Wulongzhiyangwan (WLZYW) is a Chinese prescription medicine for the treatment of pruritus, but its mechanism has not been clarified. The purpose of this study was to explore the mechanism of WLZYW in pruritus through network pharmacology analysis and experimental validation. The active components and corresponding targets of WLZYW were obtained from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database. Pruritus-related targets were obtained from the GeneCards, TTD (Therapeutic Target Database), and DrugBank databases. The key compounds, core targets, main biological processes and signaling pathways related to WLZYW were identified by constructing and analyzing related networks. The binding affinity between WLZYW components and core targets was validated by AutoDock Vina software. In this study, RBL-2H3 cells were used to construct a degranulation model to simulate histamine-dependent pruritus. 10 chemical constituents, 235 targets and 3606 pruritus-related targets of WLZYW were obtained. Subsequently, 26 core targets were identified through analysis, VEGFA and AKT1 were the main candidates. A pathway enrichment analysis showed that overlapping targets were significantly enriched in the PI3K/AKT signaling pathway. A molecular docking analysis revealed tight binding of VEGF to three core compounds, kaempferol, luteolin and quercetin. Experiments showed that WZLYW inhibited mast cell degranulation, regulated VEGFa mRNA and protein expression levels by inhibiting PI3K/AKT and ERK1/2 signaling pathway activation. The mechanism of WZLYW in pruritus may be regulating VEGFa expression. Network pharmacology assays suggested that WLZYW downregulates VEGFa expression by regulating the PI3K/AKT and ERK1/2 signaling pathways in pruritis treatment. It often leads to great personal stress and financial burden. The pathogenesis of pruritus is generally classified into histaminergic and nonhistaminergic pathways2. The former is mediated by histamine secreted primarily by mast cells, basophils and keratinocytes3. Mast cell activation causes histamine pruritus involves cell degranulation following the cross linking of immunoglobulin E (IgE) with high-affinity IgE receptors (FcεRI). Then, other influencing factors are released, such as histamine, prostaglandin, NGF, VEGFa, TNF-α and leukotriene4. When the patient is diagnosed with chronic pruritus of unknown etiology, treatment should be in principle according to T helper 2 (Th2) type inflammation5.Itch is a common sensory experience that is prevalent in patients with inflammatory skin diseases, as well as in those with systemic and neuropathic conditions. Itching that lasts 6 weeks or longer is considered chronic and its estimated lifetime prevalence is 22.6%Zaocys dhumnades), Fang feng (Saposhnikovia divaricata), Shechuangzi (Cnidium monnieri (L.) Cuss.), Kushen (Sophora flavescens Aiton), Huangbai (Phellodendron amurense Rupr.), Cangshu (Atractylodes lancea (Thunb.) DC), Renshen (Panax ginseng C. A. Meyer), Mudanpi (tree peony bark), Shedanzhi (Snake bil), Bovis Calculus (Bos taurus domesticus Gmelin), and Danggui (Angelica sinensis). This formula is widely used in the treatment of pruritus in clinical practice.The Chinese Pharmacopoeia (2010 Edition) shows that Wulongzhiyangwan (WLZYW) is used to treat addictive rashes, rubella, and pruritus caused by the accumulation of heat rash on the skin, with symptoms that manifest as red spots, blistering rashes, and intolerable itching. WLZYW contains ten compounds, namely, Wushaoshe  have been used in China for thousands of years with good efficacy for certain diseases. Recently, researchers have gradually elucidated the effective components and targets of TCM. Network pharmacology is based on the similarity in structure and function between drugs, analyze disease-related genes in regulatory network of drug–component–target–disease, to explore the interactive relationships between target molecules and biological effector molecules in vivo. TCM are two or more mixed preparations, with complex regulatory networks. The ingredients in WLZYW formulation act on different targets and pathways, but some may act on the same target. In other words, the ingredients in the formula are synergistic. Network pharmacology integrates multidisciplinary technologies and contents, such as multidirectional pharmacology, computational biology, network analysis and related findings. The comprehensive network analysis of drug effects can be achieved by multitarget research strategiesIn this study, we simulate the action network of WLZYW on the basis of manufacturing drug component target pathway network, selected the core drug components and the most likely drug targets, and validated the findings through molecular docking. The workflow diagram is shown in Fig. Ten drugs in WLZYW formula correspond to 59 complex components, and 235 drug targets were selected by OB  ≥ 30% and DL (Drug Likeness) ≥ 0.18 following the drug-like principle . The corresponding secondary protein interaction network was obtained Fig. A, with 2nds Fig. . With an. The cor10. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for the 26 target genes in the PPI core network, resulting in 2489 GO enriched terms , which used by researchers to understand all aspects of biomedicine, from protein synthesis to the roles in health and disease. Among the 26 potential targets, AKTI and VEGFa showed the highest degree of centrality (degree = 59) and were closely related to the pathways indicated in KEGG analysis. Therefore, we searched the three-dimensional structure of AKT1 and VEGFa in PDB as the protein receptor in docking experiments. The interaction targets of WLZYW with AKT1 were identified by PPI network, and the corresponding compound ligands were luteolin, quercetin, and kaempferol. PubChem software was used to score and select the ligand structure with the smallest free energy. Docking of AKT1 and VEGFa receptor and their corresponding small-molecule drug ligands were performed by AutoDock Vina (opensource molecular docking program). Based on semiflexible docking, the interaction energy results are shown in Table Kaempferol, luteolin, and quercetin bound effectively to the active pockets of VEGFA protein. Their binding energies were − 6.8, − 7, and − 6.9 kcal/mol, respectively Table . The resKaempferol, luteolin and quercetin effectively bound to the active pockets of AKT1 protein with binding energies of − 9.2, − 9.8 and − 10.1 kcal/mol, respectively, all were lower than − 7.0 Table D. Luteol0.5 µg/mL DNP-IgE was used to induce RBL-2H3 cells for 24 h, and then, DNP-BSA (100 ng/mL) was added as the secondary stimulation to cause degranulation and release inflammatory mediators. WLZYW (1 µg/mL to 100 µg/mL) was not toxic to RBL-2H3 cells , Shechuangzi-Cnidium monnieri (L.) Cuss., Kushen-Sophora flavescens Alt., Huangbai-Phellodendron amurense Rupr., Cangshu-Atractylodes Lancea (Thunb.) DC, Renshen-Panax ginseng C. A. Meyer, Mudanpi-Tree Peony Bark, Shedanzhi-Snake bil, Bovis Calculus-Bos taurus domesticus Gmelin, and Danggui-Angelica sinensis. They were searched in TCMSP database11 for drug screening, with OB ≥ 30% and OL ≥ 0.18, and obtained the targets name of all compounds. Symbol matching of target names were performed by human-derived gene information from Uniprot database for multi-database information docking.WLZYW contains ten herbs, namely, Wushaoshe-12: the keyword was pruritus, 16 pruritus-related targets were found. (2) GeneCards: the keyword was pruritus, 3596 pruritus-related targets were found. (3) DrugBank Online13: the keyword was pruritus, 9 pruritus-related targets were found.The retrieval of pruritus-related targets resulted in the collection of different genes associated with pruritus from the following resources. (1) Therapeutic Target Database (TTD)14. Finally, targets with score less than average were removed, after two times the core targets were selected.Pruritus targets and WLZYW targets were intersected by R 4.0.2 to generate coincident targets. Core target screening and analysis were performed by STRING databaseProtein–protein interaction (PPI) data were obtained from Mutual Interaction Gene/Protein Search Tool. The coincident targets were entered into a database with species restricted to Homo sapiens, and confidence values were higher than 0.4. The hypergeometric test was performed via the phyper function of R 4.0.2.Network construction was performed by two networks in this study. WLZYW compounds were connected with corresponding targets to establish compound-target network, network of pruritus targets was constructed by searching pruritus-related targets in different databases. The compound-targets network was crossed with the pruritus-related targets network to construct PPI network. Genes that did not cross were removed. A potential target–protein interaction network was constructed, and analyzed using modules to advance the core network. The networks were all made by Cytoscape 3.8.10, a software package used in the visualization and analysis for networks. For easily interpret large biological datasets, biological information is often visualized as graphs, such as nodes and edges. Nodes represent biomolecules, and edges between nodes represent certain relationship.15. The GO database includes BP, CC, and MF terms, were used to identify possible biological mechanisms based on high-throughput genomic or transcriptomic data16. KEGG is a reference knowledge base of biological interpretation for genome sequences and other high-throughput data. In addition, the bio-conductor cluster Profiler , enrich plot, and pathview were used to analyze potential target modules for GO and KEGG pathway analysis. These five R packages were also applied in the enrichment analysis of gene clusters17.DAVID , functional enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis for the targets networks of pruritus genes corresponding to WLZYW were performed. In addition, Pathview was used to obtain visualized KEGG imagesThe SDF formats of compounds were downloaded from Pubchem data and imported into Chemdraw 3D. The energy was minimized using MM2 module to obtain the lowest energy, and saved as mol2 file. VEGFA and AKT protein structures were downloaded from the Protein Data Bank (PDB) database with PDB IDs of 6zfi and 7nh5, respectively. Subsequently, Pymol was used for visualization respectively. Mgtools 1.5.6 was used to dehydrate, hydrogenate, calculate charge and combine non-polar hydrogen, the ligands and receptors were respectively stored as PDBQT. Autodock vina 1.1.2 was used to dock ligand with receptor, and the parameters were shown below. The high rated were visualized by PYMOL and Discovery Studio.−1 penicillin and 100 μg mL−1 streptomycin. The cells were cultured in a 37 °C, 5% CO2 incubator. RBL-2H3 (0.5 × 106 cells/mL) grew in multi-well plates. After the cells fused to 75%, added DNP-IgE  for 24 h, then washed with PBS. Cells and supernatants were collected after stimulation with 100 ng/mL DNP-BSA .RBL-2H3 cells were purchased from Procell (CHN). RBL-2H3 were cultured in DMEM medium containing 10% fetal bovine serum, 100 U mL5 cells/well and incubated for 24 h at 37 °C and 5% CO2. Cells were exposed to 1–1000 μg/mL WLZYW and further incubated for 24 h. The medium was replaced with 100 µL fresh medium containing 10% CCK8 , and cells were incubated for 1.5 h. The OD (450 nm) absorbance value in each well was determined by spectrophotometer . Cell viability rate (%) was calculated.CCK8 assay was performed to detect cell viability and proliferation. RBL-2H3 cells were seeded in the 96-well culture plates with 1 × 10Cells were seeded in 6-well plate culture dish. When cells fused to 75%, added DNP-IgE  for 24 h, washed with PBS, and replaced with WLZYW (10 μg/mL or 50 μg/mL). Cells were collected after stimulation with 100 ng/mL DNP-BSA for 10 min. Total RNA was extracted from cells using RNeasy Mini Kit , and 1000 μg template RNA was transcribed into cDNA by Superscript III RT-PCR Kit . Then, the cDNA was qRT-PCR via SYBR GREEN . The primer sequences are: TNF-α, Forward-CATCCGTTCTCTACCCAGCC; Reverse-CATCCGTTCTCTACCCAGCC. VEGFa, Forward-ACGAAAGCGCAAGAAATCCC; Reverse-GCAACGCGAGTCTGTGTTTT. GAPDH, Forward-TGAGATCAACGTGTTCCAGTG; Reverse-ACCAGATGAAATGTGCCCC.RBL-2H3 cells were sensitized with IgE (0.45 µg/mL) for 24 h and then treated with WLZYW (10 or 50 µg/mL) for 30 min. After cells were attacked by 100 ng/mL DNP-BSA for 4 h, supernatant was collected and the levels of TNF-α and VEGFa  were determined with ELISA kit according to the instructions.The proteins extracted from cells were measured with BCA Protein Assay Kit . 25 μg protein samples were boiled at 90 °C for 7 min, then loaded and subjected to electrophoresis in 12% FuturePAGE  gels at 160 V for 20 min. The membranes were probed with anti-PI3K/AKT antibody , anti-PI3K/AKT antibody , anti-p-ERK1/2 antibody , anti-ERK1/2 antibody , and mouse monoclonal antibody against tubulin  overnight at 4 °C. IRDye 680RD Donkey anti-Mouse (LI-COR USA) and IRDye 800CW Donkey anti-Rabbit (LI-COR USA) was used as secondary antibody. The visualization of protein bands was accomplished by Li-cor Odyssey infrared imaging system (LI-COR USA).t test, one-way ANOVA and Dunnett’s multiple comparisons test were used. In all analyses, p values less than 0.05 were considered significant.All experiments were repeated at least three times independently. Date was presented with mean ± SD, and statistical analysis was performed using Prism 8 . 18. Unfortunately, targeted treatments are limited, current treatments usually only relieve itching or reduce irritation caused by external factors, do not cure. Such as glucocorticoids were used to treat inflammatory skin pruritus19. In addition, patients need to prolong prescribing, which may lead to drug resistance and adverse side effects. Due to its complex mechanism, pruritus is rarely studied. In this study, in order to explore the therapeutic mechanism of WLZYW in pruritus, network pharmacology was used to find the key agents and target pathways by constructing ingredients-targets-pathway and other networks.Pruritus is the most common complaint encountered by dermatologists, and it causes psychological stress and insomnia. Moreover, in patients with atopic dermatitis, it results in skin barrier damage. Pruritis aggravates the progression of skin disease and increases sensitivity to antigens20. The activation of these molecules enhances genes expression, the most important is VEGF21. Binding of VEGFR to VEGF ligand will directly activate phosphotyrosine residues, such as Sck, PLC-γ, PI3K, Akt, and MAPK22. VEGFa is a versatile polypeptide that mediates endothelial cell specific responses, including induced proliferation and vascular leakage. Krause23 reported significant improvement in simple pruritus patients treated with bevacizumab . Moreover, serum levels of VEGF were significantly higher in patients with pruritus23, and correlated with severity assessed by physicians. VEGFa can effectively enhance vascular permeability, with molar efficacy approximately 1000-fold greater than that of histamine24. In addition, mast cells constitutionally express VEGF as preformed heparin-binding factor25 and can secrete this protein to respond to triggers, including IgE and antigen4.Now the mechanism of itching is thought that degranulation via Lyn in mast cells or basophils results in the phosphorylation and recruitment of Syk, which in turn activates the substrate LAT and NLAT signaling molecules, such as PI3K/AKT, ERK1/2, PLC, PKC, transcription factors, and NF-κB26. Flavonoid derivatives act as antivascular agents by interfering VEGF/VEGFR2 pathway27. Kaempferol inhibits VEGF secretion in a time-dependent manner by regulating the signal cascades of PI3K/AKT, MEK and ERK29. Quercetin can inhibit the secretion of angiogenic factor30. Kaempferol and quercetin effectively inhibit the development of IgE-mediated allergic inflammation31. Kaempferol prevents MC-mediated allergic diseases by attenuating Lyn activation32. ERK1/2 is regulated dose-dependently by luteolin, induced by inhibition of AKT and MAPK pathways33. Luteolin can inhibit the FcεRΙ- and MrGPrx2-mediated allergic reaction in vivo and in vitro, and reduce the serum levels of histamine, TNF-α, MCP-1, IL-8 and IL-13 in mice34.After screening drug targets network, 16 core targets were identified following multiple analyses of 160 common targets between WLZYW and pruritus. The results indicated that kaempferol, luteolin, and quercetin might be the key ingredients in WLZYW able to exert therapeutic effects against pruritus. All three are natural flavonoids found in many plants with therapeutic properties, such as antioxidant, anticancer, and anti-inflammatory35. LY294002 (Akt phosphorylation antagonist) can effectively treat atopic dermatitis in mice and significantly inhibit scratching behaviors36. These results suggested that Akt can be targeted in the treatment of pruritus. RBL-2H3 cell line is a useful in vitro model for studying the degranulation of mast cells induced by IgE–FcεRI37. ERK1/2 regulates the phosphorylation of cPLA2, as well as the production and release of arachidonic acid, which are the major factors of pruritus38. Through in vitro experiments, both concentrations of WLZYW significantly inhibited PI3K/AKT and ERK1/2 signaling pathways, and the degree of degranulation in the model reduced. To understand the relationship between two pathways and VEGFa, wortmannin (1 µM) and U0126 (2 µM) were added to inhibit the model. Western blot and PCR experiments proved WLZYW induced down-regulation of TNF-α and VEGFa mRNA and protein levels. In conclusion, WLZYW downregulates VEGFa expression by inhibiting the phosphorylation of PI3K/AKT and ERK1/2 pathways.GO and KEGG pathways enrichment results indicated that WLZYW might exert effects on pruritus by regulating PI3K-Akt (has04151) signaling pathway. Pathview revealed that WLZYW act not only on AKT, but also on the upstream and downstream of AKT pathway, as well as on VEGF and ERK1/2. Recently, in vivo experiments have confirmed the effects of p-AKT and p-ERK in chronic pruritus, enhance the expression of melatonin and improve the quality of sleepOur results suggest that the active components of WLZYW regulate mast cell activation in many ways, thereby inhibiting the release of sensitizing mediators caused by degranulation, which is main mechanism in the treatment of pruritus. The limitation of this study is that in vivo experimental verification was not carried out. In addition, the etiology of pruritus is complex, and it is difficult to simulate perfectly with only one model.Network pharmacology can demonstrate the pharmacological effects of WLZYW on pruritus via multiple components, multiple targets and multiple pathways. The molecular docking results suggest that the main pathway is PI3K/AKT, the main target is VEGFa. In vitro, WLZYW inhibits DNP-IgE/BSA-induced RBL-2H3 degranulation and regulates VEGFa expression by inhibiting PI3K/AKT and ERK1/2 pathways.Supplementary Legends.Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.Supplementary Legends.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.Supplementary Table 7.


Chromobacterium violaceum, has gained extensive research interest in recent years due to its huge potential in the pharmaceutic area and industry. In this review, we summarize the latest research advances concerning this pigment, which include (1) fundamental studies of its biosynthetic pathway, (2) production of violacein by native producers, apart from C. violaceum, (3) metabolic engineering for improved production in heterologous hosts such as Escherichia coli, Citrobacter freundii, Corynebacterium glutamicum, and Yarrowia lipolytica, (4) biological/pharmaceutical and industrial properties, (5) and applications in synthetic biology. Due to the intrinsic properties of violacein and the intermediates during its biosynthesis, the prospective research has huge potential to move this pigment into real clinical and industrial applications.Violacein, a purple pigment first isolated from a gram-negative coccobacillus  Furthermore, the violacein extraction selectivity using [N4444]Cl–SDS is notably superior to that achieved using the commercial nonionic surfactant Triton X-114 [Another yeast species, bstances , which ae colors . Subseque colors . As the e colors . MeanwhiIn recent years, researchers in synthetic biology and metabolic engineering have frequently utilized the violacein pathway to evaluate their designs. This is mainly due to the easily detectable hues of violacein and the intermediates on the pathway as the reporters, as well as the proper number of genes in the cluster, similar to other systems metabolic engineers typically meet to manipulate. The applications of violacein in synthetic biology are summarized in vioABE, whose product prodeoxyviolacein (PDV) or its derivative is visibly green and highly red fluorescent , which yielded a 95% reduction of PDV compared with a 63% reduction with the canonical PTS1 tag (SKL). Then, the optimized tag ePTS1 was fused to β-glucosidase, vioA, B, or E, respectively, to estimate the permeability of the peroxisomal membrane, which indicates the membrane has a cut-off limit of small molecules with molecular weight between 571-733 Da (S. cerevisae to sequester the toxic truncated norcoclaurine synthase (tNCS), from Coptis japonica, which catalyzes the first committed step for the biosynthesis of benzylisoquinoline alkaloids (BIAs). They further identified peroxisome protein capacity as a limiting factor and induction of larger peroxisomes was achieved by the addition of oleate, leading to further improvement of BIA production [In the applications as reporters, the Dueber group fully characterized yeast peroxisome for pathway compartmentalization to improve the biocatalytic efficiency by using orescent . By expr1-733 Da . They aloduction .E. coli [et al. constructed the combinatorial library of violacein pathway genes in S. cerevisiae with different promoter strengths, and applied and trained a linear regression model. This model enabled the prediction of optimized production based on a few measurements only comprising 3% of the total library, which should also be useful in engineering new pathways in other systems [S. cerevisiae. From the initial screen of 24 random strains, this method allowed identification of a combination with 2.42-fold improved violacein production among 3125 possible designs. Furthermore, this method also reliably predicted the synthesis of very pure violacein, avoiding the branch pathway for the by-product deoxyviolacein formation [S. cerevisiae through a toolkit having specialized transcription factors and promoters that are designed to control the expression of vioC and vioD genes in the violacein biosynthetic pathway. These DNA parts with efficient performances can lead to the development of fine genetic circuits, that can be utilized in several biotechnological applications [Synthetic biologists favor the use of the violacein biosynthetic pathway also due to the proper number of five genes in the cluster. For example, the violacein pathway has been used as the reporter to evaluate the efficiency of gene assembly toolkits developed based on Golden Gate assembly in E. coli  and yeasE. coli . Lee et  systems . Similarormation . In anotormation . In anotications .et al. constructed a T7 promoter mutant library in E. coli by site-directed mutagenesis, from which promoters of different strengths were obtained. The combinatorial library of vioA, B, C, D, and E was then built up using these promoters, and violacein production can be directly evaluated in a high-throughput manner, based on the violet color. Such fine-tuning method for multiple genes, called ePathOptimize, can be straightforwardly applied to other multi-step pathways to balance metabolic flux [In metabolic pathway engineering, the vivid violet hue of violacein or deoxyviolacein has been utilized to evaluate genetic mutants for improved production by high-throughput ways. For example, Jones lic flux . In anotlic flux .e.g., lycopene, carotenoid, pyocyanin as reported have been used to derive whole-cell biosensors that give visible output signals in response to environmental pollutants [Finally, the accumulation of heavy metals in the environment including water bodies, soil, and foods caused by various anthropogenic activities of humans has become a matter of concern with increasing risk factors and morbidity . Geneticllutants . A wholellutants . Anotherllutants .This review focuses on the bacterial secondary metabolite, violacein, which has shown promising potential in various biotechnological applications. In this compilation of recent research on violacein, we have demonstrated the increased interest in natural and recombinant producers of violacein. Similarly, there is a lot of interest in its various properties of industrial, biological, and research significance. The structure and synthetic biology perspective of violacein has also been discussed. Therefore, there is wide room for future research in increasing its yield, fermentation, and applications.


Anguilla anguilla. Tandem mass tag (TMT)-labelling proteomics with the liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for performing quantitative identification of the proteins. In addition, the quantitative protein amount was detected by parallel reaction monitoring (PRM) analysis. A total of 3486 proteins were identified, of which 2935 were quantified. When a protein fold change was greater than 1.3 or less than 0.76, it indicated a differentially expressed protein (DEP). Overall, 187 up-regulated proteins and 126 down-regulated proteins were detected, and most of the DEPs were enriched in the CAMs pathway, intestinal immune pathway, herpes simplex virus 1 infection pathway, phagosome pathway and p53 signaling pathway. The results of the DEPs detected by PRM were highly consistent with the results of the TMT-labelled quantitative proteomic analysis. The findings of this study provide an important research basis for further understanding the pathogenesis of AngHV.Anguillid herpesvirus 1 (AngHV) is an important viral pathogen affecting eel. This study was designed to investigate the potential molecular mechanisms and immune response elicited at the protein levels in the skin mucus of AngHV-infected  A. japonica in 1990 by Sano et al. , the max number of missed cleavages as 0, the peptide length was set as 7–25, the fixed modifications as alkylation on Cys. For the transition settings, precursor charges were set as 2, 3; the ion charges as 1; and ion types as b, y. Moreover, the productions were set from ion 3 to the last ion, and the ion match tolerance as 0.02 Da.analysis . The samIn conclusion, a total of 187 up-regulated proteins and 126 down-regulated proteins were found in the skin mucus of the eels infected with AngHV. Most of the identified DEPs were enriched in the CAMs pathway, intestinal immune pathway, herpes simplex virus 1 infection pathway, phagosome pathway and p53 signaling pathway. Overall, the results provided important information for the elucidation of pathogenesis of AngHV and understanding the various defense mechanisms of infected eels.


Lysine (Lys) is essential for skeletal muscle growth and protein synthesis in mammals. However, the regulatory network underlying Lys-regulated skeletal muscle development is unknown. To determine whether any cross-talk occurs among mammalian targets of rapamycin complex 1 (mTORC1) and Lys in the regulation of muscle satellite cells (SCs) proliferation, we applied the treatment rapamycin (a mTORC1 inhibitor) and MHY1485 (a mTORC1 activator) on Lys-added or -deficient SCs. The results show Lys deprivation significantly decreases SCs viability, protein synthesis, and cell cycling, increases autophagy and apoptosis, and inhibits the mTORC1 signaling pathway. Restoration of Lys content significantly attenuates this effect. mTORC1 signaling pathway activation during Lys deprivation or mTORC1 signaling pathway inhibition during Lys addition attenuates the effect of Lys deprivation or addition on SCs viability, protein synthesis, cell cycling, autophagy, and apoptosis. In conclusion, Lys could improve SCs proliferation, and inhibit SCs apoptosis and autophagy, via the mTORC1 signaling pathway. Muscle growth is driven by muscle stem cells, which can eventually form the most basic structures of skeletal muscle. Muscle SCs are mainly found in the basement membrane and myofibrils of muscle fibers . SkeletaLys is the first limiting amino acid (AA) for most mammals, which could promote muscle protein synthesis and hypertrophy of muscle fibers ,10. Lys mTORC1 is a conserved serine/threonine (Ser/Thr) kinase, which is recognized as the main AA receptor . In receIn the current work, we compared our research with previous studies ,14,15,162 for 2 h. Then, later, the suspensions were transferred onto new plates, and differential apposition was repeated 3–4 times to remove a large number of fibroblasts, labeled as P0 generation cells, and SCs started to culture for about 24 h. The culture was maintained until confluency reached about 80%, and the cells were then washed twice with sterile phosphate buffer solution , dissociated by 0.25% trypsin–EDTA. The cells were passaged onto new plates at a ratio of 1:2. As the number of passages increased, the cells continued to proliferate.Single muscles were first isolated from the dorsolumbar muscles of 3 day old rabbits (3 rabbits), excess fat and connective tissue were cut off with ophthalmic scissors, and the muscles were cut into minced meat and digested in 0.3 mg/mL collagenase I  and 0.25% trypsin–ethylene diamine tetraacetic acid (Trypsin–EDTA)  for 60 min before the addition of Dulbecco’s modified eagle medium/F-12  containing 10% fetal bovine serum  to terminate the digestion. Tissue debris with cell suspension was collected by passing the cells through a 70 μm cell sieve, centrifuged at 1200 rev/min for 8 min at room temperature. The supernatant was then removed, and the cells were initially resuspended by adding complete medium to obtain a cell suspension. Cells were suspended in selection medium containing 15% FBS, 10 ng/mL basic fibroblast growth factor (BFGF), 10 ng/mL epidermal growth factor (EGF), 2 mM/L L-glutamine, and 1% penicillin–streptomycin in DMEM/F12 . The cell suspensions were inoculated into T25 culture flasks and cultured at 37 °C in 5% COP0 generation cells were inoculated into six-well microplates for immunofluorescence and induction of differentiation identification. Anti-desmin, anti-myogenic differentiation (MyoD), anti-paired box 7 (Pax7), and anti-myosin heavy chain (MYHC) were used; all antibodies came from Cell Signaling Technology . Immunofluorescent detection was performed as previously described .Cells adhered to the wall after inoculation for 24 h, then starved for 12 h in the medium without Lys and FBS, and divided into three groups: cultured in the medium with 0.92 mmol Lys for 120 h ; cultured in the medium with 0.02 mmol Lys for 120 h ; cultured in the medium with 0.02 mmol Lys for 48 h, and then cultured in the medium with 0.92 mmol Lys for 72 h . The cell proliferation was measured at 24, 48, 72, 96, and 120 h, and the cell samples were collected at 120 h .Cells adhered to the wall after inoculation for 24 h, and were then starved for 12 h in the medium without Lys and FBS, and divided into five groups: cultured in the medium with 0.92 mmol Lys for 120 h ; cultured in the medium with 0.02 mmol Lys for 120 h ; cultured in the medium with 0.02 mmol Lys for 48 h; and then cultured in the medium with 0.92 mmol Lys for 72 h ; cultured in the medium with 0.02 mmol Lys for 48 h, and then cultured in the medium with 0.92 mmol Lys and 1 μM rapamycin for 72 h ; cultured in the medium with 0.02 mmol Lys for 48 h, and then cultured in the medium with 0.02 mmol Lys and 1 μM MHY1485 for 72 h . Cell proliferation was measured at 120 h and the cell samples were also collected at 120 h .For the Cell Counting Kit-8  method, 20 μL of water-soluble tetrazolium salt was added to each well, incubated for 1 h at 37 °C in a constant temperature incubator, and then removed and restored to room temperature. The OD of each well was measured at 450 nm using a microplate reader .Cell cycle progression and apoptosis were measured using PI/RNase standing buffer  and PE Annexin V Apoptosis Detection Kit I , respectively, with flow cytometry . The results were analyzed with ModFit LT 5.0 software .Total protein was extracted from SCs using a radioimmunoprecipitation assay (RIPA) lysis buffer , and the protein concentrations were determined using a bicinchoninic acid assay (BCA) protein assay Kit . The extracted proteins (50 ng/sample) were solubilized in 40 mL of sodium dodecyl sulfate (SDS) loading buffer  and then resolved by electrophoresis  on 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) gels prior to electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes . Standard markers for protein molecular masses were purchased from Thermo . The proteins were closed in fast closing solution at 37 °C for 30 min and incubated with primary antibodies at 1:2000 dilution ; anti-phospho-P70S6K1, Thr389; anti-ribosomal protein S6 (S6); anti-phospho-S6, Ser235/236; anti-eukaryotic translation initiation factor 4E binding protein 1 (4EBP1); anti-phospho-4EBP1, Ser65; anti-eukaryotic translation initiation factor 4E (eiF4E); anti-muscle atrophy F box protein (Atrogin1); anti-muscle specific finger protein 1 (MuRF1); anti-microtubule-associated protein 1 light chain 3 beta (LC3B); anti-apoptosis-related cysteine peptidase 3 (caspase-3); anti-tubulin; anti-β-actin; and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), all from Proteintech . The membranes were then washed with Tris-buffered saline containing tween , and incubated with a 1:1000 dilution of a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody  at 37 °C for 1 h. The proteins were visualized using Beyo ECL reagents  as the reference . The intTo detect protein synthesis, we used a non-radioactive technical method called surface sensing of translation (SUnSET) . In our p < 0.05.The data were expressed as the mean ± SD and analyzed using one way ANOVA with SAS software. Multiple comparisons between the groups were performed with Tukey tests. Differences between treatments were considered statistically significant when In the present study, we isolated SCs from 3 day old rabbit dorsal girdle muscles and identified SCs by the induced differentiation method and immunofluorescence before the start of the experiment. The results of induced differentiation show that SCs appear as distinct orange–red lipid droplets after oil red O staining, and SCs successfully differentiate into adipocytes. The results of immunofluorescence show that almost all anti-desmin, anti-MyoD, anti-Pax7, and anti-MYHC are co-localized with 4′,6-diamidino-2-phenylindole (DAPI)-labeled nucleic acids, which is evidence that these cells are SCs .p < 0.05) (p < 0.05), and significantly recovers with adequate Lys supplementation (p < 0.05) (p > 0.05) (p < 0.05).Compared with the control group, the SCs proliferation rate is significantly reduced between 24 and 120 h of continuous Lys deficiency, which is significantly restored when Lys is added ( < 0.05) A. The SU < 0.05) B. The ex > 0.05) C. Lys co > 0.05) D. The lep < 0.05), and in the second gap/mitotic phase (G2/M) is significantly decreased (p < 0.05) compared with the control group. Compared with the Lys deficiency group, the proportion of SCs in G0/G1 is significantly reduced (p < 0.05), and in G2/M is significantly increased (p < 0.05) in the Lys addition group. Lys level has no significant effect on cell proportion in the S phase (p > 0.05)  is significantly increased ( > 0.05) A–F.p < 0.05). Compared with the Lys deficiency group, cell apoptosis rate and caspase-3 protein expression decreases in the Lys addition group (p < 0.05) (p < 0.05). The LC3-II/LC3-I protein ratio in the Lys addition group is significantly lower than in the Lys deficiency group (p < 0.05)  A–E. The  < 0.05) F.p < 0.05). MHY1485 treatment in absence of Lys significantly restores cell proliferation compared to the Lys deficiency group (p < 0.05) (p < 0.05). In contrast, MHY1485 treatment in the Lys deficiency condition significantly increases SCs protein synthesis compared with the Lys deficiency group (p < 0.05) . Compared with the Lys deficiency group, MHY1485 treatment in Lys deficiency significantly increases the protein expression of p-mTOR P-S6K1, P-S6, P-4EBP1, and eiF4E (p < 0.05) (Proliferation of SCs in the Lys addition + rapamycin group is significantly diminished compared to the Lys addition group ( < 0.05) A. Rapamy < 0.05) B. Rapamy < 0.05) C.p < 0.05). In contrast, MHY1485 treatment in the Lys deficiency group significantly increases the cell ratio in G0/G1, and decreases the cell ratio in G2/M compared with the Lys deficiency group. The rapamycin and MHY1485 treatments have no significant effect on cell ratio in the S phase (p > 0.05)  .p < 0.05). In contrast, the MHY1485 treatment in the Lys deficiency condition significantly decreases the apoptosis rate and caspase-3 expression compared with the Lys deficiency group (p < 0.05) (p < 0.05). In contrast, the Lys deficiency + MHY1485 group has a significant decrease in the LC3-II/LC3-I protein ratio compared with the Lys deficiency group (p < 0.05) (Rapamycin treatment in the Lys addition condition significantly increases apoptosis rate and apoptotic protein caspase-3 expression compared with the Lys addition group ( < 0.05) A–G. The  < 0.05) H.In the current study, we investigated the effect of Lys deficiency or addition on SCs proliferation, apoptosis, and autophagy, and whether the mTORC1 signaling pathway is involved. Our data demonstrate that (1) Lys deficiency decreases SCs proliferation and protein synthesis, and induces cell autophagy and apoptosis, which is attenuated when Lys is added; (2) the activation of the mTORC1 signaling pathway attenuates the effect of Lys deficiency on SCs proliferation, protein synthesis, autophagy, and apoptosis; (3) the inhibition of the mTORC1 signaling pathway attenuates the effect of Lys addition on SCs proliferation, protein synthesis, autophagy, and apoptosis. The results suggest that Lys stimulates cell proliferation and inhibits cell apoptosis via the mTORC1 signaling pathway in SCs.Lys plays an essential role in skeletal muscle growth by acting as a cell signaling molecule . SimilarIn addition, there is a large amount of protein synthesis during cell proliferation . We examApoptosis and autophagy are two forms of programmed cell death , but autIn line with a previous study , Lys defmTORC1 is a conserved Ser/Thr kinase that is recognized as a major receptor for AA . mTORC1 To further determine the potential role of the mTORC1 signaling pathway in SCs during Lys-regulated autophagy and apoptosis, the mTORC1 signaling pathway was blocked by rapamycin treatment in the condition of Lys addition, and the mTORC1 signaling pathway was activated by MHY1458 treatment in the condition of Lys deprivation. Activation or inhibition of the mTORC1 signaling pathway attenuates the effect of Lys deprivation and Lys addition, respectively, on cell proliferation, protein synthesis, and the cell cycle. Similar to previous studies, activation or inhibition of the mTORC1 signaling pathway regulates the rate of protein synthesis, which further affects cell proliferation ,40. ThesmTORC1 is an important suppressor of autophagy and a regulator of cell metabolism. The role of the mTORC1 signaling pathway and its involvement in regulation of apoptosis and autophagy remains controversial . In one Our findings demonstrate that Lys improves the proliferation, cellular protein synthesis, and the cell cycle in SCs, and inhibits autophagy and apoptosis. Moreover, the mTORC1 signaling pathway mediates these processes. These effects are ultimately be reflected in the growth and hypertrophy of skeletal muscle .


Shenlingbaizhu powder (SLBZP), one of the classic Earth-cultivating and gold-generating prescriptions of traditional Chinese medicine, is widely used to treat various diseases. However, the pharmacological mechanisms of SLBZP on bronchial asthma (BA) and allergic colitis (AC) remain to be elucidated. R software. The key targets were screened by using the plug-in “cytoHubba” of Cytoscape software, and the “active compound-key target” network was constructed. Molecular docking analysis was performed using AutoDock software. The miRTarBase database was used to predict microRNAs (miRNAs) targeting key targets, and the key target-miRNA network was constructed.  Network pharmacology and molecular docking technology were used to explore the potential mechanism of SLBZP in treating BA and AC with the simultaneous treatment of different diseases. The potential active compounds of SLBZP and their corresponding targets were obtained from BATMAN-TCM, ETCM, SymMap TCM@TAIWAN, and TCMSP databases. BA and AC disease targets were collected through DisGeNET, TTD, GeneCards, PharmGKB, OMIM, NCBI, The Human Phenotype Ontology, and DrugBank databases. Common targets for drugs and diseases were screened by using the bioinformatics and evolutionary genomics platform. The analyses and visualizations of Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of common targets were carried out by  Through screening, 246 active compounds and 281 corresponding targets were obtained. Common targets were mainly enriched in 2933 biological processes and 182 signal pathways to play the role of treating BA and AC. There were 131 active compounds related to key targets. The results of molecular docking showed that the important active compounds in SLBZP had good binding ability with the key targets. The key target-miRNA network showed that 94 miRNAs were predicted.  SLBZP has played the role of treating different diseases with the same treatment on BA and AC through the characteristics of multicompound, multitarget, and multipathway of traditional Chinese medicine, which provides a theoretical basis for explaining the mechanism and clinical application of SLBZP treating different diseases with the same treatment in BA and AC. Asthma generally refers to bronchial asthma (BA). BA, one of the most common chronic noncommunicable diseases in children and adults, is characterized by variable respiratory symptoms and variable airflow limitation, which is the result of complex gene-environment interactions, and is heterogeneous in clinical manifestations and the type and intensity of airway inflammation and remodeling . The goaof Peaceful Benevolent Dispensary and composed of 10 Chinese medicines including renshen (Panax ginseng C. A. Mey.), fuling (Poria cocos (Schw.) Wolf.), baizhu , baibiandou (Lablab Semen Album), shanyao (Rhizoma Dioscoreae), lianzi (Semen Nelumbinis), yiyiren (Coicis Semen), sharen (Amomum aurantiacum H. T. Tsai Et S. W. Zhao), jiegeng (Platycodon grandiforus), and gancao (licorice), has the effects of replenishing qi, strengthening spleen, excreting dampness, and stopping diarrhea [Shelingbaizhu powder (SLBZP), from the Prescriptions diarrhea . Previoudiarrhea , 14.Network pharmacology, targeting biological networks, analyzes the connections between drugs, targets, and diseases in these networks. A comprehensive and systematic research on network pharmacology conforms to a holistic view, which is the main characteristic of many traditional medicines. Studies have shown that many traditional medicines exhibit synergistic effects by acting on multiple targets and pathways at different levels through network pharmacology . This mehttp://bionet.ncpsb.org.cn/batman-tcm/index.php/Home/Index/index) [http://www.tcmip.cn/ETCM/index.php/Home/Index/) [http://www.symmap.org/) [http://tcm.cmu.edu.tw/review.php?menuid=3) [http://lsp.nwu.edu.cn/tcmsp.php) [https://www.drugbank.ca/) [https://www.uniprot.org/?tdsourcetag=s_pcqq_aiomsg) [The active compounds of SLBZP were separately obtained from these databases: BATMAN-TCM (x/index) , ETCM (h/Index/) , SymMap ap.org/)  and Tradenuid=3) . Then, tmsp.php) . Meanwhiank.ca/)  and UniP_aiomsg)  to get thttp://www.disgenet.org/web/DisGeNET/menu/search) [https://db.idrblab.org/ttd/) [https://www.genecards.org) [https://www.pharmgkb.org/) [https://omim.org/) [https://www.ncbi.nlm.nih.gov/gene) [https://hpo.jax.org/app/) [The target genes related to BA were obtained with the keyword “bronchial asthma” and the species set as “Homo sapiens” from these 8 databases: DisGeNET (/search) , TTD (htrg/ttd/) , GeneCarrds.org) , PharmGKkb.org/) , OMIM (him.org/) , NCBI (hov/gene) , The Humrg/app/) , and Druhttp://bioinformatics.psb.ugent.be/webtools/Venn/). At the same time, a Venn diagram was drawn to obtain the common targets of the active compounds of SLBZP for treating BA and AC.The targets related to active compounds, BA, and AC were matched and mapped by using the bioinformatics and evolutionary genomics platform .The enrichment analysis and visualization of Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were carried out for the common targets of SLBZP in treating BA and AC with the species set as “Homo sapiens” and the threshold set as rofiler” , and “gghttp://www.cytoscape.org) [The obtained common targets were imported into Cytoscape software  , and thehttps://www.rcsb.org/) [https://pubchem.ncbi.nlm.nih.gov/) [According to the above analysis results, the key target proteins and the important active compounds were molecularly docked. The protein structures of the targets were obtained from the RCSB PDB database (sb.org/) . The 2D ih.gov/)  and werehttps://mirtarbase.cuhk.edu.cn/%7EmiRTarBase/miRTarBase_2019/php/index.php) is used to predict upstream miRNAs targeting key targets [The miRTarBase database , 75 for cellular component (CC), and 171 for molecular function (MF). The results of GO enrichment analysis showed that the common targets of SLBZP in treating BA and AC mainly involved response to oxidative stress, response to molecule of bacterial origin, membrane region, membrane microdomain, signaling receptor activator activity, receptor ligand activity, and other biological functions . 182 sigThe 149 common targets obtained above were screened by “cytoHubba”, a plug-in of Cytoscape software, and then the 20 key targets with the highest degree value were obtained . These 2π-σ interactions with amino acid residues A chain TYR248 and LEU188, formed π-π stacked interactions with amino acid residue A chain HIS226, and formed π-alkyl interactions with amino acid residues A chain VAL223 and LEU188. These forces reduced the binding energy and increased the affinity, which played an auxiliary role in the binding of compound ligand molecules to the residues of target protein structures.Based on the above analysis results, the 5 important active compounds  and the key targets were docked by AutoDockVina software. The docking results are shown in 94 miRNAs were predicted from 6 key targets by the miRTarBase database. Cytoscape software was used to construct a key target-miRNA network , among wThe theory of traditional Chinese medicine believes that the spleen is the foundation of acquired life and that the spleen is not harmonious and causes all kinds of diseases. Therefore, it has always paid attention to regulating the spleen to protect the five internal organs. The pathogenesis of spleen deficiency is involved in the occurrence and development of BA and AC. SLBZP, one of the classic Earth-cultivating and gold-generating prescriptions, can not only treat BA and AC with simultaneous treatment of different diseases but also protect the spleen to prevent and promote recovery. This study aimed to explore the action mechanism of SLBZP in treating BA and AC with simultaneous treatment of different diseases by using network pharmacology and molecular docking, so as to provide references for more in-depth experimental research and wider clinical applications.GO annotation results showed that the biological functions involved in common targets were mainly response to oxidative stress, response to molecule of bacterial origin, membrane region, membrane microdomain, signaling receptor activator activity, receptor ligand activity, and so on. In addition, the main enrichment pathways of common targets were PI3K-Akt signaling pathway, proteoglycans in cancer, MAPK signaling pathway, IL-17 signaling pathway, TNF signaling pathway, apoptosis, Th17 cell differentiation, and other pathways related to inflammation, cancer, apoptosis, and immunity. Studies pointed out that, during the onset of asthma, both PI3K-Akt signaling pathway and MAPK signaling pathway were active , 36. ManThe active compound-key target network of this study showed that the five active compounds of quercetin, luteolin, beta-carotene, kaempferol, and naringenin, and the 6 key targets of PTGS2, CASP3, AKT1, JUN, TP53, and VEGFA were particularly important. Moreover, the results of molecular docking also verified that these five active compounds had good binding characteristics with their corresponding important key targets, indicating that they played vital roles in SLBZP for treating BA and AC with simultaneous treatment of different diseases and had critical potential research values.κB signaling pathways to protect ARPE-19 cells from the proliferation of IL-6, IL-8, sICAM-1, and MCP-1 stimulated by IL-1β, thereby alleviating the inflammatory response [κB signal transduction, which may help alleviate the inflammatory response associated with Cox2 expression [κB activity in a mouse model of asthma [Studies suggested that quercetin was known for its antioxidant activity in free radicals scavenging and antiallergic properties . It is cresponse . Kaempfepression –63. Narif asthma –66. The β-catenin signaling in the intestine, JUN is not only involved in inflammatory response and tumorigenesis but is also related to the inflammatory response in mice with LPS-induced macrophages and DSS-induced colitis [κB and promotes chronic inflammation and inflammation-related colorectal cancer, which is also related to the occurrence and development of inflammatory bowel disease [PTGS2, as the most critical target in the network, is one of the key factors of cell response to inflammation and has long been considered to play a key role in the pathogenesis of respiratory inflammation, including asthma , 68. In  colitis , 74. TP5 colitis . It ofte disease –78. VEGF disease . However disease –82. The α, IL-1β, and IFN-γ, and promoted the production of IL-10, thereby reducing allergic inflammation [The key target-miRNA network shows that hsa-miR-16-5p, hsa-miR-101-3p, hsa-miR-143-3p, hsa-miR-199a-5p, hsa-miR-30d-5p, hsa-miR-30c-5p, hsa-miR-30e-5p, hsa-miR-302d-3p, hsa-miR-203a-3p, hsa-miR-200b-3p, hsa-miR-125a-5p, hsa-miR-15a-5p, hsa-miR-504-5p, and hsa-miR-150-5p all target and regulate multiple key targets, which may have important upstream regulatory effects and are of great significance for the occurrence, development, and treatment of BA and AC. Studies suggested that baseline airway secretion signatures of hsa-miR-302d-3p and hsa-miR-612 were detected during rhinovirus (RV) infection that was the most common cause of asthma exacerbation and the most important early risk factor for asthma development after childhood in children, which was helpful to develop novel strategies for treating and monitoring respiratory conditions in all age groups . The lowammation . Downregammation . Once agIn conclusion, network pharmacology and molecular docking technology demonstrated that SLBZP in treating BA and AC with simultaneous treatment of different diseases was a complex process involving multiple compounds, multiple targets, and multiple pathways. It may involve important active compounds and key targets represented by quercetin, luteolin, beta-carotene, kaempferol, naringenin, PTGS2, CASP3, AKT1, JUN, TP53, and VEGFA, may be related to inflammation, cancer, apoptosis, and immune-related pathways, and may involve the targeted regulation of multiple upstream miRNAs. These can provide references for future clinical and experimental studies.


Primary hypertension is understood as a disease with diverse etiology, a complicated pathological mechanism, and progressive changes. Gedan Jiangya Decoction (GJD), with the patent publication number CN114246896A, was designed to treat primary hypertension. It contains six botanical drugs; however, the underlying mechanism is uncertain. We utilized network pharmacology to predict the active components, targets, and signaling pathways of GJD in the treatment of primary hypertension. We also investigated the potential molecular mechanism using molecular docking and animal experiments. The Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), the Protein Database (UniProt), and a literature review were used to identify the active components and related targets of GJD's pharmacological effects. The GeneCards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and DrugBank databases were utilized to identify hypertension-related targets. Based on a Venn diagram of designed intersection targets, 214 intersection targets were obtained and 35 key targets for the treatment of hypertension were determined using the STRING data platform and Cytoscape software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of key targets revealed that the relevant molecular action pathways of GJD in the treatment of hypertension include the Toll-like receptor, MAPK, PI3K-Akt, and renin-angiotensin signaling pathways. A GJD active ingredient-key target-pathway connection diagram was created using Cytoscape software, and 11 essential active components were selected. Molecular docking was then used to verify the binding activity of key targets and key active ingredients in GJD to treat primary hypertension. The results of this study indicate that AGTR1, AKT1 with puerarin, EDNRA with tanshinone IIA, MAPK14 with daidzein, MAPK8 with ursolic acid, and CHRM2 with cryptotanshinone had high binding activity to the targets with active components, whereas AGTR1 was selected as target genes verified by our experiment. HPLC was utilized to identify the five active ingredients. Experiments in high-salt rats demonstrated that GJD might decrease the expression of AGTR1 in the kidney and thoracic aorta while increasing the expression of eNOS by preventing the activation of the renin-angiotensin pathway, thereby reducing lowering systolic and diastolic blood pressure. Hypertension is well-defined as a high systemic arterial blood pressure induced by the environment, polygenic heredity, and several risk factors. It has a great influence on the structure and function of blood vessels and often leads to brain, renal, and heart complications. It is also a major risk factor for cardiovascular diseases such as coronary heart disease, left ventricular hypertrophy, and arrhythmia . HyperteTraditional Chinese medicine (TCM) is a combination of a complex herbal formula that has been used for thousands of years to treat countless diseases and increase life expectancy in various parts of the world. The complex interaction of several bioactive components in herbal preparations or medicine results in the synergistic therapeutic effect of TCM . When trIn recent years, network pharmacology, based on bioinformatics and systems biology, has been widely used in TCM research to clarify the TCM treatment of complex ailments from a multicomponent, multitarget, multipathway approach . The pri2+ [Gedan Jiangya Decoction  includes six botanical drugs: Uncaria rhynchophylla (Miq.) Miq. ex Havil. (Gouteng), Salvia miltiorrhiza Bunge (Danshen), Pueraria lobata (Willd.) Ohwi. (Gegen), Eucommia ulmoides Oliv. (Duzhong), Prunella vulgaris L. (Xiakucao), and Achyranthes bidentata Blume (Niuxi). Previous studies have revealed that isorhynchophylline and rhynchophylline in Uncaria rhynchophylla can reflexively and directly inhibit the vasomotor center, dilating peripheral blood vessels as exertion antihypertensive effects by inhibiting the influx of extracellular Ca2+ , 17. Stu2+ –21. Prun2+ –24.Previous studies have mostly concentrated on a single medication or ingredient, making it challenging to precisely represent the mechanism of GJD's multichannel, multitarget, and multicomponent compound. Therefore, this research seeks to use the study methods of molecular docking and network pharmacology to produce the GJD active ingredient-key target-pathway, analyze the molecular mechanism of GJD in treating hypertension, and provide a reference for future medical clinical experiments and theoretical research.https://old.tcmsp-e.com/index.php) [https://www.uniprot.org/) [The TCMSP database (dex.php)  was usedot.org/)  is then https://www.genecards.org/), the OMIM database (https://omim.org/), the TTD database (http://db.idrblab.net/ttd/) [https://go.drugbank.com/) [We used the GeneCards database (et/ttd/) , and thenk.com/)  to colleThe GJD-related and primary hypertension-related targets were imported into the R language, and the intersection of the targets was drawn as a Venn diagram. Then, import the intersection targets into the STRING 11.0 data platform , settinghttps://metascape.org) [P < 0.01 as the screening condition; GO function analysis and KEGG pathway enrichment analysis are performed for key targets; and the enrichment results are drawn into a bubble plot in the R language.We import key targets into the Metascape data platform (ape.org) , select The active components corresponding to the key targets were screened out, and the drugs, key targets, and KEGG pathways were transferred into Cytoscape program for analysis, and the network diagram of the GJD active ingredient-key target-pathway was obtained. Use the “Analyze Network” of the Cytoscape toolbar to calculate it, and select the active ingredients with a degree value greater than 2 times the median as the key ingredients in treating primary hypertension.http://www.rcsb.org/) [https://pubchem.ncbi.nlm.nih.gov/) [Using the PDB database (sb.org/)  to downlsb.org/)  and expoih.gov/) ; save itih.gov/)  was usedGJD's composition is as follows: Uncaria rhynchophylla (Miq.) Miq. ex Havil. (Gouteng), 10 g; Salvia miltiorrhiza Bunge (Danshen), 25 g; Pueraria lobata (Willd.) Ohwi (Gegen), 30 g; Eucommia ulmoides Oliv. (Duzhong), 15 g; Prunella vulgaris L. (Xiakucao), 15 g; and Achyranthes bidentata Blume (Niuxi), 20 g. The botanical drugs described above were purchased from Heilongjiang Xiushengtang Pharmaceutical Co., Ltd. First, mix the botanical drugs  with 60% aqueous ethanol solution in a material liquid ratio of 1 g : 10 ml, soak for 0.5 hour, heat and reflux the soaking solution for 1.5 hours (keep the solution slightly boiling during the reflux process), and filter with six layers of degreasing gauze; after that, in the same way, reheat and reflux with 10 times the amount of 60% aqueous ethanol solution for 1.5 hours and filter; then, to get extracts of Salvia miltiorrhiza, Pueraria lobata, Eucommia ulmoides, Prunella vulgaris, and Achyranthes bidentata, combine two filtrates, distill ethanol under reduced pressure using a rotary evaporator, and then dry under reduced pressure and vacuum. Second, soak Uncaria rhynchophylla in a 70% aqueous ethanol solution in a 1 g : 10 ml ratio for 0.5 hour, then heat it to 65~75°C, and soak it for 2 hours. Filter it through six layers of degreasing gauze, then soak it in 10 times amount of 70% aqueous ethanol solution for 2 hours, filter it, then use the above method to get the extract of Uncaria rhynchophylla. Finally, combine the two extracts to make GJD.μm). Puerarin , tanshinone IIA , daidzein , ursolic acid , and cryptotanshinone  were purchased from National Institutes for Food and Drug Control. Take a dosage of puerarin, tanshinone IIA, daidzein, ursolic acid, and cryptotanshinone, and dissolve it in methanol. Standard solutions with concentrations of 0.3, 0.25, 0.5, 0.5, and 0.3 mg/ml were prepared. Before HPLC analysis, 1 g of GJD extract powder is mixed with 10 ml of methanol and filtered through a 0.22 μm filter. The mobile phase was acetonitrile (A) -0.1% aqueous phosphoric acid solution (B). The following is the gradient elution program: 0-14 min, 30-37% A; 14-17 min, 37-55% A; 17-29 min, 55-67% A; 29-40 min, 67-89% A; 40-48 min, 89-97% A; and, 48-54 min, 97-100% A; column temperature: 30°C; flow rate: 1.0 ml/min. The wavelength of detection was 210 nm. The injection volume was 10 μl.This analysis was performed using the Waters Alliance e2695, Waters 2998 PDA Detector, and Diamonsil® C18 column  rats (body weight 170 ± 10 g) were acquired from Liaoning Changsheng Biotechnology Co., Ltd. (license number SYXK (Hei) 2018-007) and maintained at a constant temperature (22 ± 2°C), humidity (55 ± 5%), and lighting (12 : 12 hr light-dark cycle). One week before the experiment, rats were given unrestricted access to food and water to acclimatize to their surroundings. All experimental protocols were approved by the Animal Ethics Committee of Heilongjiang University of Chinese Medicine, with approval number 2020031203.The SD rats were randomly divided into 3 groups, 7 rats in the control group, 7 rats in the model group, and 7 rats in the GJD group. The rats in the control group were fed with standard chow. In contrast, the rats in the other groups were fed with 8% NaCl high-salt feed  05073) for 8 weeks with free access to water. The body weight of the rats in each group was recorded every two weeks. The systolic blood pressure (SBP) and diastolic blood pressure (DBP), as well as heart rate (HR), were monitored every two weeks by the noninvasive blood pressure measurement system ALC-NIBP  using the tail-cuff method. Each rat was measured three times, and the average value was recorded. A long-term 8% high-salt diet for 8 weeks caused hypertension in rats ; rats wiFor four weeks, each rat in the GJD group received 1.5 ml of GJD extract 1.07 g/kg/d by gavage, whereas the other groups received the same amount of 0.9% normal saline by gavage. The body weight of the rats in each group was recorded every two weeks. Rats' SBP, DBP, and HR were recorded every two weeks using the noninvasive blood pressure measurement system (ALC-NIBP). Each rat was measured three times, and the average value was recorded.All rats were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital (45 mg/kg), and blood samples were obtained from the abdominal aorta and put in disposable blood collection tubes. Centrifuge at 3500 rpm for 15 minutes at 4°C, then extract serum and put it in a centrifuge tube. Hearts were removed and washed in cold saline. The heart's left ventricle is obtained by resecting the atrial free wall, major arteries, and right ventricle along the atrioventricular junction. Left ventricular mass index (LVMI) is calculated by the following formula: LVMI = left ventricular weight (mg)/body weight (g). At the same time, thoracic aorta and kidney tissues were taken and frozen at -80°C for detection using RT-qPCR, Western blot, and kits.The ELISA Kit was used to measure the expression levels of renin, Ang II, ALD, and NO in serum and Ang II in renal tissue  kit was purchased from Nanjing Jiancheng Bioengineering Institute). The procedure was carried out in strict accordance with the kit's instructions.ΔΔCT- method was used to calculate and analyze the data, with GAPDH acting as an internal control. The following primers were used for PCR amplification The total RNA was extracted from kidney and thoracic aorta tissues using Trizol reagent (Takara), reverse transcribed into cDNA using PrimeScript RT Kit (Takara), and then subjected to real-time PCR reaction using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara), and the gene expression level was quantified using QuantStudio™3 Real-Time PCR system. The 2μm on the PVDF membrane . The membrane was sealed with 5% nonfat milk at room temperature for 1 hour before incubating with primary antibody overnight at 4°C. The membrane was washed three times with TBST and incubated with a secondary antibody at room temperature for 1 hour, and the protein was identified using an ECL reagent . Primary antibodies include AGTR1 , eNOS , and β-actin .The total protein was obtained from the kidney and thoracic aorta using RIPA lysis buffer with PMSF and phosphatase inhibitor , and protein concentration was measured using a BCA protein detection kit . SDS-PAGE was used to separate the whole protein, which was subsequently transferred to 0.45 P < 0.05 was regarded as statistically significant. The statistical analysis findings were created using the GraphPad Prism 7.0 program.All data in this experiment were statistically analyzed using the SPSS 26.0 software; measurement data were represented as mean ± standard deviation (mean ± SD), and one-way analysis of variance (ANOVA) was used to compare multiple groups. For variance homogeneity of pairwise comparisons between groups, the LSD test was employed, and Tamhane's T2 (M) test was used for variance heterogeneity. We obtained 32 active ingredients of Uncaria rhynchophylla, 59 active ingredients of Salvia miltiorrhiza, 4 active ingredients of Pueraria lobata, 26 active ingredients of Eucommia ulmoides, 11 active ingredients of Prunella vulgaris, and 17 active ingredients of Achyranthes bidentata through ADME screening and the TCMSP database, and according to the literature review, there is one active ingredient of Salvia miltiorrhiza , 38, twoAs shown in The key targets were imported into the Metascape data platform, and the results of the GO enrichment function analysis showed that the targets were mainly involved in biological processes such as blood vessel development, vasculature development, cardiovascular system development, blood vessel morphogenesis, and angiogenesis; the main related molecular functions are transcription factor binding, cytokine receptor binding, receptor regulator activity, receptor-ligand activity, cytokine activity, and nitric-oxide synthase regulator activity.κB pathway, and renin-angiotensin pathway which are strongly correlated with hypertension. According to the enrichment analysis results, according to the LogP value from small to large, the top 30 items of GO-BP, GO-CC, GO-MF, and KEGG were selected, respectively, and visualized by using the R language, as shown in A total of 149 pathways were obtained from the KEGG pathway enrichment analysis. Among them are the Toll-like receptor signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, NF-The GJD active ingredient-key target-pathway network diagram was constructed using Cytoscape see , and thr−1 represented 98% of the targets, indicating that most of the targets had strong binding activity to the active ingredients, demonstrating the reliability of the network pharmacology prediction results. Among them, AGTR1, AKT1 with puerarin, EDNRA with tanshinone IIA, MAPK14 with daidzein, MAPK8 with ursolic acid, and CHRM2 with cryptotanshinone all have a strong correlation with GJD in the treatment of primary hypertension. The target protein's and active ingredient molecules' docking results are shown in HPLC detected five key active ingredients of GJD, including puerarin, tanshinone IIA, daidzein, ursolic acid, and cryptotanshinone. Supplementary Material  shows thP < 0.001), indicating that GJD has a significant antihypertensive effect. In addition, the HR of the rats in the GJD group was also lower than that in the model group. (P < 0.05). By calculating the left ventricular mass/body weight of the rats in each group, after 4 weeks of treatment, the LVMI in the model group was significantly higher than that in the control group (P < 0.001), P < 0.05). We also observed no difference in the body weight of rats between the groups during the same period  in comparison to the control group, and the expression of NO was significantly decreased (P < 0.001). Compared with the model group, the expressions of renin, Ang II, and ALD in the serum of the GJD group were significantly decreased , and the expression of NO was significantly increased (P < 0.01). The expression of Ang II in kidney tissue of rats in the group was also significantly decreased (P < 0.001). The results indicate that GJD may reduce blood pressure by inhibiting the activation of the renin-angiotensin-aldosterone system and upregulating NO expression.The renin-angiotensin-aldosterone system plays an extremely important in the pathogenesis of primary hypertension, and NO is an important vasodilator factor in the body, and their expression changes in serum can reflect the development trend of primary hypertension. As shown in After preliminary verification based on network pharmacology analysis and molecular docking, this study found that AGTR1 and eNOS play key roles in GJD's antihypertension. The results of RT-qPCR revealed that the expression of ACE and AGTR1 mRNA in the GJD group's thoracic aorta and kidney was inhibited. In contrast, the expression of eNOS mRNA was increased. Western blot investigations showed that the GJD group's expression of AGTR1 protein in the thoracic aorta and kidney was significantly lower than that of the model group, whereas eNOS expression was significantly higher. The result is shown in Figures Network pharmacological analysis of Chinese medicines is becoming a research tool for studying the influence of Chinese medicines on diseases, intending to reveal complicated components and unexplored targets –46. The In this study, the network pharmacology results showed that 214 possible GJD targets for the treatment of hypertension were identified. Moreover, 35 hub targets were also uncovered in the PPI network diagram of potential targets, including STAT3, AKT1, APP, MAPK1, TP53, EP300, JUN, RELA, CXCL8, TNF, and EDN1. Based on the diagram of the GJD active ingredient-key target-pathway network, 11 components that were identified as active  are correlated with major targets of GJD in the treatment of hypertension, and it can be utilized as major components that play a pivotal role.κB, and renin-angiotensin signaling pathways are major targets of GJD in the treatment of hypertension. Ang II is the most powerful vasoconstrictor molecule in RAAS, and it causes hypertension through oxidative activation and inflammatory response. Ang II may also change myocardial and blood vessels through controlling ALD production [According to the KEGG analysis, the Toll-like receptor, MAPK, PI3K-Akt, TNF, NOD-like, NF-oduction . In this−1, indicating the binding of AGTR1 to puerarin is very active. Therefore, we performed experimental validation of the AGTR1 target.A molecular docking verification was conducted on 35 key targets and 11 active ingredients. The results indicate the AGTR1, AKT1 with puerarin, EDNRA with tanshinone IIA, MAPK14 with daidzein, MAPK8 with ursolic acid, and CHRM2 with cryptotanshinone had a high binding activity to targets with active ingredients. The smaller the binding energy, the more stable the ligand-receptor binding conformation, and the greater the possibility of interaction [When it comes to hypertensive individuals, antihypertensive medication is likely to lower the occurrence and death from disorders such as cardiovascular and cerebrovascular. Many clinical studies have pointed out that lowering the systolic blood pressure (SBP) by 10 mmHg or the diastolic blood pressure (DBP) at least by 5 mmHg may reduce the risk of mortality by 10%-15%, resulting in a 35% of reduced risk of stroke, 20% of lower risk of coronary heart disease, and 40% of lower risk of heart failure . There aMany studies have been carried out recently to examine the association between excessive salt intake and elevated blood pressure; how increased salt consumption causes hypertension is not yet exclusively understood. Several studies have confirmed that high-salt foods damage the heart, renal, and vascular tissue and change the function of the renin-angiotensin system mechanism, thereby changing the levels of Ang II, ALD, renin, and AGTR1 in high-salt rats , 50, 51.The current study acknowledges that the GJD reduces the SBP, DBP, heart rate, and LVMI in high-salt hypertensive model rats and alters blood pressure-related biomarkers in rat serum. Renin, Ang II, and ALD expressions in the GJD group are also considerably reduced, but NO expression is highly increased. The results of RT-qPCR and WB indicate that GJD could lower the expression of AGTR1 mRNA and protein in the kidney and thoracic aorta of high-salt hypertension model rats, whereas it enhances the expression of eNOS mRNA and protein in the kidney and thoracic aorta, which concurs with the results of network pharmacology and a molecular docking prediction. Hence, this study confirms a few key targets and particular pathways.As such, it can be concluded that the network pharmacology and molecular docking methods were utilized to investigate the intervention mechanism of GJD in primary hypertension. The research uncovered that the key active ingredients of GJD in managing primary hypertension are puerarin, tanshinone IIA, daidzein, ursolic acid, and cryptotanshinone, which are mainly expected to contain a regulatory function in conjunction with AGTR1, AKT1, EDNRA, MAPK14, MAPK8, and CHRM2. We identified these 5 key active ingredients in GJD by HPLC. Based on the results, the high-salt rat model experiment confirmed that GJD has an obvious antihypertensive effect by significantly reducing systolic and diastolic blood pressure, constrains the overactivation of the renin-angiotensin pathway, reduces the expression of AGTR1 in the kidney and thoracic aorta, and enhances the expression of eNOS. The current study discovered that GJD could be a potential therapeutic drug for primary hypertension, establishing a new strategy for the investigation and mechanism research of traditional Chinese medicine in treating hypertension.


Ulcerative Colitis (UC) is a chronic nonspecific inflammatory disease of the colon and rectum. Fructus Mume (FM) and Rhizoma Coptidis (RC) exert effects on inflammatory and immune diseases. We evaluated the hypothesis of the FM and RC (FM-RC) herb pair remedy in alleviating dextran sulfate sodium (DSS)-induced colitis, through network pharmacology-based analyses, molecular docking, and experimental validation.The Traditional Chinese medicine systematic pharmacology analysis platform(TCMSP) and Swiss database were used to predict potential targets of FM-RC and the GeneCards database was utilized to collect UC genes. Cytoscape software was used to construct and analyze the networks, and DAVID was utilized to perform enrichment analysis. AutoDock software was used to dock the core chemical components of the FM-RC herb pair with key UC targets. Animal experiments were performed to validate the prediction results and general conditions and body weight were observed. Pathological changes in colon tissue were observed by staining with hematoxylin and eosin. The levels of TNF-α, IL-8, IL-17, and IL-4 in serum and colon tissue were detected by ELISA. −1, indicating the core active components had strong binding activities with the core targets. FM-RC herb pair relieved pathological indicators and reduced the concentration of TNF-α, IL-8, and IL-17 and increased IL-4 levels in the serum and colon tissues of UC rats.Eighteen effective components of the herb couple were screened, and their potential therapeutic targets in the treatment of UC were acquired from 110 overlapped targets. GO and KEGG analyses revealed that these targets were highly correlated with protein autophosphorylation, plasma membrane, ATP binding, cancer pathways, the PI3K-AKt signaling pathway, and the Rap1 signaling pathway. Molecular docking established the core protein interactions with compounds having a docking energy < 0 kJ·molCollectively, FM-RC herb pair administration alleviated UC. These beneficial effects targeted MAPK1 signaling related to inflammation and immunity, which provided a basis for a better understanding of FM-RC in the treatment of UC. Ulcerative Colitis (UC) is a nonspecific gastrointestinal inflammatory disease characterized by diarrhea, mucus, and blood in the stool . Its patTraditional Chinese herbal medicine has been widely used to treat UC for many years . Wumei PNetwork pharmacology and molecular docking are new technologies based on systems biology and databased molecular correlation analysis in the exploration of new drugs and prediction of drug targets , 19. ZhoTherefore, the aim of our study is to further consider the detailed mechanism of FM-RC herb pair in the treatment of UC systematically through network pharmacology, molecular docking and experimental validation in vivo, thereby providing new theoretical basis for Chinese medicine in the treatment of UC.https://tcmsp-e.com/).We searched for the terms ‘Fructus Mume’ and ‘Rhizoma Coptidis’ in the Chinese Medicine Database and Analysis Platform (TCMSP) website. The bioavailability (OB) > 30% and drug similarity (DL) > 0.18, and main pharmacokinetic parameters, were used to screen the active components of FM-RC using the pharmacokinetic indexes of absorption, distribution, metabolism, and excretion (ADME) . The OB https://pubchem.ncbi.nlm. nih.gov/) database to confirm the molecular structure, and was exported as a two-dimensional structure in an SDF file. The results were imported into the Swiss database (http://www.Swisstarget prediction.ch/) to predict the chemical targets of FM-RC. The UniProt database (https://www.uniprot.org) was used to limit data to ‘human species’, and the target names were normalized to obtain the official name and standard gene name of the compound targets.The chemical composition of the drugs was determined through the PubChem (https://www.genecards.org/) with the keywords ‘ulcerative colitis’ [Data from target genes associated with UC were collected from GeneCards (colitis’ . Genes fhttp://www.bioinformatics.com.cn/) [The screened FM-RC targets and UC targets were imported into Bioinformatics (com.cn/) , and theA visual network was constructed using Cytoscape 3.7.2 software to reflect the complex relationship between active compounds and filtrated targets to reflect the relationship between included targets and active compounds . The nodhttps://string-db.org/). Species were set as ‘Homo sapiens’ and the ‘minimum required interaction score’ was set to 0.7 to ensure readability of the PPI network. This step hid nodes that were not related to each other, allowing the construction of the protein interaction diagram. The corresponding data files were imported into Cytoscape 3.7.1 software (http://www.cytoscape.org/), and the nodes were filtered with the median parameters of degree and betweenness centrality in Cyto NCA plug-in to obtain the core targets of UC treatment with FM-RC.The protein–protein interaction (PPI) network was derived based on the STRING database (www.bioinformatics.com.cn), Gene Ontology (GO) enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to extract the biological functions of target proteins and cancer-related pathways. In addition, a histogram and bubble graph were drawn to visualize the data.By bioinformatics technology, with bioinformatics data analysis (https://pubchem.ncbi.nlm.nih.gov/) and converted to a mol 2 format file using Chem 3D software. The PDB format structures of SRC (PDB ID:2BDF), MAPK1(PDB ID:4NIF) and PIK3CA (PDB ID:4JPS) were downloaded from the RCSB Database (https://www.rcsb.org/). Pymol software was used to remove solvent molecules and ligands, and AutoDock Tools 1.5.6 software was used to hydrogenate, calculate charge, assign atomic types, and save the data in the PDBQT format.To validate the effectiveness of the screened compounds and targets, molecular docking was performed between the top 3 active compounds and the top 3 core targets. Compound structures in SDF format were downloaded from the PubChem database , all samples were vortexed for 30 s, sonicated for 5 min in an ice-water bath, incubated at − 40 °C for 1 h, and centrifuged at 12,000 rpm at 4 °C for 15 min. A 500 μl of the supernatant was passed through a 0.22 μm filter membrane and then transferred to ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis.LC–MS/MS analysis was performed on an UHPLC system  with a Waters UPLC BEH C18 column (1.7 μm 2.1*100 mm). The sample injection volume was set at 5 μL. The flow rate was set at 0.5 mL/min. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was as follows: 0—11 min, 85—25% A; 11—12 min, 25—2% A; 12—14 min, 2—2% A; 14 – 14.1 min, 2—85% A; 14.1—15 min, 85—85% A; 15—16 min, 85—85% A. An Q Exactive Focus mass spectrometer coupled with an Xcalibur software was employed to obtain the MS and MS/MS data based on the IDA acquisition mode. During each acquisition cycle, the mass range was from 100 to 1500, and the top three of every cycle were screened and the corresponding MS/MS data were further acquired. Sheath gas flow rate: 45 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 350 ℃, Full ms resolution: 70,000, MS/MS resolution: 17,500, Collision energy: 15/30/45 in NCE mode, Spray Voltage: 4.0 kV (positive) or -4.0 kV (negative). Materials identification of peaks containing MSMS data was performed using the secondary mass spectrometry database  and the corresponding cleavage law matching method.SPF rats  were purchased from Huaxing Experimental Animal Farm, Huiji District, Zhengzhou City, animal license number SCXK (Yu) 2019–0002. All animals were housed in a room with constant temperature (25 ± 2 °C), relative humidity (50 ± 5%°C), half-day light–dark cycles, and free access to food and distilled water. Animal feeding and use were carried out in accordance with the relevant regulations of the Animal Ethics Committee of Anhui University of Chinese Medicine, ensuring that experiments were carried out in full compliance with the ‘Administrative Regulations on Laboratory Animals’ issued by the State Science and Technology Commission and the ‘Implementation Rules for the Management of Medical Laboratory Animals’ issued by the Ministry of Health.Prunus mume Sieb. et Zucc., and the rhizome of Coptis chinensis Franch. The following devices and reagents were used: mesalamine , the dextran sulfate sodium , Electronic balances , high-speed tissue grinder (Servicebio), desktop high-speed refrigerated centrifuge (Great Dragon), microplate detector (BioTeK), tumor necrosis factor-α (TNF-α), rat IL-4 ELISA kit, rat IL-8 ELISA kit, and rat IL-17 ELISA kit (Servicebio).FM-RC, the origin of Sichuan Province, were identified by Associate Professor Ou Jinmei of Anhui University of Chinese Medicine as the near-ripe dried fruit of n = 8 each): Control group (Con), DSS group (DSS), Mesalazine group (Mes), and FM-RC group (FM-RC). Rats in the control group received sterile water for 9 days and in other groups were given free access to a 3% DSS solution for 9 days to establish the UC model. On the second day of modeling, the Mes group received 0.42 g/kg Mes, intragastrically for 8 consecutive days, the FM-RC group received FM-RC1.26 g/kg, intragastrically for 8 consecutive days, while the normal group and the model group received sterile water. The herb pair accounts for a large proportion of FM, the dosage of the rats was determined according to the dosage of FM. The maximum dosage of FM recorded in the Chinese Pharmacopoeia is 12 g, based on the dose of the drug converted from the body surface area between humans and rats, it was determined that the dose of FM-RC to be administered to rats was 1.26 g/kg. The maximum dose of Mes for human is 4 g, and the dose for rats was calculated to be 0.042 g/kg in the same way. Body weight and disease activity index were monitored daily during administration, including weight loss, fecal hardness, and blood in the stool. DAI = (weight change score + stool trait score + blood in stool score) / 3. The DAI scoring criteria are shown in Table After a 7-day adaption period, 32 SPF rats  were randomly divided into four groups  for 10 min, and the upper serum fraction was obtained. TNF-α, IL-4, IL-8, and IL-17 in serum were determined using the Rat inflammation panel kit. The resulting data were analyzed by one-way ANOVA using GraphPad Prism 6.0. followed by Tukey’ s test for multiple comparisons.The colon tissues of rats in each group were added with 0.9% normal saline at a ratio of weight (mg): volume (μm) = 1:9, mechanically homogenized under an ice bath to prepare a 10% homogenate, and centrifuged  for 10 min. The supernatant fraction was obtained. TNF-α, IL-4, IL-8, and IL-17 in colon tissue were measured using the Rat Inflammation Panel kit. The resulting data were analyzed by one-way ANOVA using GraphPad Prism 6.0. followed by Tukey’ s test for multiple comparisons.The six component indexes of FM and 13 components of RC were retrieved from the TCMSP database. A total of 18 compounds were retrieved from the TCMSP database under the screening conditions of OB ≥ 30% and DL ≥ 0.18, of which quercetin was the common compound of the FM-RC , 56 were related to the cellular component (CC), and 265 were related to the biological process (BP). According to the increasing order of the P-value, the top 10 proteins were selected and a bar graph was drawn, as shown in Fig. Using the DAVID database, GO enrichment analysis was performed on 110 potential targets and set P < 0.05. The first 20 pathways were selected and incorporated into a bubble chart. The larger the bubble, the more genes enriched in the GO entry  and the top 3 key active ingredients , as shown in Table To control the quality of the herbal extract, the FM-RC extracts samples were analyzed using UHPLC-MS/MS. The total positive and negative ion chromatograms of FM-RC demonstrated the chemical composition of all compounds, Berberine, Palmatine, Citric acid, Epiberberine and Quercetin were found in FM-RC extracts, and semi-quantified by relative peak area Table . The secTo further explore the effects of FM-RC herb pair extracts on UC in vivo, we established the DSS–induced rats UC model and treated rats with FM-RC simultaneously, as shown in Fig. In the present study, the ELISA assay was used to assess inflammatory cytokine secretions in DSS-treated rats. Elevated protein levels of TNF-α, IL-8, and IL-17 in serum were observed in the DSS group compared to the control group. FM-RC significantly suppressed up-regulation of these inflammatory cytokines Fig. a–c. FurtConsistent with the inflammatory cytokines in the serum results, the levels of inflammatory cytokines in the colon tissues showed that TNF-α, IL-8 and IL-17 in the model group exhibited higher levels compared to those of the control group  provides systematic, comprehensive biological annotation information for large-scale genes or proteins, and provides the most significantly enriched biological annotations. The DAVID web server was adopted to conduct GO enrichment analysis for the candidate target protein obtained after network merging. Subsequently, KEGG pathway enrichment analysis was conducted to explore biological pathways where relevant proteins were covered. A P value ≤ 0.05 was considered signifcant, and enriched GO terms were identifed by the hypergeometric test. A bubble plot of bioprocess and pathways were drawn by bioinformatics data analysis (www.bioinformatics.com.cn). Ultimately, the three bioactive compounds  and genes  with the highest differential expression within the constraints and the apoptosis-associated pathway were identified. Quercetin is a flavonoid molecule [From the TCMSP database, Swiss, and other databases, a total of 18 potential active ingredients of FM-RC, 110 common targets in the herb pair and UC were identified. Subsequently, we further screened essential genes and core-dependent pathways through the PPI network. The Database for Annotation, Visualization and Integrated Discovery (DAVID, molecule . Ye et amolecule  studied molecule . Rajendrmolecule  studied molecule , 37. Themolecule –40. PIK3molecule . We thenAccumulating evidence has shown that overactivation of the inflammatory response contributes to the initiation and development of IBD or DSS-induced colitis. TNF-α, IL-8, and IL-17 are considered key pro-inflammatory cytokines that lead to the development of UC , 43. TNFIn this study, we combined the results of network pharmacology, molecular docking, and experimental in animal models at three levels. Among the bioactive ingredients associated with UC in FM-RC, quercetin and MAPK showed the highest degree of molecular docking. We also verified the effects of inflammatory factors associated with the MAPK pathway on the animal model of UC.However, this study also presented some limitations. First, the data on various drugs, genes, and proteins were not comprehensive, and appropriate computing software has not been developed. Second, the bioactive ingredients of FM-RC should be verified individually by experiments. Finally, it is necessary to explore the direct regulation of bioactive ingredients on MAPK1 in future studies.Through network pharmacology and molecular docking, the study preliminarily revealed the main active components, targets, and signaling pathways of FM-RC therapy UC, Meanwhile, we performed an animal experiment to confirm that the herb pair of FM-RC interferes with UC through the MAPK1 signaling pathways associated with inflammation and immunity. Specifically, FM-RC herb pair regulates the immune function by affecting the expression levels of TNF-α, IL-8, IL-17, and IL-4. These results provide a scientifific basis for the prevention and treatment of UC.