Gubitong Recipe (GBT) is a prescription based on the Traditional Chinese Medicine (TCM) theory of tonifying the kidney yang and strengthening the bone. A previous multicentral randomized clinical trial has shown that GBT can effectively relieve joint pain and improve quality of life with a high safety in treating osteoarthritis (OA). This study is aimed at elucidating the active compounds, potential targets, and mechanisms of GBT for treating OA.  The network pharmacology method was used to predict the key active compounds, targets, and mechanisms of GBT in treating OA. An OA rat model was established with Hulth surgery, and the pathological changes of articular cartilage were observed to evaluate the effects of GBT. Chondrocytes were stimulated with LPS to establish in vitro models, and key targets and mechanisms predicted by network pharmacology were verified via qRT-PCR, ELISA, western blot, and immunofluorescence. The Contribution Index Model and molecular docking were used to determine the key active compounds of GBT and the major nodes affecting predicted pathways. 2 are key targets of GBT in treating OA. KEGG results showed that PI3K/AKT, Toll-like receptor, NFκB, TNF, and HIF-1 are the key signaling pathways. An in vivo experiment showed that GBT could effectively suppress cartilage degradation of OA rats. In vitro experiments demonstrated that GBT can inhibit the key targets of KEGG-related pathways. Molecular-docking results suggested that luteolin, licochalcone A, and β-carotene were key targets of GBT, and the mechanisms may be associated with the NFκB signaling pathway. Blockage experiments showed that the NFκB pathway is the key pathway of GBT in treating OA.  A total of 500 compounds were acquired from related databases, where 87 active compounds and their 254 corresponding targets were identified. 2979 OA-related genes were collected from three databases, 150 of which were GBT-regulating OA genes. The compound-target network weight analysis and PPI results showed that IL-6 and PGEκB pathway is the most key mechanism in treating OA. This study verified that GBT can effectively protect articular cartilage through multitarget and multipathway, and its inhibitory effect on the NF Osteoarthritis (OA) is a degenerative disease characterized by degeneration of the cartilage, which is closely related to age, joint trauma, obesity, and genetic factors , 2. PrevAccording to Traditional Chinese Medicine (TCM) theory, OA is considered to be a bone arthralgia syndrome (Gubi) disease attributed to deficiency of kidney yang. Clinical investigation shows that most OA patients, especially elderly patients, have kidney-yang deficiency syndromes. Gubitong Recipe (GBT) is a prescription formulated based on the TCM theory of tonifying the kidney yang and strengthening the bone. Our previous randomized clinical trials (RCT) have shown that GBT can more effectively decrease the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score and clinical symptoms of the visual analogue scale (VAS) of OA patients in comparison with glucosamine hydrochloride, reduce the amount of diclofenac sodium use, and has no obvious side effects .Network pharmacology is a new research method, which elucidates the potential mechanism of specific disease through database analysis and target prediction , 8. MoleDrynaria fortunei (Kunze) J.Sm. , Epimedium brevicornum Maxim. , Psoralea corylifolia L. , Eucommia ulmoides Oliv. , Cibotium barometz (L.) J.Sm. , Bolbostemma paniculatum (Maxim.) Franquet. , Sinomenium acutum (Thunb.) Rehd. et Wils. , Spatholobus suberectus Dunn. ) were identified from the following four databases: Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform (TCMSP) ..29].As a specific marker of mature chondrocytes, type II collagen is often selected as a biomarker for chondrocytes . ChondroThe isolated chondrocytes were first subjected to immunofluorescent staining using a type II collagen antibody to determine whether or not the chondrocytes possessed the characteristic of expressing and secreting type II collagen . Further5 cells/mL and then planted at 100 μL per well on a 96-well plate. 0 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 250 μg/mL GBT solutions were added to each well, and cells were cultured for 12 h. After discarding the medium, the cells were washed twice with PBS. 100 μL of DMEM medium was added to each well, and cells were cultured for 2 h before 20 μL MTS was added. The chondrocytes were then incubated for another 30 min. A microplate reader  was used to measure the optical density (OD) of each well at 490 nm. The following formula was used to calculate cell viability: viability (%) = 100 × (OD of treated sample − OD of medium)/(OD of control sample − OD of medium).Cell viability was detected with the MTS assay. Chondrocytes were diluted to a concentration of 1 × 10μg total RNA was used to construct a transcription system  and was then transcribed into cDNA according to the manufacturer's instruction.A HiPure Total RNA Mini Kit was used to extract and purify total RNA using an adsorption column and collection pipes. A NanoDrop spectrophotometer  was used to detect RNA concentration and quality. 1 μL of the reaction system included SYBR Green real-time PCR master mix (10 μL), cDNA (2 μL), gene forward and reverse primer , and distilled water (7.2 μL). Real-time PCR was performed on a QuantStudio Real-Time PCR cycler with the following protocol: preheated at 95°C for 60 s, then heated at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s as a cycle; a total of 40 cycles were completed, and finally, the CT value was obtained. The relative expression was analyzed based on the 2-Ct method.20 Before the experiment, 10% SDS-PAGE gel was configured with a PAGE Gel Fast Preparation Kit based on the kit's instructions. Specific information and dilution rates of all antibodies are presented in μL RIPA lysate (containing 1% PMSF and 1% phosphatase inhibitor), gently blown for 5-10 times, and lysed on ice for 25 min. The lysate was centrifuged , and supernatants were collected. A bicinchoninic acid (BCA) assay kit was used to detect total protein concentration. According to the concentration test results, samples were adjusted to the same protein concentration with the RIPA lysate. A 5x loading buffer (dilution 4 : 1) was added, then boiled for 5 min, and then centrifuged .First, cells were rinsed twice with PBS. Each well was added with 250 μg of proteins was added into each well of the SDS-PAGE gel and labeled with a marker protein. Electrophoresis was performed at 150 V until the bromophenol blue reached the bottom of the gel. The PVDF membrane was activated with methanol for 40 s before transfer. A Mini Gel Holder Cassette, filtrate paper, PVDF membrane, and SDS-PAGE gel were prepared to make a sandwich transfer system and electrophoresis for 55 min at 70 V. Proteins in the gel were transferred to PVDF membranes and then incubated with a blocking solution for 1 h at room temperature. The PVDF membranes were then washed with TBST, added with diluted primary antibodies, and incubated at 4°C overnight. After that, the PVDF membranes were washed with TBST for five times, added with diluted secondary antibodies, and incubated for 1.5 h at room temperature. All incubations were performed on an oscillating shaker. Finally, the PVDF membranes were washed for five times with TBST, and the samples were detected with a chemiluminescence system using ECL luminous fluid.40 β, IL-18, TNF-α, and PGE2 in the supernatant were determined using a commercial ELISA kit according to the manufacturers' instructions . The OD value of each standard and sample pore was detected at 450 nm wavelength with a microplate reader, with a reference wavelength of 650 nm. A model of standard curve was constructed using CurveExpert 1.4 software, and the concentrations of the standard pore sample were calculated using the OD value.Cell culture media were collected after completion of cell intervention and then centrifugated , and supernatants were obtained for ELISA detection. The concentrations of IL-6, IL-1κB p65 of dilution at 1 : 200) was added and incubated at 4°C overnight. After discarding the primary antibody, Alexa Fluor 488-conjugated goat antirabbit IgG (H+L) (1 : 100 dilution) was added and incubated at room temperature for 1 h; then, the plate was covered with a fluorescent mounting medium with DAPI. An inverted fluorescence microscope  was used to observe the location of the antibody and DAPI to determine the location of the target protein and nucleus.After being washed with PBS, the cells were fixed with 4% paraformaldehyde. PBS/0.1% Triton X-100 was used to penetrate the cytomembrane. After being blocked with an animal-free blocking solution for 1 h at room temperature, the primary antibody  were injected into dishes for 1 h incubation after the cells were fixed and the membrane was broken. Finally, these cells were washed with 1x buffer, followed by the addition of Alexa Fluor 488-conjugated goat antirabbit IgG (H+L) (1 : 100 dilution) to cells (1 : 50 dilution). After 30 min incubation under a light-free circumstance, these prepared cells were assayed with a flow cytometer .Chondrocytes and SW1353 cells were subjected to digestion of t test was used to assess the differences between the two groups. ANOVA and Dunnett's test were used for comparisons of multiple groups (GraphPad Prism 4). A p value < 0.05 was considered to indicate a statistically significant difference.Continuous variables are presented as the mean values ± standard deviation (SD). Student's In this study, a network pharmacology module and in vivo and in vitro experiments were used to elucidate the therapeutic mechanisms of GBT for the treatment of OA . FirstlyUsing a systematic search of compounds in GBT from public databases, a total of 500 compounds were retrieved through TCMSP, YaTCM, TCMID, and Traditional Chinese Medicine Database@Taiwan databases. The number of compounds contained in each botanical drug of GBT predicted with databases is represented on Supplementary Table A total of 254 targets corresponding to 87 active ingredients of GBT were acquired from TCMSP databases. OA-related genes were recognized from three online databases, 2885 of which were obtained from GeneCards, 368 of which were obtained from DisGeNET, and 102 of which were obtained from OMIM. After eliminating the duplicated genes, 2979 genes were identified. 150 of the intersection genes of GBT-related genes and OA-related genes were obtained, which were defined as therapeutic genes of GBT against OA.β, and ESR1. In the onset and progression of OA, these core genes are mainly involved in the expression of inflammatory mediator , the activation of inflammatory signaling pathways , cartilage matrix degradation (MMP9), synovial neovascularization (VEGFA), and chondrocyte apoptosis (CASP3 and CCND1).The PPI network was built to analyze the therapeutic genes of GBT against OA, which showed that IL-6 had the most relative connection with other genes, indicating that it was the most critical gene target in this network. Detailed information of the PPI network is provided in β-carotene (GBT199) contribute to 33.3% connective degree with intersection genes (A compound-target (CT) network was established on the basis of connections between active compounds and intersection genes, whose results showed that 78 of the 87 active compounds were involved in the network . Furtheron genes . Meanwhion genes .p < 0.05 were considered statistically significant. The top 40 pathways of KEGG results were visualized with a dot plot  and enable it to enter the nucleus and activate the transcription of related inflammatory mediators [κB signaling pathway can promote the transcription and translation of the NOD-like receptor protein 3 (NLRP3), COX-2, IL-1β, IL-6, IL-18, TNF-α, and HIF-1α. Simultaneously, the formation of inflammasome that NLRP3 is involved in is essential to the activation of the NOD3 pathway, and the inflammasome can activate pro-IL-1β and pro-IL-18 and release into the extracellular, promoting further inflammatory response. In addition, TNF-α and HIF-1α are key to the activation of the TNF and HIF pathways, respectively. In conclusion, the NFκB pathway is the core of the 5 significant KEGG-predicated pathways above. A pathway hypothesis diagram was drawn based on KEGG results  signaling pathway can induce the ubiquitination and phosphorylation of Iof IκB-α –42. Degrediators , 44. Pre results .p < 0.05. Biological functions were divided into three parts, including biological process, cellular compounds, and molecular function  and related literature for quality control . The keySafranin O/fast green and hematoxylin-eosin-stained pathological sections of rat knee joints were observed and photographed under a light microscope. Results showed that rats in the normal group had smooth cartilage surfaces. Chondrocytes in the normal group were regularly arranged with a continuous and complete tide line, and the staining was evenly distributed Figures . Rats inImmunofluorescence results showed that chondrocytes isolated from the knee cartilage of suckling rats exhibited strong positive for type II collagen Figures . Flow cyμg/mL of the GBT solution remarkably inhibits the cell proliferation in comparison with 200 μg/mL of the GBT solution having no effect. Therefore, 200 μg/mL of the GBT solution is referred to as the high-dose intervention concentration, 100 μg/mL of the GBT solution is considered as the medium-dose intervention concentration, and 50 μg/mL is referred to as the low-dose intervention concentration  is the key target of treating OA with GBT, and COX-2 enzymes can catalyze the synthesis of PGEμg/mL, 100 μg/mL, and 200 μg/mL of GBT were used to intervene the chondrocytes, and 100 μmol/mL of dexamethasone was set as the positive control group. After being pretreated with the above drugs for 1 h, the chondrocytes were stimulated with 100 ng/mL of LPS for 12 h. The contents of PGE2 and IL-6 in the supernatant were detected using the ELISA method. The results suggested that LPS could enhance secretion of PGE2 and IL-6 in chondrocytes. Pretreatment with GBT and dexamethasone can significantly reduce the contents of PGE2 and IL-6 in the supernatant of the LPS-induced chondrocytes, and GBT showed a dose-dependent effect , confirming the activation of the NFκB pathway. Although the contents of TLR4 and MyD88 did not change significantly after LPS stimulation, the coimmunoprecipitation results showed that the content of MyD88 bound to TLR4 increased, suggesting the activation of the TLR4 pathway. In addition, GBT can reverse the change of IκB-α in the cytoplasm and NFκB p65 in the nucleus which was induced by LPS and reduce the proportion of the nuclear translocation of NFκB  and ASC, the key protein in the NOD pathway, and the contents of IL-1β and IL-18 in the cell culture supernatant, which were the downstream inflammatory factors regulated by the NLRP3 inflammasome. Results showed that the expressions of NLRP3, IL-1β, and IL-18 increased after LPS stimulation. 200 μg/mL of GBT could reverse the above changes, but the content of ASC was not affected by LPS and GBT  [β (PDB ID:4KIK) [κB-α/NFκB p65 (PDB ID:1K3Z) [κB pathway, TNFR1 (PDB ID:7K7A) [α inhibitor (PDB ID:4Z1V) [Molecular docking was conducted with AutoDockTools to assess the binding energy between key compounds from GBT and core proteins of key pathways predicted by KEGG analysis. Core proteins include PI3K (PDB ID:1E7V)  of PI3K/ID:5TQW) , IKK-β (ID:4KIK) , IκB-α/NID:1K3Z)  of the NID:7K7A)  of the TID:4Z1V)  of the Hβ-carotene (GBT199) were selected as key active compounds in accordance with the compound-target network result. Proteins that possess crucial roles in the PI3K/AKT pathway, Toll-like receptor pathway, NOD-like receptor pathway, TNF pathway, and HIF-1 pathway were chosen as key targets. The binding affinity (KD) was calculated using Vina software, and subsequently, a heat map was constructed , luteolin (GBT28), kaempferol (GBT19), licochalcone A (GBT306), naringenin (GBT20), and structed . The resκB pathway through signal transduction. The activated NFκB pathway then affected the HIF pathway and TNF pathway through a transcriptional regulator. The above process suggested that the NFκB pathway is the key to GBT in the treatment of OA. The supposition was further strengthened by molecular docking, which showed that the key molecules in GBT had higher binding energy by combining with the key nodes in NFκB.Literature research showed that the PI3K/AKT pathway, NOD-like pathway, and Toll-like pathway activated the NFκB pathway was blocked to verify the significant roles of NFκB in the treatment of OA with GBT. JSH-23, a blocker that specifically blocked the nuclear translocation of NFκB, has been used in our previous experiments. Results showed that JSH-23 could significantly inhibit LPS-induced IL-6 and PGE2 expression. The concomitant use of JSH-23 and GBT was not superior to the use of JSH-23 alone, confirming that the NFκB pathway is the key to the inhibitory effects of GBT on the inflammatory reaction of chondrocytes  are all triggers for chondrocytes to initiate inflammatory response . Inflammnt of OA . The expin vitro , 62. In  effects . In addims of OA , 65.2, PGE2 and IL-6 were predicted to be the core molecules of GBT in the treatment of OA in this study. The prediction was further verified through in vitro experiments. After stimulating chondrocytes with LPS, the secretion of PGE2 and IL-6 increased significantly, while GBT could inhibit the expression of these compounds and showed a dose-dependent effect. It follows from the above results that the anti-inflammatory effect of GBT relies on its inhibitory effect on key OA-related inflammatory factors.In this study, network pharmacology revealed that PTGS2 (COX-2) is a key gene in the process of OA being treated with GBT, and IL-6 is the core protein regulated by these genes. Since COX-2 regulates the catalytic synthesis of PGEκB-α in the cytoplasm was reduced and NFκB p65 in the nucleus was increased. The immunofluorescence assay also indicated that NFκB p65 was transferred from the cytoplasm to the nucleus, suggesting the activation of the NFκB pathway [κB pathway was significantly inhibited, and the expression of TNF-α and HIF-1α regulated by NFκB significantly decreased.In the subsequent research on the predicted pathway, after stimulation of chondrocytes with LPS, I pathway . After Gβ suggesting the abnormal activation of the NLRP3 pathway by LPS [β and reduce the amount of MyD88 binding with TLR4, suggesting that the PI3K/AKT signaling pathway, Toll-like receptor 4 signaling pathway, and NLRP3 signaling pathway aberrantly activated by LPS are inhibited.The increase of p-PI3K and p-AKT indicated that the PI3K/AKT pathway was aberrantly activated . Althougy by LPS . GBT canα, licochalcone A and IKK-α, β-carotene and IκB-α/NFκB p65 had a higher binding energy, suggesting that the NFκB pathway might be the critical mechanism to the process of OA being treated with GBT.Molecular docking is an in silico approach to predict and evaluate the binding affinity of an active compound against a ligand. This study utilized the method to predict key compounds of GBT and core proteins of predicted pathways –73. In oκB pathway was blocked using a specific blocker in further experiments and subsequently intervened using GBT. The results showed that after blocking the NFκB pathway, the effect of GBT on the key targets PGE2 and IL-6 was significantly diminished. The above results suggested a crucial role of the NFκB signaling pathway in the treatment of OA with GBT.Therefore, the NFIn this study, we confirmed the multitarget effect of GBT in the treatment of OA through network pharmacological prediction and experimental verification. GBT can inhibit several inflammation-related signaling pathways and ultimately reduce the expression of inflammation-related mediators, indicating a key role of anti-inflammatory effect of GBT in the treatment of OA. Previous studies have shown that cartilage inflammatory response is an essential agent of joint pain and stiffness in OA patients; suppression of this inflammatory response alleviates these symptoms, which supports the discovery that GBT exerts an anti-inflammatory effect on OA in this study . Furtherβ-carotene. A thorough review of the literature revealed that these ingredients are usually insoluble in water. In clinical applications, we usually decoct botanical drugs to produce an oral solution of GBT, which may perhaps cause excessive loss of key components. In future experiments, we may be able to optimize the extraction method and produce an oral solution of larger key ingredient contents so as to promote clinical efficacy.At the same time, we identified core compounds in the treatment of OA with GBT through network pharmacology including quercetin, luteolin, kaempferol, licochalcone A, naringenin, and


Orobanche in Europe or Asia and Striga in Africa. The genes involved in the early stages of strigolactones biosynthesis are known in several plants. The regulatory structure and the latter parts of the pathway, where flux branching occurs to produce alternative strigolactones, are less well-understood. Here we present a computational study that collects the available experimental evidence and proposes alternative biosynthetic pathways that are consistent with that evidence. Then, we test the alternative pathways through in silico simulation experiments and compare those experiments to experimental information. Our results predict the differences in dynamic behavior between alternative pathway designs. Independent of design, the analysis suggests that feedback regulation is unlikely to exist in strigolactone biosynthesis. In addition, our experiments suggest that engineering the pathway to modulate the production of strigolactones could be most easily achieved by increasing the flux of β-carotenes going into the biosynthetic pathway. Finally, we find that changing the ratio of alternative strigolactones produced by the pathway can be done by changing the activity of the enzymes after the flux branching points.Strigolactones mediate plant development, trigger symbiosis with arbuscular mycorrhizal fungi, are abundant in 80% of the plant kingdom and help plants gain resistance to environmental stressors. They also induce germination of parasitic plant seeds that are endemic to various continents, such as  Striga (Strigolactones (SLs) are a group of plant hormones that have pleiotropic effects. They were initially discovered as a germination stimulant of the parasitic plant Striga . Later, Striga  and contStriga .Strigolactones were identified in most cereal crops’ root exudates and play a crucial role in host-parasite interactions . SLs indOrobanche, and STR mediates the germination of Striga. These are two of the most critical parasitic plants whose germination is mediated by the SLs present in root cereal exudates. While Striga parasites are endemic in African soils and affect 70% of the continent’s cereal crops, Orobanche parasites are problematic in the Middle East, Europe, and North America, having important effects on leguminous crops  and strigol (STR). In addition to their roles in plant development, ORO mediates the germination of us crops . With liontinent .Due to the critical roles of STR and ORO in plant development and crop yields, it is crucial to elucidate the biosynthetic steps for the various SLs and understand their regulation. All-trans-β –carotene (BCAR) is the initial substrate for the SLs biosynthesis pathway. The initial catalytic steps that go from BCAR to carlactone (CL) are well-characterized in cereals . The indMathematical modeling provides a set of tools that can be helpful to differentiate between alternative reaction structures of a pathway. When significant amounts of quantitative data are available, statistical methods and optimization can be used to find relationships between variable and assign probabilities to alternative reaction structures . If, on in silico experiments to identify potential differences in their dynamic behavior of the alternative models. The differences between the two pathways in relation to producing ORO and STR can be used to propose additional experiments and leverage this information to further clarify the structure and regulation of SLs biosynthesis.In the middle ground where, as is our case, some information is available, differential equation models provide a good formalism to work with for structure comparison. In general, alternative models for the same pathway can be constructed . Then, tThe available experimental information is consistent with two alternative structures for the SLs biosynthesis pathway, as described in the Section “Introduction.” A conceptual representation of each alternative is illustrated in cis-β-carotene (CISB)  . Then, cWe know less about the individual steps that convert CL into the subsequent intermediates of SL biosynthesis. The simplest scenario  consistef) and which goes to STR [(1 − ω) × f]. The model also assumes that ORO and STR diffuse away from the production site at a rate that is proportional to their respective concentrations. The proportionality constants for this diffusion are defined by k.Here, we introduce a parameter 0 ≤ ω ≤ 1. In the absence of additional experimental information, this parameter determines which percentage of flux Arabidopsis thaliana (CYP711A1/AtMAX1) and Oryza sativa (CYP711A2/Os900 and CYP711A3/Os1400)  . Moreove/Os1400) . In para/Os1400) . Further/Os1400) . In maizf*) and which is used to produce STR [(1 − ϕ) × f*].Because ABA8HD can potentially catalyze the alternative conversion of CLA into either ORO or STR, we introduce a parameter 0 ≤ ϕ ≤   1. This parameter determines which percentage of flux The mathematical model was built based on the two alternative pathways presented in S] and enzyme [i]. The parameters in the equation are the turnover number kcati, and the Michaelis-Menten constant KMi, where i is any of the enzymes in the pathway . Note that Vmaxi, the maximum reaction rate, is equivalent to the product of kcati and [i]. Equation 1 shows the system of ordinary differential equations (ODEs) that describe the dynamics of the core pathway:All the reactions are described using a Michaelis-Menten approximation Next, Equation 2 describes the dynamics of the series of processes shown in Then, Equation 3 describes the dynamics of the series of processes described in By combining Equations 1, 2, we obtain the ODEs that describe the dynamic behavior of Model AB. Combining Equations 1, 3, generates the ODEs that characterize the dynamic behavior of Model AC. Given that the pathway isn’t fully established, data for parameter estimation were obtained from different plants in several databases. We started with BRENDA  and compNegative feedback by pathway intermediates or products regulates the flux going through most biosynthetic pathways. The most common form of feedback regulation is overall feedback, also known as end-product inhibition, where the final product of the pathway inhibits the reaction of the first enzyme. In addition, when a biosynthetic pathway is branched, it is also typical that the metabolite at the branching point inhibits the first reaction of the pathway and that the final product of each branch inhibits the reaction of the branch and, sometimes, activates the first reaction of the opposite branch . To the fcll, [ORO]foro1, and [STR]fstr1 into the expression To describe potential feedback regulation in the system and in the absence of specific mechanistic information, we use the power-law formalism. This formalism allows us to mathematically approximate the dynamic effect of a variable on a process . We consforo2 or [STR]fstr2 to the expressions that describe the direct production of ORO or STR, respectively. The feedback parameters are allowed to range from –1 to 0 . This excludes cooperativity. Sometimes, kinetic orders beyond one are necessary for some qualitative behaviors to appear.We also considered the possibility of other potential feedback interactions. First, we allowed ORO and STR to inhibit the reaction catalyzed by MAX1 (this enzyme uses CL to produce ORO and STR). This was implemented by multiplying either [ORO]This leads to the ODEs described in Equations 4, 5:Furthermore, we also considered the possibility that ORO and STR could regulate the fluxes directly after the branching points from CLA in model AC. In the same model, we also consider the possibility that CLA could negatively regulate the initial reaction of the pathway catalyzed by D27. The dynamics of these feedbacks are described by Equation 6:We evaluated the quality of the resulting models. In brief, biological systems are robust to changes in parameter values, and models that are representative of physiological situations should have low sensitivities to most parameters . In addiSteady state stability can be determined by analyzing the eigenvalue of the Jacobian matrix. First, we get the partial derivative of each equation with respect to dependent variables to get the Jacobian matrix. Next, we solve for the eigenvalue of the Jacobian matrix in which it is stable for negative eigenvalue and unstable for positive eigenvalue.By definition, sensitivity analysis measures how much a variable changes if one of the parameters in the system is varied by a certain amount . The robSensitivity analysis identifies regions in the model where minor inaccuracies in parameter values can lead to almost unpredictable results. Consequently, this analysis also allowed us to identify components of the model that may be problematic due to unusually high sensitivity values . In addiMathematically, the sensitivity of a variable to a parameter can be calculated fromWe performed a sensitivity analysis on all our models, as described in https://www.dropbox.com/s/9u77nj5mgp12e1w/Supplementary%20Data%20S1.rar?dl=0 as a Mathematica notebook.The model was constructed using COPASI  and EasyWe built the alternative models using parameter values collected from various plants . As such–4 (wild-type maize) to 10–1 . The models using the basal parameter values can only produce half of the experimentally measured BCAR. Thus, to better approximate reality, the flux accounting for the production of BCAR (which is represented by k0) must be increased in the models. Yet, increasing k0 by an amount that produces the appropriate levels of BCAR leads to an unstable steady state  remains unclear. We combined available experimental information with mathematical modeling to investigate how possible alternative structures for the pathway would affect the dynamics of SL biosynthesis. Building the models required understanding the essential features of SL biosynthesis. The first part of the pathway is well-established . Still, Both models predict that the most significant effect on the production of ORO and STR is achieved by modifying the influx of BCAR into the pathway , 2, rathCommonalities between the dynamic behavior stop here. The details about the best strategies for modulating differential production STR and ORO differ between models AB and AC. Model AB predicts concentration ranges for STR and ORO that can be similar to each other. In model AB it is possible to divert all flux toward the synthesis of either STR or ORO . In contAnother difference is that model AB points to changing the specificity of MAX1 as the primary determinant of the ratio between STR and ORO being produced , 7. ModeThese differences can be used in future research to experimentally differentiate the alternative pathways. Once the correct pathway is identified, the model can then be used to rank potential genetic modifications that could change the ratio of STR/ORO and decrease the risk of inducing germination of local parasitic plants.An interesting aspect of SL biosynthesis is that, while many biosynthesis pathways have negative feedback from their final product to the first enzyme of the pathway , this feedback seems to be absent in SL biosynthesis. When this was investigated, it became apparent that if biochemical regulation exists, it is superseded by circadian regulation . Still, Surprisingly, our results strongly suggest that inhibitory feedback regulation does not exist in the SL biosynthetic pathway. The existence of that feedback regulation would create unstable steady states that would make it hard for the plants to develop properly. In addition, they would decrease the concentration of pathway substrate to levels well below those observed experimentally by Carotenoids are the substrate of the SL biosynthesis pathway. They are also precursors of other developmental hormones, of photo-protection and photosynthetic pigments . Thus, fResults obtained from the mathematical model implied that modulating strigolactone levels in cereals is possible by increasing the substrate levels of the pathway. Changing the ratio of STR/ORO produced could be achieved by modulating enzyme activities after the flux branching that leads to each strigolactone type. Our results strongly suggest that feedback regulation does not exist in the pathway, as it is not possible to reach experimental BCAR concentrations while simultaneously reaching a stable steady state. In addition, the next step in our study is to couple strigolactone biosynthesis to root development and build a combined model considering the pleiotropic effects of strigolactone to root growth and development.The original contributions presented in this study are included in the article/AL and RA designed the experiments and wrote the first version of the manuscript. AL performed the experiments with the assistance of RA and OB. All authors analyzed the results, contributed to their discussion, and approved the final version.


Background: Oligoasthenozoospermia is the leading cause of male infertility, seriously affecting men’s health and increasing the societal medical burden. In recent years, obesity-related oligoasthenozoospermia has attracted increased attention from researchers to find a cure. This study aimed to evaluate the efficacy of Hua-Tan-Sheng-Jing decoction (HTSJD) in treating obesity with oligoasthenozoospermia, determine its active ingredients and identify its mechanism of action.Methods: The ingredients of HTSJD were determined by combining the ultra-performance liquid chromatography with mass spectrometry (UPLC-MS/MS) and systems pharmacology approach. The common pathogenesis of obesity and oligoasthenozoospermia and the potential mechanism of HTSJD against obesity with oligoasthenozoospermia were obtained through target fishing, network construction, and enrichment analyses. Further, molecular docking of the key ingredients with the upstream receptors of the key signaling pathways of the potential mechanism was used to predict their affinity. Finally, high-fat-induced obesity with oligoasthenozoospermia rat model was constructed to determine the effects of HTSJD on semen concentration, sperm motility, body weight, and serum lipid metabolism. The key proteins were validated by immunohistochemistry (IHC).Results: A total of 70 effective components and 847 potential targets of HTSJD (H targets) were identified, of which 743 were common targets related to obesity and oligoasthenozoospermia (O-O targets) mainly enriched in the pathways related to inflammation, oxidative stress and hormone regulation. Finally, 143 common targets (H-O-O targets) for HTSJD against obesity with oligoasthenozoospermia were obtained. Combining the hub genes and the results of Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of H-O-O targets, PI3K-AKT and MAPK signaling pathways were identified as the key pathways. Molecular docking results showed that Diosgenin, Kaempferol, Quercetin, Hederagenin, Isorhamnetin may act on the related pathways by docking EGFR, IGF1R and INSR. The animal-based in vivo experiments confirmed that HTSJD improves the sperm quality of high-fat diet-fed rats by reducing their body weight and blood lipid levels, influencing the PI3K-AKT and MAPK signaling pathways and altering the corresponding protein expressions.Conclusion: HTSJD treats obesity with oligoasthenozoospermia by up-regulating the PI3K-AKT signaling pathway and down-regulating the MAPK signaling pathway, which are at the crossroad of obesity and oligoasthenozoospermia. With the increase in the prevalence of unhealthy lifestyles such as excessive sugar and fat intake, reduced physical activity, and sedentary lifestyles, the obesity rate has rapidly increased in today’s society. Currently, nearly one-third of the world’s population is classified as overweight or obese, which is double the reports from 1980 . In ChinHedysarum Multijugum Maxim (Huangqi), Atractylodes Lancea (Cangzhu), Citrus Reticulata (Chenpi), Poria Cocos (Fuling), Alisma Orientale (Zexie), Coicis Semen (Yiyiren), Cinnanmomi Cortex (Rougui), Amomum villosum Lour (Sharen), Cyperi Rhizoma (Xiangfu), Plantago Asiatica L (Cheqianzi), and Polygonati Rhizoma (Huangjing). The formula resolves phlegm, dispels dampness, benefits the kidney, and enhances sperm production. Previous studies have shown that HTQSD significantly reduces the body mass index (BMI) of obese people, improving lipid metabolism and their quality of life  in Chinese medicine. HTSJD is made by adding and subtracting Huatan Qushi decoction (HTQSD), which consisting of  of life . Besides of life  and enha of life . Additio of life . It is a of life .Although clinical and fundamental studies have established the good effects of formulas and ingredients in herbal medicine, it has been impossible to identify their action mechanism due to the complexity of the mechanism of action of herbal medicines. Therefore, this study investigated the mechanism of action of HTSJD in the treatment of obesity-related complications such as oligoasthenozoospermia based on a combination of network pharmacology and molecular docking, combined with animal experiments . The finHTSJD was purchased from Beijing Tcmages Pharmaceutical Co., Ltd. . The batch numbers for each herb are summarized in A total of 2.6 g of the Chinese medicine granule mixture was weighed, 10 ml of hot water added and brewed for 20 min. Next, the mixture was sonicated for 5 min followed by a 10min centrifugation at 12000 r/min. The supernatant was filtered through a 0.45 um membrane, and the filtrate was injected into a hybrid quadrupole time-of-flight liquid chromatography mass spectrometer, an ultra-high performance liquid chromatography  connected to a mass spectrometer  with a Shimadzu InertSustain C18 column . The column temperature was set at 35°C and 1 ml/min flow rate. The mobile phase consisted of acetonitrile (A) and 0.1% HCOOH-H2O (B).Mass spectrometric detection was performed using electrospray ionization (ESI) in the positive and negative ion modes.https://tcmsp-e.com/). The bioactive components were selected based on oral bioavailability and drug-likeness properties. The common screening criteria were oral bioavailability ≥20% and drug-likeness ≥0.1. The molecular ingredients identified by UPLC-MS/MS and the bioactive properties obtained from the TCMSP database collectively formed the active ingredients of HTSJD. The upset plot of HTSJD active ingredients was drawn using the TBtools software .The compounds in the 11 herbs composing HTSJD were obtained from the TCMSP  and TCMSP were used to predict the potential targets for HTSJD bioactive ingredients. The identified potential protein targeted by the HTSJD bioactive ingredients was converted to a gene using the UniProt Knowledgebase .Finally, the active ingredient-target protein network was constructed using the Cytoscape 3.8.2 . Edges connected the nodes, and the HTSJD key active ingredients were obtained by screening the nodes with degree values greater than the median.http://db.idrblab.net/ttd/), DisgenetGene (https://www.disgenet.org/), Drugbank database (https://www.drugbank.ca), and OMIM (http://omim.org/). Deduplication was performed after normalizing the targets numbers according to UniProt Knowledgebase.Oligoasthenozoospermia and obesity were used as the search keywords from TTD (The common targets when treating oligoasthenozoospermia and obesity were obtained after the intersection of the HTSJD and diseases targets.https://string-db.org/) database to construct the protein interaction network. The species was limited to “Homo sapiens” and the confidence score was 0.9. The PPI interactive networks were then constructed and visualized using the Cytoscape 3.8.2 software.The O-O and H-O-O targets were respectively submitted into the String   network was constructed using the Cytoscape software.The Gene Ontology (GO) enrichment analysis, including biological processes (BP), molecular function (MF) and cellular ingredients (CC), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the R package (clusterProfiler)  to predihttps://pubchem.ncbi.nlm.nih.gov/), minimizing the energy using the Chem3D software. The protein crystal structures of macromolecular receptors were retrieved from the Uniprot/Protein Data Bank  and saved in PDB format. The water molecules and proligands were removed from the proteins using Pymol , and the proteins were modified by hydrogenation and charge balance using AutoDock Tools . The pills were ground into powder and dissolved in distilled water, and 11 g of the powder was added per 100 ml of water.In HTSJD preparation, the Huangqi, Cangzhu, Chenpi, Fuling, Zexie, Yiyiren, Rougui, Sharen, Xiangfu, Cheqianzi, and Huangjing herbal granules were mixed in the ratio of 20:15:15:15:15:20:6:10:10:15 and dissolved in distilled water.R is the drug dose used in this experiment, DH is the clinical human drug dose, W is the human body weight, taken here as 70kg, and F is the dose conversion factor for rats and humans, calculated from the body surface area, taken as 6.3.Dose calculations for the HTSJD and WZYZP were as follows.The final concentration of WZYZP suspension was 0.11 g/ml. Hua-Tan-Sheng-Jing decoction (HTSJD) was formulated into three concentrations: low, medium, and high, consisting of 0.66 g/ml, 1.32 g/ml, and 2.64 g/ml HTSJD, respectively. The medium concentration was the human equivalent dose, and the other two were half and twice the equivalent dosage, respectively.ad libitum access to water and food. The experimental protocol was reviewed and approved by the Animal Care and Use Committee of Beijing University of Chinese Medicine of China .Healthy, specific pathogen-free male Wistar rats (mass: 200 ± 20 g) were purchased from the Laboratory Animal Centre, Si Beifu, with a Laboratory Animal Certificate of Conformity (SCXC (Jing) 2016–0,002). Their housing conditions were: temperature, 24.0 ± 1.0 C; relative humidity, 55–65%; and a 12-h light/dark cycle. The rats were given n = 8), namely, the control group (C), model group (M), WZYZP group (W), low-dose HTSJD group (LHTD), medium-dose HTSJD group (MHTD) and high-dose HTSJD group (HHTD) using the random number table method.After 5 days of adaptive feeding, 48 rats were assigned into six subgroups . Groups C and M were gavaged with distilled water group W was intragastrically administered with WZYZP suspension, while groups LHTD, MHTD, and HHTD groups were intragastrically administered with low, medium, and high concentrations of HTSJD, respectively. The intragastric volume per rat was 10 ml/kg per day.Each rat was weighed every 7 days during the experiment. At the end of the experiment, rats were narcotized by 1% pentobarbital, and their body length  was measured. Lee’s index was calculated to evaluate the degree of obesity:Serum samples were collected into plain tubes using the abdominal aortic method. The concentrations of HDL-C (mm-0667R1), LDL-C (mm-20864R1), TG (mm-0610R1), and TC (mm-0611R1) in serum were measured using ELISA kits .The rat’s cauda epididymis was minced in 500 µL phosphate-buffered saline (pH 7.2) and incubated for 5 min at 37°C to allow sperm release. Ten microliters of sperm suspension was added to the pre-warmed sperm counting plate, and the WL-9000  sperm quality testing system was used for analysis. Five fields of view were selected and the test was completed within 2 min.One rat testis per rat was removed and fixed in Bouin’s solution for 24 h. The testes were then dehydrated in graded ethanol, embedded in paraffin, and sectioned (4 µm in thicknesses). The sections were stained with hematoxylin-eosin (HE) dye and observed under a light microscope .Next, the sectioned paraffin-embedded slides of rats’ spermary per group were deparaffinized and rehydrated with xylene and gradient alcohol. The slides were then incubated with primary antibodies  overnight at 4°C following an antigen repair and closure. Next, the slides were incubated with a secondary antibody labeled with horseradish peroxidase (HRP) at room temperature for 1 h. The slides were then treated with 3,3-diaminobenzidine (DAB) and hematoxylin, dehydrated with alcohol, and sealed with neutral gum. Finally, slides’ images were captured by the Olympus BX53 fluorescence microscope (40x).t-test or Tamhane’s T2-test. Data were presented as mean ± standard error of the mean (SEM). p < 0.05 was considered statistically significant. All results were presented using GraphPad Prism 9 software.All statistical analyses were performed using the SPSS software . The differences between groups were analyzed using a one-way analysis of variance (ANOVA), and the means were compared by Dunnett’s The UPLC-MS/MS system identified 370 compounds , includiAfter excluding duplicates, the database identified 203 bioactive ingredients from the HTSJD. Traditional Chinese medicine (TCM) prescriptions have thousands of ingredients, but only a few ingredients have satisfactory pharmacokinetics and pharmacodynamic characteristics that ultimately determine efficacy. The UPLC-MS/MS obtained molecules were compared with database molecules to identify corresponding ingredients. Non-corresponding molecules were eliminated, resulting in 70 HTSJD active ingredients. The 70 active ingredients were ranked by degree value. Ingredients with >41.5 median values were considered as key active ingredients. Therefore, the study identified 33 key active ingredients with corresponding Chinese herbs . Figure After screening and de-duplication, we obtained 847 potential targets of the 70 bioactive ingredients and mapped them to human homologous genes using the UniProt database. The constructed herb-ingredient-target network  had 928 Pooling data from several databases, the target numbers for obesity, oligoasthenozoospermia and O-O were obtained as 1,095, 9,124 and 743, respectively .The O-O target data of protein-protein interaction from the String was imported into the Cytoscape software, which generated a PPI network with 514 nodes and 2,278 edges . TP53, KThe 743 O-O common targets were analyzed for GO functional and KEGG pathway enrichment using clusterProfiler, and the p. adjust-values and target counts ranked the results. Consequently, 1933 enriched GO functional terms were identified, including 1784 biological processes (BP), 81 molecular functions (MF), and 68 cellular components (CC). The intersection of 847 drug targets with the above O-O targets generated 143 H-O-O common targets . The proThe GO functional and KEGG pathway enrichment analyses were performed on the 143 H-O-O common targets to further explore the mechanism of HTSJD action using the clusterProfiler R package. Based on the identified hub genes and enrichment results of the H-O-O targets, we focused on the PI3K-AKT and MAPK signaling pathways. The mechanism of HTSJD treatment against obesity with oligoasthenozoospermia by targeting PI3K-AKT and MAPK signaling pathways is demonstrated in Molecular docking of the key ingredients with the upstream receptors of the PI3K-AKT and MAPK signaling pathways was used to predict their affinity.The protein crystal structures  were subThe best conformation was further analyzed to demonstrate the binding mode. p < 0.001, p < 0.001, p < 0.01, p < 0.001). MHTD improved the disorder indicators caused by the high-fat diet (p < 0.001) and substantially normalized the indicators.We established a rat model of obesity with oligoasthenospermia by high-fat diet feeding . After 1The testis germinal tubules of some rats in the model group showed structural changes . The chain vivo, inhibiting the obesity and sperm quality decline caused by the high-fat diet.We examined the expression and phosphorylation levels of important proteins on relevant pathways based on network pharmacological enrichment analysis. The aim was to investigate the mechanism of HTSJD intervention in obesity-induced oligoasthenozoospermia. The relative expression of PI3K/p-PI3K and AKT/p-AKT in the model group was lower than the control group . MoreoveOligoasthenozoospermia consists of oligozoospermia and asthenozoospermia and is the main cause of male infertility . AbnormaBased on traditional Chinese medicine, obese individuals prefer fatty, sweet, thick and greasy, less active, and sedentary habits, which are the pathogenesis factors in phlegm-dampness accumulation leading to a decline in sperm quality . HTSJD rin vivo validation.Given the complexity of Chinese herbal formulae, we explored the action mechanisms and active ingredients of HTSJD for treating obesity with oligoasthenozoospermia using UPLC-MS/MS, network pharmacology, molecular docking, and animal experiments. Our main focus was on the HTSJD mechanism on weight loss and sperm quality maintenance; thus, the common target against obesity and oligoasthenozoospermia (O-O targets) were used as the basis for the work. Finally, 143 H-O-O common targets were obtained. According to the PPI analysis on H-O-O targets, 14 hub genes were obtained. Combining the hub genes and the results on GO function and KEGG enrichment analysis, PI3K-AKT and MAPK signaling pathways were identified as the key pathways. PI3K and MAPK signaling pathways are major post-receptor cell signal transduction pathways in EGFR, IGF1R, and INSR . They reA combination of drug targets and immunohistochemical techniques were used to validate the expression and phosphorylation of PI3K, AKT, JNK, and p53, and the key proteins in the PI3K-AKT and JNK MAPK signaling pathways in the testicular tissues of model and drug groups. Our results revealed that PI3K and AKT protein expression and phosphorylation in rat testis were significantly up-regulated, while JNK and p53 were significantly down-regulated, in the drug groups compared to the model group. PI3K-AKT and MAPK signaling pathways are key in obesity and spermatogenesis inhibition . ObesityOverall, our findings confirmed the relationship between obesity and sperm quality. Besides, the bioinformatics and molecular biology analysis revealed the efficacy and mechanism of HTSJD in treating obesity with oligoasthenozoospermia. The underlying mechanism entails resolving phlegm and dispelling dampness, benefiting the kidney and promoting sperm production. This study explores the common target of obesity and oligoasthenozoospermia as the entry point for in-depth research, providing a new research idea on their pharmacological mechanism. Network pharmacology and molecular docking are effective and reliable methods for studying multi-compound, multi-target drugs such as Chinese medicinal compounds. However, this method has limitations, including excessive reliance on database and software analysis, which may deviate the actual results due to imperfect database or software algorithms. Therefore, collecting data from as many databases as possible is the key to bringing the results closer to the real situation. This experiment integrated animal and molecular biology experiments based on information biology, which validated the data. In the follow-up experiments, clinical trials and cellular experiments are needed to explore the corresponding efficacy and mechanisms.HTSJD treats high-fat diet-induced obesity and oligospermia by activating the PI3K-AKT signaling pathway and inhibiting the JNK MAPK signaling pathway, which alleviates obesity and blood lipids, increasing sperm motility and semen concentration, preventing inflammatory infiltration within the testes.


Rabdosia angustifolia and Perilla frutescens. At present, it is known that isoegomaketone has a wide range of pharmacological activity, but there has been no thorough investigation of its potential targets. As a result, we examined the potential targets of isoegomaketone using the network pharmacology approach. In our study, the TCM Database@Taiwan was utilized to search for the chemical formula. The pharmacological characteristics of isoegomaketone were then evaluated in silico using the Swiss Absorption, Distribution, Metabolism, and Excretion (Swiss ADME) and Deep Learning–Acute Oral Toxicity (DL-AOT) methods, and the potential isoegomaketone target genes were identified using a literature study. Additionally, using the clusterProfiler R package 3.8.1, the Gene Ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of target genes were performed. In order to obtain the protein interaction network, we simultaneously submitted the targets to the STRING database. After this, we performed molecular docking with respect to targets and isoegomaketone. Finally, we created visual networks of protein–protein interactions (PPI) and examined these networks. Our results showed that isoegomaketone had good drug-likeness, bioavailability, medicinal chemistry friendliness, and acceptable toxicity. Subsequently, through the literature analysis, 48 target genes were selected. The bioinformatics analysis and network analysis found that these target genes were closely related to the biological processes of isoegomaketone, such as atherosclerotic formation, inflammation, tumor formation, cytotoxicity, bacterial infection, virus infection, and parasite infection. These findings show that isoegomaketone may interact with a wide range of proteins and biochemical processes to form a systematic pharmacological network, which has good value for the creation and use of drugs.Isoegomaketone is a water-soluble natural ketone compound that is commonly present in  Rabdosia angustifolia and Perilla frutescens [Perilla frutescens and has great drug development potential. However, the potential targets of isoegomaketone and its potential for further development are not yet fully understood. At present, the extraction of potential targets from existing research and the use of computer methods to predict the potential development directions has become the main method [Traditional Chinese medicine contains a large number of active drug molecules . Throughutescens . Recent utescens ,6,7,8,9.n method ,11,12. CAs a result, we applied the network pharmacology approach to comprehensively examine the pharmacological effects of isoegomaketone. Firstly, the Swiss Absorption, Distribution, Metabolism, and Excretion (Swiss ADME) and Deep Learning–Acute Oral Toxicity (DL-AOT) methods were used to evaluate in silico the drug properties of isoegomaketone. Furthermore, we provided candidate target genes through literature analysis. Additionally, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and molecular docking were performed on these potential target genes. Finally, we thoroughly demonstrated the probable targets of the compound by creating the isoegomaketone pharmacological interaction network.http://tcm.cmu.edu.tw/ (accessed on 25 March 2022)), which houses sources of chemical and pharmacological information on isoegomaketone [http://www.swissadme.ch/ (accessed on 25 March 2022)), which supports drug development, enables researchers to calculate the physicochemical descriptors and predict the ADME parameters, pharmacokinetic features, drug-like nature, and medicinal chemistry friendliness of one or more small compounds [An important database resource that is free to use and readily available is the TCM Database@Taiwan ) is a tool used to evaluate the acute oral toxicity (AOT) of small molecules [The DL-AOT prediction server ), which enables users to supplement the knowledge already known about PPI with computational predictions [One of the online databases that compiles all publicly available sources of knowledge of PPI is STRING 11.0 , molecular functions (MF), and cellular components (CC) . It is phttps://pubchem.ncbi.nlm.nih.gov/ (accessed on 26 March 2022)) in mol2 format, was imported into the AutoDockTools 1.5.6 software. After checking its spatial structure, adding atomic charges, assigning atomic types, and changing all flexible bonds to rotatable by default, it was saved in pdbqt format as a docking ligand. The 3D structures of target-gene-associated proteins were downloaded from the Uniprot database (https://www.uniprot.org/ (accessed on 26 March 2022)) and the pdb database (https://www.rcsb.org/ (accessed on 26 March 2022)), and they were then saved in pdb format. AutoDockTools 1.5.6 software was used to carry out the removal of origin ligands, removal of water molecules, hydrogenation, charge addition, and various optimizations. The grid box was set as the default value and the final optimized proteins were saved in pdbqt format as docking acceptors. Molecular docking was performed using AutoDock Vina 1.1.2. The protein structure was set during molecular docking to be a rigid macromolecule, and the Genetic Algorithm Parameters algorithm was used. PyMOL was used to illustrate the outcomes for the group in which each protein had the lowest binding energy.The 2D structure of isoegomaketone, downloaded from PubChem , cellular components (CC), and molecular functions (MF) according to GO enrichment analysis. BP enrichment mainly involved the following target genes: neuron death (20/48), regulation of neuron death (18/48), cellular response to chemical stress (17/48), aging (17/48), response to radiation (17/48), cellular response to abiotic stimulus (16/48), cellular response to environmental stimulus (16/48), neuron apoptotic process (15/48), response to light stimulus (15/48), and positive regulation of neuron death (13/48). The following target genes were mostly associated with CC enrichment: transcription regulator complex (8/48), mitochondrial outer membrane (6/48), organelle outer membrane (6/48), outer membrane (6/48), phosphatidylinositol 3-kinase complex (4/48) and protein–DNA complex (4/48). Target genes for MF enrichment included the following in particular: protein serine kinase activity (11/48), DNA-binding transcription factor binding (11/48), DNA-binding transcription activator activity (11/48), and MAP kinase activity (5/48). All the results are shown in p value was the lipid and atherosclerosis pathway.We performed KEGG enrichment analysis of these target genes concurrently. KEGG pathway enrichment analysis revealed that 48 possible target genes were enriched and that there were significant relationships between the target genes and 194 signal pathways (FDR ≤ 0.05). The top 20 pathways with the highest enrichment ratios are demonstrated in In general, the likelihood of interaction decreases with the energy of conformational stability of the ligand and receptor. There is yet no single standard for the target screening of active compounds, and, at present, binding energy ≤ −5.0 kcal/mol is typically used as the basis for screening. The findings of molecular docking revealed that 6 of the 12 chosen target proteins exhibited an affinity for isoegomaketone that was less than −5.0 kcal/mol. All docking results are shown in Currently, network pharmacology is being used with increasing frequency in the development and utilization of novel drugs. It converts the “single target, single drug” concept of drug research into a “network target, multicomponent treatment” model . As a reDrug-likeness is recognized as a guideline for chemical library screening optimization in the area of drug development . LipinskAs indicated in Most of them are associated with anti-inflammatory, anti-tumor, and cytotoxic responses. They might, thus, be the main targets of isoegomaketone’s pharmacological activity.To elucidate the potential biological activity of isoegomaketone based on gene functions and signaling pathways, we further conducted GO analysis and KEGG pathway analysis of these key targets with the clusterProfiler R package 3.8.1. According to the analysis of BP items, isoegomaketone is closely associated with the following: neuron death, regulation of neuron death, cellular response to chemical stress, aging, response to radiation, cellular response to abiotic stimulus, etc. The results of KEGG pathway enrichment analysis show that the lipid and atherosclerosis pathway is highly enriched. It should be mentioned that no previous studies have clarified the relationship between isoegomaketone and atherosclerosis. The result indicates that isoegomaketone may play a comprehensive role in preventing and treating atherosclerosis, as pathways such as the lipid and atherosclerosis pathway and fluid shear stress and atherosclerosis pathway are involved. In addition, the anti-inflammatory activity of isoegomaketone may also be involved. Further in vitro and in vivo experimental validation could be carried out. Moreover, we found that 9 of the top 20 enriched KEGG pathways were related to bacterial, viral, and parasite infections, such as hepatitis B, Kaposi sarcoma-associated herpesvirus, Epstein–Barr virus, human cytomegalovirus, human immunodeficiency virus 1, tuberculosis, Chagas disease (American trypanosomiasis), measles, and toxoplasmosis. According to these findings, isoegomaketone could possess antiviral, antibacterial, and parasitic action. Some pathways are related to inflammatory diseases—for example, the AGE-RAGE signaling pathway in non-alcoholic fatty liver disease and diabetic complications—which is in accordance with a previous in vivo study . TherefoHowever, there are limitations to our research. The most significant is that, as mentioned above, our target screening was obtained through a large number of previously published studies. These provide high-quality, real-world evidence, based, in part, on in vivo and in vitro studies ,22,23,24Our research revealed that isoegomaketone has a wide range of pharmacological effects. At the same time, we investigated isoegomaketone’s potential targets, which may be used to further create secure and efficient anti-atherosclerosis, anti-inflammatory, anti-tumor, cytotoxic, antiviral, antibacterial, and anti-parasite medications. Our work offers a new perspective on the study, creation, and clinical use of isoegomaketone.


Gynura divaricata (GD) functions in the treatment of diabetic foot (DF).To predict and validate the potential mechanism by which The main chemical constituents of GD were identified by reviewing the literature, the traditional Chinese medicine database platform (TCMIP) and the BATMAN-TCM platform. DF disease targets were identified with the GeneCards database, and the compound-target network was constructed by using the intersection of drugs and disease. The STRING platform was used to construct the protein–protein interaction (PPI) network, and Cytoscape 3.7.2 software was used to visualize the results. Moreover, the Metascape database was used for Gene Ontology (GO) enrichment analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking of the active ingredients of GD and core protein targets of DF was performed using AutoDock software. Finally, the predicted results were preliminarily verified with experiments.A total of 140 potential targets of GD were identified and associated with DF. According to the PPI network analysis, GD accelerated DF wound healing, and the mechanism may be related to proteins such as AKT1, TP53, IL6, CASP3, TNF, and VEGFA. GO and KEGG enrichment analyses indicated that GD may play a role in the treatment of diabetic foot by affecting various signaling pathways. Molecular docking results showed that the proteins AKT1, TP53, IL6, CASP3, TNF, and VEGFA were closely associated with the components of GD. The animal experiments showed that GD reduced the levels of IL-6 and TNF-α and increased the mRNA and protein expression of VEGFA in rats with DF.GD regulates multiple targets and multiple pathways to promote wound healing in DF.The online version contains supplementary material available at 10.1186/s10020-023-00605-w. Diabetic foot (DF) is a common chronic complication of diabetes  is a perennial herb that is native to Singapore and is now abundant in the southwestern and southeastern coastal areas of China  network. In addition, the core targets were used to study the target and target pathway of the active ingredients of GD by using network pharmacology, gene ontology (GO), and biological pathway (KEGG) functional enrichment analyses. Finally, we performed a molecular docking analysis and some experiments to verify our predictions.http://tcmspw.com/), BATMAN-TCM (http://bionet.ncpsb.org.cn/batman-tcm/), and published literature .Figure https://www.genecards.org/) was searched to identify potential disease-related targets by using “Diabetic Foot” as a keyword. According to experience, target points with a Z-Socre > 1.0 were selected as potential targets in DF.The Genecards database (https://cn.string-db.org/) was used to construct a target protein–protein interaction (PPI) network  was used to further explore the complex mechanism by which GD functions in the treatment of DF. Enrichment results were considered significant at P < 0.01, and the analysis results were visualized.Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) describe and annotate screened genes and contribute to the interpretation of system-level data , and PyMOL software was used to remove ligands and water molecules. The.sdf file of the active ingredients of GD was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Autodock Tools 1.5.6 software was used to add atomic charges and assign atomic types. All the flexible bonds were set as rotatable Running AutoDock Vina for molecular docking. The lower the energy is when the ligand–receptor interaction conformation is stable, the more likely the effect is to occur.The core protein structure of the PPI network was downloaded from the PDB database  solution (40 mg·kg-1) one time. Intravenous blood was collected to monitor fasting blood glucose levels ≥ 16.7 mmol/L, and polydipsia, polyphagia and polyuria were considered indications that the diabetes model had been successfully established , and the specific steps were carried out according to the instructions of the kit.Total protein was extracted and collected from ischemic foot tissue, and the concentration of the total protein was measured. The proteins were separated by 8% SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking in 5% skim milk powder for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C and a secondary antibody for 1 h the next day. After washing, the proteins were detected using the ECL Plus chemiluminescence system. The blots were analyzed using ImageJ, and protein expression levels were normalized to the GAPDH expression levels.−ΔΔCt method was used to evaluate fold changes in expression relative to the control. The primers specific for the target genes are listed in Additional file Briefly, total RNA was extracted using TRIzol reagent , and reverse transcription was conducted using RevertAid™. Messenger RNA expression levels were determined by RT–PCR using the SYBR GreenPremix Ex TaqTM kit  following the manufacturer's instructions. GAPDH was used as an internal control. The 2P < 0.05 indicates that the difference is statistically significant.SPSS 17.0 statistical software was used for statistical analysis. The experimental data are expressed as the mean ± SD. One-way ANOVA was used for comparisons between groups. The LSD method was used for comparisons between groups. For data with a non-normal distribution or uneven variance, a nonparametric test of multiple independent samples was used. Searches of the TCMSP and BATMAN-TCM databases and published literature revealed that GD contains 25 traditional Chinese medicine active ingredients. Among these active ingredients, 17 compounds had no known potential targets, while 8 compounds had many targets. Some molecules are targets of many kinds of compounds  network. Information about drug components and effective targets as well as other information about GD was imported into Cytoscape 3.7.2 software to construct a drug-active ingredient-effective target network diagram , 106 cellular components (CC), and 138 molecular functions (MF). The results were ranked according to We selected the key genes AKT1, TP53, IL-6, CASP3, TNF-α and VEGEA that were identified in the previous steps and carried out molecular docking with uridine and quercetin, the main pharmaceutical components in GD. The docking energy results are shown in Fig. P < 0.001). The medium and high doses of GD improved the diabetic foot wound healing rate at each time point , while the low-dose extract had no significant effect on the wound healing rate. In addition, as we expected, all dose groups of GD could reduce the blood glucose of diabetic rats  is a common complication of long-term diabetes mellitus, and it is associated with a high incidence of lower extremity ulceration and amputation


To explore the rules of TCM medication in the treatment of constipation in network pharmacology.  Collect and screen the clinical intervention literature on TCM for constipation from China's national knowledge infrastructure, Wanfang and VIP databases established a database of TCM for constipation, applied R software (3.3.1) to analyze the pattern of prescriptions for TCM for constipation, and summarized the core prescription. The effective active compounds and action targets in the core prescription were screened by Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Traditional Chinese Medicine Integrated Databases (TCMID), constipation-related targets were derived from the DisGeNET and GeneCards databases, Protein-protein interaction network (PPI) was drawn by STRING database, and enrichment analysis was conducted by the Clusterprofiler package in R software (3.3.1). Finally, molecular docking was used to validate the binding ability of candidate compounds to potential targets.  Two hundred sixteen target prescriptions were screened through data mining, involving 226 herbs. Association rule analysis results suggested that the “Angelicae sinensis-Radix-dried rehmanniae-Cistanche deserticola-Atractylodes macrocephala-Astragali Radix” was a strong affinity for medicine. Network pharmacology analysis of the core prescription resulted in the screening of 115 candidate compounds, such as quercetin, kaempferol, mangostin, eugenol A, and beta-sitosterol; 131 potential targets, such as PTGS2, PTGS1, and CHRM3; and 160 signaling pathways, such as lipid and atherosclerosis, proteoglycans in cancer, hepatitis B, Kaposi's sarcoma-associated herpesvirus infection, and PI3K/AKT pathways. Molecular docking showed that PTGS1-formononetin, PTGS2-kaempferol, and CHRM3-kaempferol were all well bound and well matched.  This study provides a new method and ideas for clinical applications of integrated Chinese and western medicine in treating constipation. Constipation is one of the most common digestive system diseases in China. It mainly is manifested as the reduced number of defecation, laborious defecation, dry stool knot, and frequent cathartic use. Recently, the incidence of constipation in China has increased, and the long-term frequently serious defecation disorder reduced the quality of life. Currently, the main clinical treatment of constipation is the laxative and stimulant drug , but theTCM treatment emphasizes a holistic approach, taking into account both the symptoms and the root causes, and follows a discernment-based approach to treatment, overcoming the shortcomings of palliative treatment in Western medicine, which has unique advantages in improving the body's internal environment, reducing toxicity, and increasing effectiveness . In addiIn this study, data mining was used to analyze the application rules of TCM in the treatment of constipation. We also proposed a computational systems pharmacology method and molecular docking to determine the associated molecular mechanisms. We intended to explore TCM's drug use pattern for treating constipation by collating the published clinical intervention literature of TCM in treating constipation, analyzing the frequency of drug use, nature, and taste of prescriptions. Using R software (3.3.1) to explore the key points of treatment and prescription use and the core combination of drugs , and usiWe conducted an electronic search of the Chinese databases from their inception to 31/12/2021. We searched the Chinese literature in CNKI, Wanfang, and VIP databases. All clinical intervention literature on the internal treatment of constipation with TCM was considered. The following search terms were used in combination: “Chinese medicine,” “traditional medicine,” “herbal medicine,” “herb,” “plant,” “prescription,” “decoction,” and “constipation,” among others.The included literature for this study had to be a clinical intervention caseThe included literature was related to the internal use of TCM for constipationThe composition and dosage of the prescribed medication in the literature must be fully documentedThe literature on the experience of famous veteran Chinese medicine practitioners can also be included in this studyThe inclusion criteria were as follows:Studies with cellular, animal, and other basic studies, reviews, meta-analysis, systematic evaluations, literature data mining, and mechanistic theoretical discussionsDuplicate publication, only abstract or lack of outcome data, and no access to obtain the full textConstipation due to or in combination with other types of disease, e.g., tumour, pregnancy, radiotherapy, fractures, drug-related, and constipated irritable bowel syndromeThe combination of Western medicine taken orally or topically with acupuncture or enemas for constipation is in the literatureThe exclusion criteria were as follows:Following the search, all identified citations were collated and uploaded into EndNote (X9) citation manager, and duplicates were removed. The titles and abstracts were then screened against the inclusion criteria. Those studies meeting the eligibility criteria were retrieved in full. The full texts of selected studies were assessed in detail, and those that did not meet the inclusion criteria were excluded.Relevant data were extracted from the final inclusion literature that included the review using a standardized data extraction template developed using a Microsoft Excel workbook. Double data extraction and entry were performed to ensure accuracy.The names of herbs were standardized concerning the 2015 Chinese Pharmacopoeia (CHP)  and the The normalized TCM was quantified by dichotomizing them using the Reshape function to build a shaping databaseThe ggplot2 function was applied to the data for frequency analysis to create a bar chart of Chinese medicines plottedThe Corrplot function was used to correlate the data and extract the core prescription. Association analysis was performed using rules and rules viz functions to visually demonstrate the degree of association used in TCMK-means, PAM, and GMM algorithms to cluster the dataThis study uses This study used R software (3.3.1) to perform a statistical analysis of the literature included in the database. http://tcmspw.com/tcmsp.php) and TCM Integrative Database (TCMID) (http://www. http://megabionet.org/tcmid/) [https://www.uniprot.org/) is used to query the gene name of the target protein [The chemical components and related targets of the core prescription should be retrieved from the TCM Systems Pharmacology (TCMSP) Database (/tcmid/) , 11. The/tcmid/) , 13. The protein .Using “constipation” as the keyword, the DisGeNET and GeneCards databases searched and screened the disease target genes –17. The The active components of the core prescription and the related targets and the target genes of constipation were input into the Cytoscape 3.7.2 software to build a network map of the active component-action target network and clarify the potential interaction between the constipation target genes and the active components of the core prescription .Chemical components and intersection targets of the core prescription were imported into Cytoscape 3.7.2 software to construct the active component target network, and the topological properties of the network were analyzed; the intersection targets were uploaded to STRING database, and the species was limited to “human”; the confidence was 0.4, the free nodes were deleted, and the PPI network was constructed.P value ≤ 0.05 was used to screen biological functions and signal pathways of the core prescription acting targets.Furthermore, the Clusterprofiler package in R software (3.3.1) was used for the Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the core prescription acting targets, and the bioinformatics platform was used for visualization analysis of biological functions and pathways. To assess the credibility of the connection between the target and the compound and identify the core prescription for constipation treatment, molecular docking of the core compounds with core targets was carried out. Molecular docking can study the active components of the core prescription and its related targets in the treatment and can explain the mechanism of action and binding activity of active components and target proteins to a certain extent. Then, we run Schrodinger software for molecular docking and use PyMOL 2.1 to analyze the docking conformation visually.A total of 11,386 pieces of literature were initially searched including 2,978 on the CNKI database, 5,552 on Wanfang the database, and 2,856 on the VIP database. The following steps are as follows: (1) the literature was first checked against title and author to remove duplicates. (2) After that, the title and abstract were read for initial screening to eliminate irrelevant literature. (3) The full text of the literature was further read and rescreened to eliminate those not meeting the inclusion criteria. (4) Next, the literature was extracted from the prescriptions, and those with duplicate use of drugs and no clear prescription information were excluded.Bar graph of 31 TCM used >30 times and classified according to the 2016 edition of traditional Chinese medicine. Bar graph of 31 TCM used >30 times is classified according to the 2016 edition of Traditional Chinese Medicine. The results showed that TCM for constipation is mainly tonic for deficiency, followed by qi regulating and diarrhea medicines, and the top 10 TCM in high-frequency combinations are mostly sweet tasting and warm in nature; they belong to the spleen and lung meridian. The overall treatment reflects the principle of tonifying deficiency, guiding stagnation, and clearing the bowels, which is in line with the basic pathogenesis of constipation in TCM theory. The medicine characteristics and TCM theory “deficiency of the internal organs” are shown in Atractylodis macrocephalae-Bai Shao, rhubarb-Angelicae sinensis, Herba cistanches-firenuts, Radix Rhizoma gastrodiae-Mai Dong, and Radix Astragali-licorice  is an online biological information repository and analysis tool for extracting biological information regarding gene functional annotation and pathways enrichment. These targets existed in the nucleus, cytoplasm, and plasma membrane of cells and were involved in biological processes, such as transcriptional regulation, drug response, signal transduction, cell proliferation, and senescence. The molecular function of these genes was involved in binding proteins, enzymes, and zinc ions. In GO enrichment analysis, 2352 GO items, 2133 biological processes, 80 cellular components, and 139 molecular function-related items were identified. KEGG enrichment analysis indicated that 160 pathways were affected by the active components of the core prescription with the smallest P value. The top 20 pathways included lipids and atherosclerosis, proteoglycans in cancer, hepatitis B, Kaposi's sarcoma herpesvirus infection, and PI3K-AKTB pathways. Based on this information, the core prescription compound-target-pathway network association was established.In order to clarify the characteristics of the relevant targets of the key active components of the core prescription, GO and KEGG pathway enrichment analyses were performed in The active component target network of constipation consists of 199 compound nodes, 131 target nodes, and 1202 edges. As shown in The molecular docking results obtained according to the SP method are shown in To better understand the interactions between the compounds and target genes, we constructed an interaction network between the key target genes and corresponding active compounds in the core prescription . AccordiIn recent years, people have paid more and more attention to the modernization, inheritance, and development of TCM. With the arrival of the era of big data and the rise of artificial intelligence, many software and platforms have emerged for TCM prescription analysis. Many medical researchers have devoted themselves to mining literature and data, hoping to guide clinical guidance better and give full play to the advantages of TCM. This study used data mining and R software , 16 to fIn this study, in data mining in R software combined with the numerical ranking of support, confidence, and improvement, we can see that the core prescription of “Angelicae sinensis-Radix-dried rehmanniae-Cistanche deserticola-Atractylodes macrocephala-Astragali Radix” has high frequency and high comprehensive score and has good correlation. TCM treats constipation to fill and through. Angelica can nourish the blood and dryness; baishu can improve the qi and spleen; astragalus can benefit qi and aphrodisiac; angelica and astragalus can replenish qi, blood, and laxative; astragalus and baishu can play a role in rationalizing qi and laxative; cistanche can supplement kidney yang, improve muscles and activate blood, and run laxatives; raw yellow can nourish yin and moist intestines. The combination and use of the above drugs reflect the treatment of constipation to supplement qi and blood, regulating qi and guiding stagnation, nourishing Yin, and moistening dryness, which is in line with the basic pathogenesis of the “virtual standard” of constipation. As a basic Chinese medicine, the core prescription is widely found in multiple prescriptions to treat constipation, such as Runchang pills, Huangqi decoction, and Xinjia Huanglong decoction, which proves that the core prescription conforms to the clinical reality and is commonly used to treat constipation, and the data mining results are reliable.Based on data mining and collation results, this study applied network pharmacology to investigate the mechanism of action of the core prescription for treating constipation and constructed a network between the effective compounds of the core prescription and the constipation-related genes. The network map analysis determined a total of 115 active components and 131 targets involved in the network construction. The compound-target network map can be predicted that the treatment-related components are quercetin, kaempferol, hilanin, and other activities. Quercetin is a flavonol compound with multiple bioactivities and widely found in natural plants and has antioxidation and anti-inflammatory effects. Quercetin relieves the symptoms of difficult defecation, reduces the harmful flora, and reduces the intestinal mucosa . Kaempfein vivo are involved in the core prescription for constipation treatment mechanism.From the “core prescription-active, ingredient-target of action map” and the PPI network of key targets of the core prescription for constipation, the abnormal targets of the core prescription for constipation can be predicted, among which the closest relationships are with cyclooxygenase 2 (PTGS2), cyclooxygenase 1 (PTGS1), and acetylcholine receptor M3 (CHRM3). PTGS is a cyclooxygenase (COX); it has two isozymes , 31: oneMolecular docking results show that the active ingredients such as quercetin, kaempferol, and stalk flower element bind well with the three target proteins  and have a high matching degree, which reflects good molecular docking, suggesting that the core prescription may play a role through these key targets. These findings may provide further insight into the therapeutic mechanisms of core prescriptions for constipation and may facilitate future screening of potential therapeutic targets.This study of prescription medication analysis of constipation through data mining method provides more basis for the treatment of constipation, reduces the difficulty of clinical workers in drug dispensing of constipation treatment, has positive significance for the improvement of clinical efficacy, and makes the continuous improvement of the treatment of constipation in traditional Chinese medicine.