-based text mining technology can help us quickly find the genes that play a key role in the treatment of obesity and inflammation by acupuncture from the vast amount of literature information and use them for subsequent analysis. Text mining can not only keep up with the latest research results but also be a powerful tool for us to generate new hypotheses. However, since the results obtained in this research are based on the prediction of text mining and considering the automation in the literature retrieval process and the possibly hidden false-positive results in the literature , as wellκB/IL-6 pathway are associated with acupuncture affecting obesity by regulating inflammation, further revealing the importance of IL-6/STAT3 and its downstream pathway in pancreatic cancer and providing some theoretical basis for adjuvant treatment of pancreatic cancer targeting IL-6. However, in this study, we have not yet delved into the interactions of other core genes in the mechanism of acupuncture regulating inflammation and affecting obesity, but this could be a direction for subsequent research.To date, although there are quantities of clinical studies on acupuncture for the treatment of obesity, there are still few basic studies on its specific pathways, especially inflammatory pathways. However, our results provide new insights into the pathogenesis of obesity and the possible molecular mechanisms of acupuncture in the treatment of obesity and inflammation. This study found 24 coexpressed genes related to obesity, inflammation, and acupuncture based on text mining. By analyzing the GO, KEGG, and PPI networks of the genes, we found that the core gene IL-6 and TLR4/NF-


The incidence of depression has been increasing globally, which has brought a serious burden to society. Sinisan Formula (SNSF), a well-known formula of traditional Chinese medicine (TCM), has been found to demonstrate an antidepressant effect. However, the therapeutic mechanism of this formula remains unclear. Thus, the present study aimed to explore the mechanism of SNSF in depression through network pharmacology combined with molecular docking methods.Bioactive compounds, potential targets of SNSF, and related genes of depression were obtained from public databases. Essential ingredients, potential targets, and signaling pathways were identified using bioinformatics analysis, including protein-protein interaction (PPI), the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, Autodock software was further performed for conducting molecular docking to verify the binding ability of active ingredients to targets.A total of 91 active compounds were successfully identified in SNSF with the use of the comprehensive network pharmacology approach, and they were found to be closely connected to 112 depression-related targets, among which CREB1, NOS3, CASP3, TP53, ESR1, and SLC6A4 might be the main potential targets for the treatment of depression. GO analysis revealed 801 biological processes, 123 molecular functions, and 67 cellular components. KEGG pathway enrichment analysis indicated that neuroactive ligand-receptor interaction, serotonergic synapse pathways, dopaminergic synapse pathways, and GABAergic synapse pathways might have played a role in treating depression. Molecular docking suggested that beta-sitosterol, nobiletin, and 7-methoxy-2-methyl isoflavone bound well to the main potential targets.This study comprehensively illuminated the active ingredients, potential targets, primary pharmacological effects, and relevant mechanism of the SNSF in the treatment of depression. SNSF might exert its antidepressant effects by regulating the signaling pathway of 5-hydroxytryptamine, dopamine, GABA, and neuroactive ligand receptor interactions. Still, more pharmacological experiments are needed for verification. Depression has been recognized as a major global public health issue due to its significant morbidity and mortality, cognitive and behavioral changes, psychotic symptoms, and even self-injury and suicidal behaviors. According to the World Health Organization (WHO), major depressive disorder (MDD) is the third leading cause of global burden and is expected to be the first leading cause by 2030 . EvidencTo date, the pharmacological treatment of depression mainly includes selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors (MAOIs), which have been demonstrated to be effective on depressive symptoms. However, one-third to half of the patients with depression was found to be unresponsive to antidepressant agents . Many paBupleurum chinense (Chaihu), Paeoniae Radix Alba (Shaoyao), Aurantii Fructus Immaturus (Zhishi), and licorice (Gancao), with a dose proportion of 1:1:1:1. According to the theory of TCM, SNSF has been widely used in treating liver stagnation and spleen deficiency, disorders of the digestive system, as well as depression for thousands of years in China.Recently, herbal remedies and plant products have been increasingly used to treat depression . A growivia the CaSR-PKC-ERK signal pathway in rats exposed to stress  the active ingredients involved in the treatment of depression, (2) the protein regulatory goals to achieve the biological activity, (3) the biological processes or pathways by which the active ingredients treat depression, and (4) the molecular docking binding ability and key targets of the active ingredients by investigating the therapeutic effect of SNSF on MDD, in order to provide a basis for relevant connotation interpretation, theoretical studies and clinical precision application of this promising herbal formula.1, updated version, June 2nd, 2021), which is specifically designed to characterize the relationships between drugs, targets and diseases of Chinese herbal medicines , and DrugBank.7 After the deletion of duplicate genes, active compound targets were mapped to depression-related targets, and therapeutic targets of SNSF against depression were obtained using a Venny2.1 online tool.All target proteins were converted to their corresponding gene symbols in the UniProt database8) for PPI network construction. The topological properties of targets were analyzed using the CytoNCA tool (a plug-in of Cytoscape), in order to identify key target genes with high connectivity with SNSF. Three parameters, namely degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC), were estimated using the Network Analyzer tool for topological analysis. DC is a key topological parameter representing the number of nodes in the relations between one node and other nodes in a network; The degree value is used to describe the significance of potential targets and active ingredients, with ingredients with higher degrees indicating that more targets would respond to it. Meanwhile, targets with a higher degree value also indicated that they might be the primary targets in the molecular mechanism.The targets shared by the herbs and disease were then entered into the Search Tool of the Retrieval of Interacting Genes database  and BioGrid. The network was obtained through the analysis involving the subset of proteins that had physical interactions with at least one other member in the list. For networks consisting of 3 to 500 proteins, the Molecular Complex Detection (MCODE) algorithm should be applied to identify the densely connected components in the network.9 and KEGG pathway enrichment analysis10 were performed to examine the mechanism of SNSF in the treatment of depression and to clarify the biological processes involving target proteins in distinct clusters, as well as their involvement in signaling transduction. After entering the target genes and restricting the species to humans exclusively, all of the target genes were adjusted to their official gene symbols. The GO terms and KEGG pathways were regarded as significant when the p-value was lower than 0.01 after Bonferroni correction. We employed FDR multiple comparison adjustment to avoid false positives, as we conducted in our previous study (Gene ontology (GO) function annotationus study .11 and then saved as PDBQT protein receptor format files after the removal of water molecules, co-crystalline ligands, and ions by Pymol software. The 2D structures of compounds were retrieved and downloaded from the PubChem database,12 then transferred to 3D structures after energy minimization by Chem3D software. Vina software was utilized to conduct molecular docking and calculate docking affinity. The specific steps and parameters of docking were the same as those in one of our previous studies , 123 molecular functions (MF), and 67 cellular components (CC) were enriched. Most GO terms of BP were related to chemical synaptic transmission, regulation of membrane potential, blood circulation, positive regulation of protein phosphorylation, response to hormones, phospholipase C-activating G protein-coupled receptor signaling pathway, response to xenobiotic stimulus, regulation of ion transport, response to hypoxia, regulation of secretion by cell, etc. MF enrichment was mainly involved in the neurotransmitter receptor activity, G protein-coupled amine receptor activity, G protein-coupled neurotransmitter receptor, active amines, nuclear receptor binding, protein homodimerization, protease binding, acetylcholine binding, protein domain specific binding, inorganic cation transmembrane transporter activity, etc. The main terms of CC were associated with postsynaptic membrane, membrane raft, integral component of postsynaptic membrane, plasma membrane protein complex, neuronal cell body membrane, nuclear membrane, euchromatin dendritic spine, organelle outer membrane, focal adhesion, etc. The terms of BP, CC, and MF were ranked on the basis of their In the KEGG pathway enrichment, a total of 153 signaling pathways were enriched after FDR correction, with the most significant pathways including the neuroactive ligand-receptor interaction, cGMP-PKG signaling pathway, pathways in cancer, cholinergic synapse, morphine dependence, etc. After hierarchical clustering of the enriched pathways, the networks with a similarity of > 0.3 were regarded as sub-networks, with the most significant pathway used as the reference of the enriched networks. Nervous system interaction effects of the KEGG enriched pathways and putative target proteins were identified for SNSF. Serotonergic synapse pathways , dopaminergic synapse pathways , and GABAergic synapse pathways  were identified in this study. See p values were used as the functional description of the corresponding components , PTGS2(PDB ID: 3HS5), NOS3(PDB ID: 1M9M), PRKCA(PDB ID: 4RA4), SLC6A4(PDB ID: 5I6X), ESR1(PDB ID: 1L2I), MMP9(PDB ID: 2OVX), TP53(PDB ID: 2VUK), CASP3(PDB ID: 1NME), and CREB1(PDB ID: 5ZK1), were conducted molecular docking with three candidate bioactive compounds . The binding energy is generally considered to be less than −5.0 kcal/mol or −7.0 kcal/mol, indicating good or strong binding activity between the ligand and the receptor, respectively . The docMajor depression disorder severely limits the social and psychological function and lowers the quality of life of patients, and it is often associated with a variety of chronic physical diseases, including hypertension, cardiovascular diseases, cancer, diabetes, obesity, cognitive impairment and dementia . With thIn the active compounds-targets network, a total of 112 targets affected by 91 bioactive compounds in the SNSF were obtained. The core active compounds mainly included flavonoids such as isosinensetin, formononetin, nobiletin and sterols such as stigmasterol, beta-sitosterol, and sitosterol. Formononetin is a type of isoflavone that upregulates the expression of the glucocorticoid receptor and brain-derived neurotrophic factor, protects neurons, and promotes neurogenesis in the hippocampus, thereby effectively reducing chronic corticosterone-induced depression-like behavior in mice . NobiletThe PPI network analysis showed that the common targets included CREB1, NOS3, CASP3, TP53, ESR1, SLC6A4, MMP9, PTGS2, SLC6A3, and GSK3B. The cyclic adenosine monophosphate (cAMP) responsive element-binding protein 1 (CREB1), as a junction of intracellular depression-related signal transduction pathways, is key to the cAMP signaling pathway, while this pathway has been found to be altered in most patients with psychiatric disorders . FurtherThe GO function analysis showed that the therapeutic effect of SNSF in depression was mainly exhibited as chemical synaptic transmission, regulation of membrane potential, neurotransmitter receptor activity, G protein-coupled amine receptor activity, postsynaptic membrane raft, etc., while the MCODE analysis showed that neurotransmitter receptor activity, postsynaptic neurotransmitter receptor activity, and G protein-coupled amine receptor activity were the most correlated gene clusters. The KEGG pathway analysis showed that the targets of active components of SNSF were mainly concentrated in neuroactive ligand-receptor interaction, including serotonergic synapse pathways, dopaminergic synapse pathways, GABAergic synapse pathways, and cGMP-PKG signaling pathway, etc.The monoamine neurotransmitter and receptor hypothesis of depression proposed lately suggests that the imbalance of monoaminergic neurotransmission is central to the biological dimension of depression, which mainly manifests as the reduction of the synthesis and release of neurotransmitters such as norepinephrine (NE), 5-hydroxytryptamine (5-HT), and dopamine (DA) in relevant brain regions . The serIn this study, we found that stigmasterol, one of the main active compounds of Chaihu, targeted the serotonergic synapse pathways , GABAergic synapse pathways (GABRA3 and PRKACA) and dopaminergic synapse pathways . At the same time, beta-sitosterol, one of the main active components of Radix Paeoniae Alba, targeted serotonergic synapse pathways , GABAergic synapse pathways  and dopaminergic synapse pathways  in the nervous system. Genetic factors are important internal factors in the development of depression, while inter-individual differences result from genetic diversity. Notably, the genes, targets, and pathways identified in the present study are not only specifically related to depression but also may involve other psychiatric disorders. This is consistent with the largest genetic meta-analysis of research on depression to date, which involved 2.1 million people and identified a possible common genetic component between depression and other psychiatric disorders, including anorexia nervosa, attention deficit hyperactivity disorder, schizophrenia, and bipolar disorder . The finThus, we used the method of molecular docking to simulate and predict the binding of active components to targets. The results indicated that Molecular docking suggested that beta-sitosterol, nobiletin, 7-methoxy-2-methyl isoflavone bound well to the main potential targets including CREB1, NOS3, CASP3, TP53, ESR1, PTGS2, and SLC6A4. Therefore, these might be the key compounds for the effect against depression. These findings were in accord with the prediction obtained through the network pharmacology approach. Our findings might provide a guidance for further studies on the molecular targets of SNSF in the treatment of depression and the application of network pharmacology in drug development. It may also provide valuable clues for the development of novel TCM antidepressant drugs with good efficacy, low toxicity and multiple targets.Despite of the strengths, there were still some limitations to the present study. Firstly, studies on the active ingredients determination of the content of SNSF are currently incomplete, therefore, future research on content determination should be carried out. Secondly, the analysis of the main components of the TCM formula and its drug-like properties might not be sufficient enough, and the important targets were identified only on the basis of computer simulation and verification against previous studies involving this type of drugs. Thirdly, whether the role of pathways in our research was up-regulated or down-regulated was unclear. Therefore, further experiments are necessary to explore and verify more pharmacological effects and mechanisms of SNSF.In this study, we found that SNSF is a valuable TCM for treating patients with depression through multiple components, targets, and pathways. Its pharmacological mechanism could through the serotoninergic synaptic pathway, dopaminergic synaptic pathway, and GABAergic synapse pathway to alleviate depression. Hopefully, our research may provide a scientific basis for the prodrug discovery of its natural ingredients and the identification of therapeutic targets in the future.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/XC, QO, and HW contributed to the conception and design of the study. YX, JH, and TX collected the data. XC, JL, and DH performed the statistical analysis. HW wrote the first draft of the manuscript. XC, SW, and SZ critically revised the manuscript. All authors contributed to manuscript revision, read, and approved the final version of the manuscript.


Yi-Qi Cong-Ming (YQCM) decoction has been widely used to prevent age-related hearing loss (ARHL), the most prevalent neurodegenerative disease in the elderly.To explore the mechanism of YQCM decoction in the treatment of ARHL.in vitro model of ARHL by exposing auditory cells with 100 μM H2O2 for 3 h were applied. Cell viability and mitochondrial membrane potential (ΔΨM) were detected by CCK-8 and high-content analysis. γH2AX and cleaved caspase-3 were detected by Western blot.The chemical constituents of YQCM were screened from the Traditional Chinese Medicine Systems Pharmacology Database. Potential targets of YQCM against ARHL were predicted by DrugBank, GeneCards, and OMIM database. Protein-protein network and enrichment analysis were used for exploring possible molecular mechanisms. Molecular docking and an 2O2 treatment could reduce the cell viability by 68% and impaired ΔΨM, while 90 μg/mL YQCM pre-treatment could restore the cell viability by 97.45% and increase ΔΨM (2-fold higher). YQCM pre-treatment also reduced γH2AX and cleaved caspase-3 protein levels.The main compounds have good affinities with hub targets, especially AKT1, PTGS2, and CASP3. GO and KEGG analysis showed that the main biological process and key targets were related to negative regulation of the apoptotic process. HOur study suggested that YQCM prevents ARHL by modulating the apoptosis process in auditory hair cells. Moreover, this study proved that bioinformatics analysis combined with molecular docking and cell model is a promising method to explore other possible pharmacological interventions of ARHL. Age-related hearing loss (ARHL), also called presbycusis, is one of the universal features of mammalian ageing which is characterised by gradual, progressive, bilateral loss of hearing starting from a high-frequency region of the hearing spectrum , Astragali radix , Glycyrrhizae radix et rhizome , Cimicifugae rhizome (Actaea cimicifuga L. [Ranunculaceae]), Puerariae lobatae radix (Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep [Fabaceae]), Viticis fructus (Vitex trifolia L. [Lamiaceae]), Paeoniae radix alba , and Phellodendri chinensis cortex (Phellodendron chinense C.K.Schneid. [Rutaceae]). This Traditional Chinese formula has been widely used to prevent early signs of ageing in Chinese society for centuries, particularly helpful for the treatment of neurodegenerative diseases in older, such as Alzheimer's disease  and nitric oxide synthase (NOS) production in the organ of Corti and SGNs and auditory hair cells  the loss of sensory hair cells, which are responsible for the conversion of sound stimulations to neural impulses; (2) the loss of SGNs, which serve as the primary carrier of auditory information; (3) the atrophy of the stria vascularis (SV) of the cochlear lateral wall of scala media, which maintains endolymph potential Keithley . The exaThe chemical composition information of eight traditional Chinese medicines in YQCM decoction was obtained by using the Traditional Chinese medicine systems pharmacology database and analysis platform, TCMSP Version 2.3. Two parameters, oral bioavailability (OB) and drug-likeness (DL) were used to screen the above compounds, and the chemical components with both OB ≥ 30% and DL ≥ 0.18 were selected as potentially effective chemical components.https://go.drugbank.com;). We used “Age-related hearing loss” and “Presbycusis” as the keywords in the GeneCards database  and Online Mendelian Inheritance in Man database  to search potential targets of ARHL. The visualisation network of the ingredient-action target was established by Cytoscape 3.6.0. Then the Network Analyser plug-in is used to analyse the network diagram to find its main active components and important targets.Based on the identified components, the corresponding targets were predicted by DrugBank online database . The statistical significance threshold of enrichment analysis was established at p < 0.05.The common targets of disease and drug are obtained by using the Venny online tools, that is, the potential target of YQCM decoction in the prevention and treatment of presbycusis. The drug-disease common target was introduced into the STRING: functional protein association networks. The PPIs with medium confidence were included in this study. Subsequently, a PPI network for the potential target was constructed using Cytoscape. The "Network Analyser" plug-in of Cytoscape software is used to analyse the topological attributes of the PPI network. The topological parameters, including Degree, Betweenness, and Closeness, were applied for screening core targets. To explore the mechanism underlying YQCM’s effect on ARHL, the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway and GO including cellular components (CC), biological processes (BP), and molecular functions (MF) were analysed by the database for annotation, visualisation, and integrated discovery (DAVID) tools  database. Three-dimensional structures of the target were retrieved from the PubChem database and minimised using MMFF94 forcefield. The water molecules and original ligands were removed by PyMol 2.5 software and transferred to pdbqt format via ADFR suite 1.0. Subsequently, AutoDock Vina 1.1.2 was employed for molecular docking to evaluate the binding affinity between core potential targets and core compounds. The binding activity is evaluated by the binding energy (kJ/mol). The smaller the binding energy, the more stable the docking module. The minimum binding energy <−5.0 kcal/mol indicates a good binding affinity between receptor and ligand  simulation strategy was carried out to simulate the binding stability of the receptor and ligand by the AMBER 18 software package  method with long-range electrostatic interaction. The SHAKE algorithm was used to restrict the bond to the hydrogen atom . Liquid chromatographic separation was performed on an ACQUITY UPLC®HSS T3 1.8 μm 2.1 × 100 mm column maintained at 35 °C and eluted with gradient water. The mixture containing six reference components was used for the qualitative analysis: (1) puerarin, (2) ginsenoside-Rg1, (3) luteolin, (4) astragaloside IV, (5) kaempferol, and (6) formononetin.Astragali Radix, Ginseng Radix et Rhizoma, Glycyrrhizae Radix et Rhizoma, Cimicifugae Rhizoma, Puerariae Lobatae Radix, Viticis Fructus, Phellodendri Chinensis Cortex, Paeoniae Radix Alba, purchased from Kangqiao Chinese Medicine Tablet Co., Ltd, were used to prepare YQCM decoction with the proportions as 10:10:10:6:6:3:2:2. All crude drugs were soaked in 60% ethanol for 0.5 h and then extracted with 60% ethanol 2 times for 1.5 h, respectively. Next, the ethanol extract was evaporated 2.HEI-OC1 cells (the House Ear Institute-Organ of Corti 1 cell line) in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum without antibiotics. Cell cultures were maintained at 33 °C in a humidified incubator with 10% CO3 cells per well for 24 h. To investigate the cytotoxic effect on YQCM, cells were incubated with YQCM solutions with different concentrations  for 24 h. To evaluate YQCM’s protective effect under oxidative stress, cells were replaced with fresh DMEM containing different concentrations of YQCM  for 24 h. The positive control group and the YQCM-treated groups were then exposed to H2O2 (100 μM) for 3 h. After various treatments, 10 μL of the cell counting kit-8  solution was added to each well of the plate. Cell viability was quantified at an absorbance of 450 nm using a VICTOR® Multimode Microplate Reader .Cells were incubated in a 96-well plate at a density of 5 × 102O2 (100 μM) for 3 h. To monitor the mitochondrial membrane potential, the fluorescent TMRE  was added to each well for 15 min and then incubated in 1 μg/mL Hoechst 33342  for 10 min. Subsequently, the plate was washed with PBS/0.2% BSA. The TMRE fluorescent signal was immediately scanned and quantified with the High-content imaging system ImageXpress Micro microscope  using a 20 × objective to take an image covering the whole well for each well.HEI-OC1 cells were plated in a 96-well plate and exposed to different concentrations of YQCM  for 24 h as described in “Cell viability.” The positive control group and the YQCM-treated groups were then exposed to HTM Touch Imaging System  using standard chemiluminescence. Quantification of band intensity was analysed with Image Lab Software .Proteins were extracted from treated cells and put through the 12% Protein Gel electrophoresis. After that, protein samples were transferred into the polyvinylidene fluoride membrane  and blocked for 15 min with QuickBlock solution . Afterward, the blotted membrane was incubated with specific primary antibodies: anti-γH2AX , anti-cleaved caspase-3 (ASP175) , anti-β-actin  for overnight at 4 °C. Then, each membrane was washed with TBS-T buffer and incubated with a secondary antibody for 1 h at room temperature. The blots were visualised by ECL Plus Western Blotting Substrate  and imaged by ChemiDOCpost hoc test was applied for multiple group comparisons. P values < 0.05 were considered statistically significant. Data are expressed as the means ± standard deviation (SD).All statistical analyses were performed by GraphPad 7.0 . One-way analysis of variance (ANOVA) followed by Dunnett’s  chinensis cortex, 85 in Paeoniae radix alba. Among them, 206 compounds met the criteria of OB ≥ 30% and DL ≥ 0.18.Using the TCMSP database, 87 compounds were found in Astragali radix, 190 in Ginseng radix et rhizoma, 280 in glycyrrhizae radix et rhizome, and 187 in cimicifugae rhizome, 18 in Puerariae lobatae radix, 144 in Viticis fructus, 140 in PhellodendriO-methylisomucronulatol, 7-methoxy-2-methyl isoflavone  were predicted as the core targets of YQCM action on ARHL. In this network, AKT1, IL-6, MAPK3, VEGFA, CASP3, JUN, MAPK8, EGF, PTSG2, and MAPK1 were the top 10 nodes in terms of degree value .p < 0.05). These pathways are predominantly involved in the HIF-1 pathway , FoxO  and PI3K/AKT pathway . The top 10 KEGG enrichment pathway ranking p values according to the order from small to large are displayed in To understand the function and the underlying significance of the YQCM’s therapeutic effect, the above 51 core targets were entered into the DAVID platform for GO term and KEGG pathways enrichment analysis. The results showed that the targets were mainly enriched in 45 molecular functions, 301 biological processes, and 28 cellular components. The top 10 enriched terms are shown in The ten candidate therapeutic targets with high degree value, AKT1, IL6, MAPK3, VEGFA, CASP3, JUN, MAPK8, PTSG2, EGF, and MAPK1, were docked with the ten core active compounds of YQCM. The lowest binding energy of the molecular docking of the core targets and compounds was presented in the heatmap as shown in Molecular dynamics simulation was used for studying the binding stability of the top ten protein-compound with the best molecular docking scores. The MM/GBSA method was further used to calculate the binding energies. The specific binding free energy of AKT1_7-Metho-2-met_isoflavone, AKT1_β-sitosterol, AKT1_fumarine, AKT1_luteolin, AKT1_quercetin, AKT1_stigmasterol, CASP3_stigmasterol, MAPK8_fumarine, PTGS2_β-sitosterol, PTGS2_fumarine was showed in 2O2 (100 μM) was significantly reduced by 68% (2O2-treated group dropped by nearly 40% as compared with the control group (p < 0.01). On the contrary, YQCM pre-treatment attenuated the H2O2-induced reduction of ΔΨM in a concentration-dependent manner. The essential proteins responsible for DNA damage (γ-H2AX) and apoptosis (cleaved caspase-3) were measured in H2O2-induced HEI-OC1 pre-treated with or without YQCM. As shown in 2O2 increased by 2-fold and decreased by 33% after 90 μg/mL YQCM treatment. Immunoblotting results showed that exposure to H2O2 for 3 h resulted in a 50% increase in the expression of cleaved caspase-3. There is a 37% decrease in cleaved caspase-3 level at 30 μg/mL and a 50% decrease at 90 μg/mL YQCM , and improve an antioxidant enzyme peroxiredoxin 6 (Prdx6) in preventing auditory cell damage during ARHL. FoxO is a well-studied pathway affected by ototoxic drugs, which is one of the factors in increasing ROS production in ARHL. For example, the FoxO signalling pathway was activated following amikacin-induced ototoxicity which is consistent with increased apoptosis  levels were dramatically increased in the age-related hearing loss mice model  of YQCM were enriched in this process, including AKT1, GSK3B, CDKN1A, CASP3, MYC, CAT, BCL2L1, etc. Increasement of ROS production in ARHL, in addition to reduced antioxidant activity with age, will exacerbate the ROS-antioxidant imbalance (Tavanai and Mohammadkhani AX level , indicatFor the first time, network pharmacology and molecular docking strategy to were utilised to explore the underlying mechanism of YQCM in treating ARHL. Our network pharmacology data suggest that modulations of DNA damage and apoptosis might be the key protective role for YQCM decoction. Potential hub targets for YQCM such as AKT1, CASP3, and PTGS2 may be exploited as potential strategies. We used molecular docking analysis and MD simulation to validate the interaction between core targets and compounds. Additionally, the protective effect of YQCM on ARHL and its mechanism were supported by the evidence that YQCM could alleviate oxidative stress-induced auditory hair cell death via modulating DNA damage/apoptosis. Future experiments using molecular methods and animal models as well are needed to support the current findings. Nevertheless, the results of this study provide an important basis for further research on the mechanism of YQCM in ARHL. This study proved that bioinformatics analysis combined with molecular docking and cell model is a promising method to explore other possible pharmacological interventions of ARHL.


CA7, CPY1B1, and PTPN11; in particular, the expression level of CYP1B1 in the DSS + RSBDP group was up-regulated by as high as 9-fold compared to the DSS group. Western blot results indicated that the protein levels of AKR1C1, PI3K, AKT, p-AKT, and Bcl-2 were significantly down-regulated, and Bax was significantly up-regulated in the DSS + RSBDP group. Compared to the DSS and control groups, the Bax/Bcl-2 value in the DSS + RSBDP group increased 4-fold and 8-fold, respectively, which suggested that oral RSBDP promotes apoptosis of intestinal epithelial cells. In short, this study established quercetin, kaempferol, luteolin, naringenin, and sitosterol as the primary key active ingredients of RSBDP that exert synergistic therapeutic effects against IBD through modulating the AhR/CYP1B1 and AKR1C1/PI3K/AKT pathways.Ren-Shen-Bai-Du Powder (RSBDP) is currently used for inflammatory bowel disease (IBD) therapy in China. However, its potential mechanism against IBD remains unknown. In this study, we initially identified potential targets of RSBDP against IBD through network pharmacology analysis and molecular docking. Afterwards, the DSS-induced colitis mice model was employed to assess the effects of RSBDP. The results of network pharmacology indicated that a total of 39 main active ingredients in RSBDP generated 309 pairs of drug-ingredient and ingredient-target correspondences through 115 highly relevant targets of IBD. The primary ingredients  exerted functions through multiple targets that include CYP1B1, CA4/7, and ESR1/2, etc. GO functional enrichment analysis revealed that the targets related to IBD were significantly enriched in the oxidation-reduction process, protein binding, and cytosol. Per the KEGG pathway analysis, pathways in cancer, adherens junction, and nitrogen metabolism were pivotal in the RSBDP’s treatment of IBD. Additionally, molecular docking demonstrated that a set of active ingredients and their targets displayed good bonding capabilities . For the animal experiment, oral RSBDP promoted weight recovery, reduced intestinal inflammation, and decreased serum IL-1, IL-6, and IL-8 concentrations in the DSS + RSBDP group. Meanwhile, oral RSBDP significantly up-regulated the mRNA levels of  Inflammatory bowel disease (IBD) is characterized by an idiopathic chronic inflammatory state of the gastrointestinal tract and encompasses 2 distinct disease states: ulcerative colitis (UC) and Crohn’s disease (CD) . Since oPanax ginseng , Radix bupleuri , Radix Peucedani , Rhizoma Ligustic Chuanxiong , Fructus Aurantii , Rhizoma et Radix Notopterygii , Radix Angelicae Pubescentis , Poria , Radix Platycodonis , Radix Glycyrrhizae , Rhizoma Zingiberis Recens , and Herba Menthae . RSBDP has also shown notable therapeutic properties against infantile diarrhea and has been demonstrated to improve the intestinal mucosal barrier in UC rats [Drugs used frequently to treat IBD tend to be expensive and have high side effects, rendering their consistent application challenging . Convers UC rats . HoweverThe network pharmacology approach includes the use of systems biology, network analysis, connectivity, and multiple effects . It is iPrimary bioactive ingredients in RSBDP and corresponding ADME information were extracted from the TCMSP data server. In this study, a total of 39 main bioactive ingredients were identified in RSBDP and obtained the drug targets for the corresponding ingredients utilizing the SwissTargetPrediction database . After dWe imported drug–ingredient–target relationships into the Cytoscape  software  to construct a drug–ingredient–target network diagram for the treatment of IBD with RSBDP . The darWe performed topological analyses of the drug-ingredient-target network in the Cytoscape 3.7.2 software using “degree value” as a screening parameter for the active ingredients. Our results suggest that quercetin, kaempferol, luteolin, naringenin, and sitosterol were the potential key molecules for RSBDP’s treatment of IBD .We also analyzed the core targets of RSBDP for IBD treatment in the same way in the Cytoscape 3.7.2 software. The top 10 targets, namely cytochrome P4501B1 (CYP1B1), carbonic anhydrase 7 (CA7), cytochrome P45019A1 (CYP19A1), carbonic anhydrase 4 (CA4), protein tyrosine phosphatase 1 (PTPN1), estrogen receptor 2 (ESR2), multidrug resistance-associated protein 1 (ABCC1), ATP-binding transporter protein G family member 2 (ABCG2), estrogen receptor 2 (ESR1), and cyclin-dependent kinase 1 (CDK1), are also listed in This study imported the 115 obtained common targets into the SRING database to create a PPI network diagram , in whicTo further illustrate the biological functions of the 115 intersecting targets, we imported them into the DAVID database for GO and KEGG enrichment analyses. GO examination consisted of three modules: Biological process (BP), Molecular function (MF), and Cellular component (CC). We sorted the top 15 GO items in each module according to the count number  and then drew the GO function enrichment map . The tarThe KEGG pathway analysis returned 74 items, and we selected the top 20 for a visual analysis based on gene count number rank . The anaThis study selected a portion of the molecular docking results for display in this study . Five viZO-1, Occludin, and Claudin-1) were up-regulated in the DSS + RSBDP group but not statistically different from the DSS group. Serum concentrations of IL-1, IL-6, and IL-8 were lower in the DSS + RSBDP group than in the DSS group, among which the level of IL-8 was significantly lower  is one of the potential targets of RSBDP’s treatment of IBD, exerting various regulatory effects through binding to flavonoids or natural drugs and influencing the transduction of downstream signaling pathways ,28. EvidCYP1B1 h. ParticTo further reveal the mechanism of RSBDP against IBD, we integrated and analyzed the results of PPI, GO, and KEGG, focusing on the AKR1C1/PI3K/AKT pathway. Although the gene and protein sequences of mouse AKR1C3 were not available in the NCBI database, a previous study analyzed the homology of human AKR1C3 with mouse AKR1C family, and suggested that the mouseAKR1C1  and human AKR1C3 has higher homology and the similar functions . Interes−/− mice [CYP1A1 and CYP1B1 by activating AhR, which alleviates experimental colitis [CYP1B1 expression through AhR, reducing inflammation in IBD models. RSBDP also promotes the apoptosis of intestinal epithelial cells via AKR1C1/PI3K/AKT and, thus, against IBD. Although there is no direct evidence that the critical ingredients of RSBDP can alleviates the symptoms of IBD by affecting the concentration of AKR1C1, indirect evidence suggests that the addition of kaempferol inhibited the mRNA level of AKR1C1 and induced apoptosis in non-small cell lung cancer cells [In this study, we confirmed the positive effect of RSBDP on DSS-induced mice at the molecular level. However, the ingredients of RSBDP are pretty complex, and it would be fascinating to explore the therapeutic effects of critical ingredients of RSBDP in IBD. As the vital natural flavonoid ingredients of RSBDP, quercetin, kaempferol, luteolin, and naringenin have all been demonstrated to resist IBD through anti-inflammatory and an-tioxidant pathways ,15,16,17−/− mice . Hence,  colitis . Our reser cells . Finallyhttps://lsp.nwu.edu.cn/tcmsp.php, accessed on 4 September 2021) [https://pubchem.ncbi.nlm.nih.gov/, accessed on 6 September 2021) by importing the unique IDs of the retrieved compounds [Data on the main active compounds of RSBDP were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  . Oral biompounds .http://www.swisstargetprediction.ch/, accessed on 15 September 2021) for molecular docking and screened potential targets using a probability threshold ≥0.5. Meanwhile, the keywords “inflammatory bowel disease” were input into GeneCards [https://www.genecards.org/, accessed on 15 September 2021) and OpenTargets [https://www.opentargets.org/, accessed on 15 September 2021) databases to search for IBD-related proteins. The IBD targets obtained from both databases were first merged and then overlapped with the potential targets acquired by prediction from the SwissTargetPrediction database for subsequent analysis.The collated structural formulae of the main active ingredients were imported into the SwissTargetPrediction database   and set the “minimum required interaction score” for the protein-protein interaction network (PPI) analysis to 0.7 [The screened 115 targets were introduced into the STRING database  (https:/s to 0.7 . The PPIWe performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses on the targets from Molecular docking analyses of the screened key ingredients and their targets were conducted. The 3D molecular structures of the vital active ingredients and their targets were obtained from the Pubchem  and PDB In this study, Panax ginseng, Radix bupleuri, Radix Peucedani, Rhizoma Ligustic Chuanxiong, Fructus Aurantii, Rhizoma et Radix Notopterygii, Radix Angelicae Pubescentis, Poria, Radix Platycodonis, Radix Glycyrrhizae, Rhizoma Zingiberis Recens, and Herba Menthae were purchased from the outpatient department of Chengdu University of traditional Chinese medicine , and they were conformed to the quality standards of Chinese Pharmacopoeia (2015 edition). Afterward, these herbs were authenticated by Prof. Jin Pei . Finally, all herbs were crushed separately and mix the above herbs according to the ratio 2:4:2:3:2:2:2:2:2:2:1:1, then added 750 mL (1:10 g/v) of pure water and boiled for 20 min. Subsequently, the drug solution was filtered through a 0.45um filter and concentrated to 75 mL (stored at −20 °C).Six-week-old male C57BL/6 mice were maintained with five to six animals per cage and house in specific pathogen-free facility with a 12-h light and 12-h dark cycle at 22 °C.n = 5), DSS group (n = 6) and RSBDP+DSS group (n = 6). 3% dextran sodium sulfate  was added to drinking water for 9 days. For the next 5 days, 0.308 mL of saline was gavaged daily in the control and DSS groups, and 0.308 mL of drug solution was gavaged daily in the RSBDP + DSS group. All mice were euthanized at the end of the experiment. Tissues or serum were frozen in liquid nitrogen, and colon specimens were fixed in 4% paraformaldehyde.All mice were randomly divided into three groups, control group (Hematoxylin and eosin (H&E) was used to measure the degree to which the colon had been damaged and inflamed, and the epithelial proliferation was detected using Ki67 .IL-1, IL-6, and IL-8 were performed on serum using the ELISA Kit according to the manufacturer’s instructions .RNA extraction was performed using EASYspin Plus Kit  following the manufacturer’s instructions, and the HiScript Ⅱ qRT SuperMix R223  was used to transcribe total RNA into cDNA. The ChamQ Universal SYBR qPCR Master Mix Q711  was used for qRT-PCR. The primer sequences are listed in Tissues were lyzed in RIPA buffer  containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors, and the extracts were centrifuged at 14,000 rpm for 10 min at 4 °C. The 10% SDS-PAGE was used to separate total protein, then transferred onto the polyvinylidene fluoride (PVDF) membrane. Primary antibodies include AKR1C1 (A13004), PI3K (A11526), Bax (A19684), Bcl-2 (A20736), TNF-α (A11534), and β-actin (AC028) were purchased from ABclonal ; AKT (AF6261) was purchased from Affinity ; p-AKT (80455-1-RR) was purchased from Proteintech . Anti-rabbit or -mouse IgG conjugated to horseradish peroxidase was applied after secondary antibody incubation for protein detection.p < 0.05 was considered statistically significant.The experimental results are presented as means ± SEM for each group with at least three independent experiments. Data analysis using one-way ANOVA followed by LSD test. The In summary, we firstly performed the network pharmacology combined with molecular docking approach to elucidate that the anti-IBD effects and the underlying mechanism of RSBDP, and the key targets of RSBDP against IBD were partially identified by experiments, including CA7, CYP1B1, and PTPN11. In addition, western blotting results revealed multiple functions of RSBDP in the DSS-induced colitis which were exerted by anti-inflammatory and pro-apoptotic activities, relying on the AhR/CYP1B1 and AKR1C1/PI3K/AKT pathways.