Qingfei Gujin Decoction (QGD) has been shown to be effective against osteosarcoma. This research was aimed at investigating the main active ingredients and potential mechanisms of QGD acting on osteosarcoma through network pharmacology and molecular docking techniques.  The active ingredients and targets of QGD were screened from the TCMSP database, and the predicted targets were obtained from the PharmMapper database. Meanwhile, the targets of osteosarcoma were collected using OMIM, PharmGKB, and DisGeNET databases. Then, GO and KEGG enrichment analyses were performed by RStudio. PPI and drug-ingredient-target networks were constructed using Cytoscape 3.2.1 to screen the major active ingredients, key networks, and targets. Finally, molecular docking of key genes and their regulatory active ingredients was performed using AutoDockTools 1.5.6 software. β-sitosterol, and kaempferol were the main active ingredients of QGD acting on osteosarcoma, and major signaling pathways such as PI3K-Akt signaling pathway, MAPK signaling pathway, and IL-17 signaling pathway were observed. TP53, SRC, and ESR1 were identified as key proteins that docked well with their regulated compounds.  38 active ingredients were collected, generating 89 cross-targets; quercetin, luteolin,  QGD is effective against osteosarcoma through multicomponent, multitarget, and multipathway. This study was helpful for finding effective targets and compounds for osteosarcoma treatment. Osteosarcoma is the most common primary bone solid malignant tumor. Local pain, followed by localized swelling and limitation of joint movement, is the typical sign and symptom of osteosarcoma . AdolescNumerous studies have shown that traditional Chinese medicine (TCM) can not only alleviate the symptoms of tumor patients, such as fatigue, chronic pain, and cachexia, but also improve their quality of life and reduce the adverse reactions and complications caused by chemotherapy, radiotherapy, and targeted therapy . In Chinβ1, and CXCR4 expressions. [In TCM, osteosarcoma is classified as “osteoma,” “osteomyelitis,” and “indurated knee mass.” It is believed that osteosarcoma is frequently caused by insufficient endowment, a lack of genuine qi. Pathogenic qi enters the body, traveling with meridian qi and blood to the bone marrow, causing qi and blood stagnation, meridian obstruction, tendon erosion, and bone formation. On this basis, we established the “pulmonary deficiency phlegm obstruction syndrome” osteosarcoma pulmonary metastasis rat model and found that Qingfei Gujin Decoction  has a good antiosteosarcoma pulmonary metastasis effect, and its mechanism may be related to the downregulation of IL-10, TGF-essions. Network pharmacology, a new concept in TCM research, has been widely used to study complex network relationships between TCM and diseases. To better understand the potential mechanism of QGD in the treatment of osteosarcoma, we used network pharmacology and molecular docking technology to screen the active components of QGD and predict its possible targets and pathways in osteosarcoma, with the goal of providing a theoretical foundation for the treatment of osteosarcoma with TCM .https://old.tcmsp-e.com/tcmsp.php) is a one-of-a-kind pharmacological platform of Chinese herbal medicines that captures the relationships between drugs, targets, and diseases [Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (diseases . We collhttp://www.lilab-ecust.cn/pharmmapper/), an online tool for identifying potential target candidates based on molecular structure [TCMSP was used to collect targets that interact with active ingredients. Simultaneously, the predicted targets of QGD were obtained using PharmMapper (tructure . All thehttps://omim.org/) is a comprehensive, authoritative compendium of human genes. PharmGKB (https://www.pharmgkb.org) is created by the National Institutes of Health (NIH), which provides information about how human genetic variation affects response to medications [https://www.disgenet.org) is a versatile platform that can be used for different research purposes including the investigation of the molecular underpinnings of human diseases and their comorbidities, the analysis of the properties of disease genes, the generation of hypothesis on drug therapeutic action and drug adverse effects, the validation of computationally predicted disease genes, and the evaluation of text-mining method performance [Online Mendelian Inheritance in Man (OMIM) . In this network, the active ingredients were represented by circular nodes of various colors, while potential targets were represented by rectangular nodes.https://cn.string-db.org/) is an online database for searching known protein interactions. The PPI network was constructed by importing the potential genes into the search tool to retrieve interacting genes, and the organism type was selected as Homo sapiens (humans). The minimum required interaction score was set with a medium confidence = 0.9, and all other parameters were left at their default values. The “string interactions. tsv” file was downloaded in order to visualize the network and determine the intersection of the PPI network and core genes.STRING , closeness centrality (CC), degree centrality (DC), eigenvector centrality (EC), and local average connectivity-based method (LAC) were calculated using the “CytoNCA.” Firstly, the top 50% candidate genes of all values are to be subnetworks. Secondly, the candidate genes with the top 50% of values in subnetworks are selected as critical genes and established core networks.R packages including “colorspace,” “stringi,” and “ggplot2” were installed in RStudio, and a Bioconductor package that includes “DOSE,” “clusterProfiler,” and “enrichplot” was used for GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses.http://rcsb.org), the US data center for the global PDB archive, makes PDB data freely available to all users in support of a “Structural View of Biology” [AutoDockTools 1.5.6 was used to dock the key targets and their conditioning ingredients. The mol2 chemical structure of ingredients was downloaded from the TCMSP database, and all compounds were saved as ligand parameter files in pdbqt format. The Research Collaboratory for Structural Bioinformatics Protein Data Bank , cellular component (CC), and molecular function (MF). 89 potential targets of QGD in the treatment of osteosarcoma were analyzed by R package. As shown in In order to further explore the possible mechanism of QGD in the treatment of osteosarcoma, we performed KEGG pathway enrichment analysis on 89 target targets. As shown in Essential genes were selected for molecular docking with compounds that might regulate these targets , cisplatin (DDP), adriamycin (ADM), ifosfamide (IFO), epirubicin (EPI), and etoposide VP-16) are commonly used for osteosarcoma chemotherapy . The MAP are commTCM has the benefits of safety and low toxicity, and it is increasingly being used for the prevention and treatment of osteosarcoma patients in China after surgery and chemotherapy. Network pharmacology has evolved into a sophisticated method for studying the practical components and complex mechanisms of TCM and compound prescriptions. In this study, the active ingredients of QGD were collected and network pharmacology was used to validate the effect of multitarget and multichannel treatment.β-sitosterol, and kaempferol. Quercetin was a flavonol compound with a variety of biological activities which had been widely used to treat cardiovascular diseases, diabetes, tumors, and other diseases. Dana et al. hypothesized that quercetin inhibited osteosarcoma cell proliferation, migration, and invasion, inducing autophagy and apoptosis, and could play a separate or synergistic role in overcoming drug resistance in osteosarcoma cell lines [β-Sitosterol was one of the most common phytosterols. A meta-analysis revealed that consuming high levels of dietary β-sitosterol might have a positive effect on antitumor mechanisms [β-sitosterol in osteosarcoma was rarely reported, it has been shown to improve bone fragility and fracture occurrence [QGD, composed of Astragalus membranaceus, Fritillaria thunbergii, Platycodon grandiflorum, Hedyotis diffusa, and Coicis semen, has been shown to effectively prevent local recurrence and metastasis after osteosarcoma surgery. According to our study, the main active ingredients of QGD acting on osteosarcoma may be quercetin, luteolin, ll lines . Luteolill lines . Meanwhill lines . β-Sitoschanisms . Althougcurrence . Kaempfecurrence . Chen etcurrence .α (ER), had been widely studied in breast cancer. Although there were fewer studies on ESR1 and osteosarcoma, ESR1-mediated estrogen effects associated with bone mineralization have been reported [P53 was encoded by the TP53 gene on human chromosome 17 and served as a tumor suppressor gene in the human body, protecting genomic integrity. Mutation in the TP53 gene was detectable in about 50% of patients with tumor, and more than 75% of TP53 gene mutations resulted in a loss of wild-type p53 activities, thereby promoting tumorigenesis, progression, and metastasis . TP53 mureported . One of reported . Our stuPI3K/AKT was one of the most important carcinogenic pathways in human cancer, and it was frequently overactivated in osteosarcoma. It was involved in tumor occurrence and progression, including proliferation, invasion, cell cycle progression, angiogenesis, and chemical resistance. Therefore, targeting the PI3K/AKT signaling pathway was proposed as a potential treatment for osteosarcoma . The MAPIn summary, QGD had a variety of components and played an antiosteosarcoma role via multiple targets and pathways. Unfortunately, the efficacy of QGD in the treatment of osteosarcoma had not been validated at the molecular level, and further research was required to supplement the conclusion. At the same time, through network pharmacology and molecular docking technology, the role of Qingfei Gujin Decoction in different stages of osteosarcoma cannot be clearly defined. Its dose, intervention time, and exact curative effect need to be further studied in the future.β-sitosterol, and kaempferol were the main active ingredients. We discovered that the main pathways of QGD against osteosarcoma were the PI3K-Akt signaling pathway, proteoglycans in cancer, MAPK signaling pathway, chemical carcinogenesis receptor activation pathway, cell aging, IL-17 signaling pathway, and EGFR tyrosine kinase inhibitor resistance. In addition, TP53, SRC, and ESR1 were presumed to be key proteins because of their good docking with the regulated compounds. These findings may aid in the identification of effective targets and compounds for osteosarcoma treatment, as well as provide a particular reference for osteosarcoma treatment.A total of 38 active ingredients were extracted from the QGD. 526 targets and 526 disease targets were collected, as well as 89 common targets were generated. Quercetin, luteolin,


Kangxian decoction (KXD) has been used in clinical practice to treat epilepsy. The purpose of this study was to explore the active components of KXD and clarify its antiepileptic mechanism through network pharmacology and molecular docking.  The components of KXD were collected from the Encyclopedia of Traditional Chinese Medicine (ETCM) database and the literature was searched. Then, active ingredients were screened by SwissADME and potential targets were predicted by the SwissTargetPrediction database. Epilepsy-related differentially expressed genes were downloaded from the Gene Expression Omnibus database. A component-target-pathway network was constructed with Cytoscape. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein‒protein interaction network analysis revealed the potential mechanism and critical targets. Receiver operating characteristic (ROC) curves and box plots in microarray data validated the good diagnostic value and significant differential expression of these critical genes. Molecular docking verified the association between active ingredients and essential target proteins.  In our study, we screened the important compounds of KXD for epilepsy, including quercetin, baicalin, kaempferol, yohimbine, geissoschizine methyl ether, baicalein, etc. KXD may exert its therapeutic effect on epilepsy through the following targets: PTGS2, MMP9, CXCL8, ERBB2, and ARG1, acting on the following pathways: neuroactive ligand-receptor interactions, arachidonic acid metabolism, IL-17, TNF, NF-kappa B, and MAPK signaling pathways. The molecular docking results showed that the active ingredients in KXD exhibited good binding ability to the key targets.  In this study, we explored the possibility that KXD for epilepsy may act on multiple targets through multiple active ingredients, involving neurotransmitters and neuroinflammatory pathways, providing a theoretical basis for subsequent clinical and experimental studies that will help develop effective new drugs to treat epilepsy. Epilepsy is considered one of the most common serious brain disorders and affects more than 70 million people worldwide. It is not a disorder with a single presentation or cause, but a combination of symptoms with multiple risk factors and a strong genetic predisposition. Epilepsy has a bimodal distribution of prevalence, with infants and older populations at the highest risk . AnxietyTraditional Chinese medicine (TCM) has been used for thousands of years as a unique treatment modality with the advantages of clinical efficacy, few side effects, and low drug resistance. In recent years, TCM research as a new treatment strategy has gained increased attention. Chinese medicine has a long history of treating epilepsy. Different herbs can exert antiepileptic effects through various mechanisms such as enhancing GABAergic effects, modulating sodium and NMDA channels, and exerting antioxidant, anti-inflammatory, and neuroprotective properties .Acorus tatarinowii (Chinese name: Shichangpu), Pinellia ternata (Chinese name: Banxia), Panax notoginseng (Chinese name: Sanqi), Gastrodia elata (Chinese name: Tianma), Uncaria (Chinese name: Gouteng) and Pheretima (Chinese name: Dilong), which have the effect of eliminating phlegm and promoting blood circulation. The names of the plants involved are consistent with “The Plant List” (https://www.theplantlist.org/). Previous clinical and experimental studies have shown that KXD significantly prolongs the seizure incubation period, reduces seizure level, duration, and number of seizures in epileptogenic rats, and achieves antiapoptotic effects by reducing hippocampal Glu content, increasing GABA content, and promoting Bcl-2 protein expression [Kangxian decoction (KXD) is composed of six traditional Chinese medicines, including pression –9. In adpression , 11. Thipression . Moleculpression . At preshttps://www.tcmip.cn/ETCM/) [https://pubchem.ncbi.nlm.nih.gov/) [https://www.swissadme.ch/) [https://www.swisstargetprediction.ch/) [The components in the KXD were collected from the Encyclopedia of Traditional Chinese Medicine (ETCM) database (n/ETCM/) . To ensuih.gov/)  to obtaidme.ch/) . The scrdme.ch/) . Althougion.ch/)  to obtaihttps://www.ncbi.nlm.nih.gov/geo/). The expression profile data contained 34 untreated epilepsy patient samples (blood) and 51 normal samples (blood). Differentially expressed genes (DEGs) from the epilepsy samples and normal samples were screened using the limma package in R 4.0.2 software based on |logFC|>0.3, adj. P value < 0.05. Visualization of DEGs was performed using the ggplot2 package as a volcano plot. From intersecting drug target genes with DEGs using the VennDiagram package, we eventually obtained KXD and epilepsy common associated genes.The expression profile data of GSE143272 and the microarray platform GPL10558 were downloaded from the GEO database (https://string-db.org/cgi/input.pl) [KXD and epilepsy common-related genes were used to construct the PPI network using the STRING database (nput.pl) . The PPITo assess the diagnostic ability of each hub gene for epilepsy, ROC curves were plotted, and the area under the curve (AUC) values were calculated using the pROC package in R software. The differential expression of hub genes between normal and epilepsy samples was assessed using box plots.P < 0.05 was considered significant.The cluster profiler package in R software was used to perform the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes commonly associated with KXD and epilepsy. Cytoscape was used to build a network of relationships between active ingredients, common targets for KXD and epilepsy, and pathways. In the component-target-pathway network, “nodes” represent components, targets, and pathways, and “edges” represent the relationships between them. The “degree” parameter represents the number of connections between nodes in the network and is used to assess the important components and targets.https://www.rcsb.org/), and the two-dimensional structure of the molecular ligand was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The proteins and molecules were preprocessed with PyMOL 2.4.0 software and saved in pdb format. The AutoDock Tools 1.5.6 hydrogenated and calculated the charges, selected the proteins as receptors and the molecular compounds as ligands, and then set up the docking parameters. The Grid Box was used to generate the docking area, which was defined as a 40 × 40 × 40 three-dimensional grid centered on the ligand binding site, with a grid space of 0.375 Å. Finally, the docking of the receptor protein and the molecular ligand was completed using AutoDock Vina 1.1.2, and the binding energy was obtained. The optimal conformation was selected and visualized using PyMOL. The entire network pharmacology and molecular docking process are illustrated in To validate the component-target association, the key target genes were selected for subsequent molecular docking analysis. The three-dimensional (3D) structure of the target protein was downloaded from the RCSB PDB database (Acorus tatarinowii (Chinese name: Shichangpu), 11 from Pinellia ternata (Chinese name: Banxia), 10 from Panax notoginseng (Chinese name: Sanqi), 8 from Gastrodia elata (Chinese name: Tianma), 18 from Uncaria (Chinese name: Gouteng), and 6 from Pheretima (Chinese name: Dilong). Notably, some of these ingredients were present in at least two herbs. Details of the active ingredients are shown in After SwissADME screening and removing some compounds without targets, 49 active ingredients from six herbs were identified in the KXD formulation. Including 6 from According to the target screening, there were 149 targets in Shichangpu, 212 targets in Banxia, 292 targets in Sanqi, 159 targets in Tianma, and 517 targets in Gouteng in KXD. After merging and removing duplicates, 705 target genes corresponding to the 49 active ingredients in KXD were finally identified.P value < 0.05. Finally, 312 differentially expressed genes associated with epilepsy were obtained, including 189 upregulated and 123 downregulated genes, and the results are presented in a volcano plot  remains a primary unresolved therapeutic issue. Different antiepileptic drugs have been reported to cause serious side effects, with the development of pharmaco-resistance . KXD is α2-adrenoceptor antagonist with anticonvulsant effects at low doses [Quercetin and kaempferol, both phytoflavonoids, are important dietary components of fruits and vegetables and have various biological effects, including antioxidant, anti-inflammatory, and neuroprotective activities. In vivo studies have shown that these phytoflavonoids can modulate the immune response, becoming an important treatment alternative for epilepsy . GABA exow doses . Geissosow doses . Baicaleow doses , 30. Stuow doses .The PPI network identified PTGS2, MMP9, CXCL8, ERBB2, and ARG1 as key genes. Inflammatory pathways play an essential role in the pathophysiology of epilepsy . PTGS2, κB pathway is associated with inflammation and oxidative stress, plays a key role in epileptogenesis, and can regulate neuronal survival and apoptosis [Neuroinflammation-related pathways could be biomarkers and important therapeutic targets for epilepsy . Glutamapoptosis . The IL-poptosis . Cyclooxpoptosis . FinallySystems pharmacology provided a robust approach to exploring the complex interactions between the ingredients and targets of KXD. In KXD, Acorus tatarinowii combined with Pinellia ternata dissolves turbidity and dispels phlegm, while Gastrodia elata and Uncaria pacify the liver and calm the wind, and Panax notoginseng combined with Pheretima has a meridian-clearing effect. This study revealed that different herbs have the same composition and that different components can act on the same target, allowing the individual herbs in KXD to better exert synergistic or antagonistic effects.In this study, epilepsy-associated genes with differential expression were identified using microarray datasets, and the screened hub genes were validated with both ROC curves and box plots to enhance the accuracy of the prediction results. However, our study has several limitations. First, we need a more comprehensive database of TCM target genes to make the results of the network pharmacological analysis more reliable. Second, experimental validation and multidisciplinary collaboration are still needed to understand the exact mechanism of KXD in epilepsy.In this study, we explored the multicomponent, multitarget, and multichannel characteristics of KXD-mediated epilepsy treatment through network pharmacology and molecular docking. The main components of KXD for epilepsy may be quercetin, baicalin, kaempferol, yohimbine, geissoschizine methyl ether, baicalein, etc. In conclusion, KXD may exert its therapeutic effect on epilepsy through the following targets: PTGS2, MMP9, CXCL8, ERBB2, and ARG1, acting on the following pathways: neuroactive ligand-receptor interactions, arachidonic acid metabolism, IL-17, TNF, NF-kappaB, and MAPK signaling pathways. This study preliminarily illustrated the mechanism of KXD in the treatment of epilepsy, providing a theoretical basis for further experimental validation and contributing to the development of effective drugs to treat epilepsy.


Epimedium has gained widespread clinical application in Traditional Chinese Medicine, with the functions of promoting bone reproduction, regulating cell cycle and inhibiting osteoclastic activity. However, its precise cellular pharmacological therapeutic mechanism on osteoporosis (OP) remains elusive. This study aims to elucidate the molecular mechanism of epimedium in the treatment of OP based on system bioinformatic approach.R software. Epimedium-OP common targets were obtained by Venn diagram package for further analysis. The protein-protein interaction network was constructed using Cytoscape software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were carried out by using clusterProfiler package. Molecular docking analysis was conducted by AutoDock 4.2 software to validate the binding affinity between epimedium and top 3 proteins based on the result of protein-protein interaction.Predicted targets of epimedium were collected from TCMSP, BATMAN-TCM and ETCM databases. Differentially expressed mRNAs of OP patients were obtained from Gene Expression Omnibus database by performing Limma package of A total of 241 unique identified epimedium targets were screened from databases, of which 62 overlapped with the targets of OP and were considered potential therapeutic targets. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that these targets were positive regulation of cell cycle, cellular response to oxidative stress and positive regulation of cell cycle process as well as cellular senescence, FoxO, PI3K-Akt, and NF-kappa B signaling pathways. Molecular docking showed that epimedium have a good binding activity with key targets.Our study demonstrated the multitarget and multi-pathway characteristics of epimedium on OP, which elucidates the potential mechanisms of epimedium against OP and provides theoretical basis for further drug development. Following the accelerated speed of population aging in global, the incidence of OP continues to rise affecting the health and quality of life in the elderly. Currently, the prevention and treatment strategies for OP mainly include bone reproduction promoters, bone resorption inhibitors and bone minerals, such as bisphosphonates, teriparatide, calcitonin, and hormone replacement. However, these drugs may be accompanied by serious adverse effects, including an increasing risk of calculus, jaw necrosis, breast cancer, or atypical femur fracture.–5 To avoid the drawbacks of conventional therapies and provide alternative therapeutic agents, uncovering the natural products with potential bone-protecting effects is vitally important.–10Osteoporosis (OP) is a common systemic skeletal disease characterized by reduced bone mass, impaired microarchitecture and susceptible to fracture.–13 However, its precise cellular pharmacological therapeutic mechanism on OP remains elusive.Traditional Chinese Medicine (TCM) has been playing an important role in the improvement of life quality and curing disease. In recent years, more and more attentions had been paid to the Chinese herb medicine for the treatment of OP. Epimedium has a longstanding history and has gained widespread clinical application in China, with the functions of promoting bone reproduction, regulating cell cycle, inhibiting osteoclastic activity, and antioxidative stress. In addition, previous studies have reported that epimedium can effectively enhance the activity of osteoblasts, indicating its antiOP biological activities.Due to the multicomponent, multitarget and multi-pathway characteristics of the TCM in the treatment of diseases, the biological molecular mechanism is relatively complicated. Considerable utilization of manpower and material resources make it difficult to thoroughly investigate the molecular mechanism of TCM through animal and cellular studies. which analyzes the network of biological system and selects specific signal nodes to design multitarget drug molecules, including chemoinformatics, bioinformatics, network biology, and pharmacology.,16 It emphasizes the multi-channel regulation of signaling pathways, improving the therapeutic effect of drugs and reducing the side effects. Thus, these advantages contribute to improve the success rate of clinical trials of new drugs and save costs for the drug research and development. Therefore, in this study, we adopted the system bioinformatic approach through network pharmacology, molecular docking and microarray data analysis to reveal the pharmacological antiOP mechanism of epimedium. The whole flowchart is shown in Figure Network pharmacology is a new subject based on the theory of system biology,http://tcmspw.com/tcmsp.php), Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine database , and the Encyclopedia of Traditional Chinese Medicine platform . TCMSP, one of the world’s largest noncommercial TCM molecular databases, collects 499 herbs from the 2015 Chinese Pharmacopoeia and the compound ingredients of each herb, including 13,144 molecules and 29,384 compounds, in which 2 key absorption, distribution, metabolism, and excretion (ADME) properties including oral bioavailability (OB≥30%) and drug similarity (DL≥0.18) were employed to screen the candidate active compounds in epimedium. In BATMAN-TCM database, effective chemical components were further screened by setting cutoff value to 55. Then, the corresponding targets were entered into UniProt (https://www.uniprot.org/), the most comprehensive and functionally annotated database of protein sequences, and the species was selected as “Homo sapiens.”Epimedium was used as a key word inquire and screen of active ingredients in TCM systems Pharmacology Database and Analysis Platform (TCMSP: www.ncbi.nlm.nih.gov/geo), which contained 9 blood samples including 5 OP patients  and 4 cases  as control group. Differential expression analysis between 2 groups was performed to identify differentially expressed mRNAs by setting FDR < 0.05, |logfc| > 0.5 as cutoff threshold. “Limma” package of R was used to perform this process.The terms “osteoporosis” and “OP” were searched as key words. Gene expression profile of OP named GSE35958 was downloaded from Gene Expression Omnibus database (R (3.6.2 for Windows)], the intersection of the targets of epimedium and the OP-related targets were obtained as the potential targets for next step analysis. Then, protein-protein interaction (PPI) network was conducted by STRING database (https://string-db.org/), which covered approximately all functional interactions between the expressed proteins. “Homo sapiens” was chosen, and a scoring value >0.4 was selected as the medium confidence basis for protein interactions. Cytoscape  platform was available for visualizing and analyzing the interconnection network, and plug-in “Network Analysis” was used to analyze the topological properties of the network.By drawing a Venn diagram [VennDiagram of http://geneontology.org/), mainly including the biological process (BP) and molecular function (MF) terms, was applied to analyze the potential biological molecular mechanisms. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.kegg.jp/) pathway enrichment analysis was conducted to identify the systemic involvement of pathways. In this research, the GO functional annotation and KEGG pathway enrichment analysis were performed by using clusterProfiler package in R software, and the P value <0.05 was employed for further analysis.The gene ontology (GO) database  to build the PPI network for the 62 critical potential genes which consists of 42 nodes and 123 edges Fig. . The resR. A total of 1340 BP terms were enriched, and the top 25 remarkably enriched BP terms based on P value were selected for visualization, including regulation of fibroblast proliferation (GO:0048145), fibroblast proliferation (GO:0048145), positive regulation of reactive oxygen species metabolic process (GO:2000379), cellular response to oxidative stress (GO:0034599), response to decreased oxygen levels (GO:0036293), and positive regulation of cell cycle process (GO:0090068) , peptide binding (GO:0042277), kinase regulator activity (GO:0019207), and ubiquitin-like protein ligase binding (GO:0044389) , cellular response to oxidative stress , positive regulation of cell cycle process  and regulation of apoptotic signaling pathway . What’s more, the protein serine/threonine kinase activity and transcription co-factor binding were analyzed by molecular function enrichment analysis. These results suggested that antiOP mechanisms mainly involved antioxidative stress, regulation of cell cycle and apoptosis. Studies have revealed that oxidative stress plays an important role in the pathogenesis of OP.,27 Oxidative stress inhibits osteoblast differentiation and stimulates osteoclast differentiation in bone marrow, leading to OP and accelerating its progress. More importantly, oxidative stress can accelerate cell senescence and death by activating many signal transduction pathways, including inflammatory transcription pathway nuclear factor (NF-κb), JAK/STAT signaling, mitogen activated protein kinases (MAPKs), and heat shock factor (HSF). Chavan et al have found that the oxidative biological markers in postmenopausal OP women were significantly higher than those in the healthy group. The positive regulation of cell cycle and apoptosis process of epimedium could balance bone reproduction and bone loss, thus delaying the process of OP.In order to reveal the mechanism of epimedium against OP, we analyzed the candidate targets by performing GO enrichment via biological processes and molecular function. The main GO biological processes terms related to OP including positive regulation of cell cycle , FoxO signaling pathway , PI3K-Akt signaling pathway , and NF-kappa B signaling pathway . Under the background of global aging, cellular senescence has received considerable attention as a potential target in treating multiple age-related diseases such as osteoporosis. Sundeep et al have found the expression of senescent cell biomarker Cdkn2a increased consistently in mice with aging in bone-derived enriched populations of osteoprogenitors, osteoblasts, and osteocytes. In addition, researchers also found the expression levels of senescence biomarkers p16INK4A and p21CIP1 were statistically significantly increased in iliac crest of older postmenopausal women compared with younger premenopausal women. These data indicated that senescent cells accumulate in old bone in mice and humans, implicating a potential role for these cells in the pathogenesis of OP. Our result showed that epimedium could prevent and treat OP through regulating cellular senescence.The therapeutic targets of epimedium were enriched in several OP-related pathways, such as cellular senescence , which are closely related to OP. FoxOs are evolutionarily conserved transcription factors for their highly conserved DNA-binding domain. FoxOs participate in regulating many cellular processes, including cell survival, proliferation, differentiation, apoptosis, oxidative stress resistance, metabolism, inflammation, and aging.–34 Recent studies have revealed that FoxOs regulate bone cell functions and their intercellular signaling. It is reported that H2O2 accumulation is a critical adaptation for the differentiation and survival of osteoclasts. Bartell et al confirmed that FoxO proteins could inhibit the osteoclast formation and bone resorption through attenuating H2O2 accumulation. In addition, the PI3K-Akt signaling pathway plays an important role in differentiation of bone cells, especially in osteoblasts. PI3K is a lipid kinase that generates PIP3, which recruits and activates PDK-1 and Akt. Akt is a major signaling molecule activated by PI3K and plays a key role in osteoblast differentiation. Previous study has reported that blocking the PI3K/Akt signaling pathway can heavily inhibit cell proliferation, calcium accumulation and ALP activity in osteoblasts.Also, the KEGG enrichment analysis suggested that epimedium exerted antiOP through FoxO signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway . The sponsor has no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.Data curation: Keliang Wu, Linjing Han, Yingzhao, Qinghua Xiao, Zhen zhang.Funding acquisition: Xiaosheng Lin.Investigation: Keliang Wu, Zhen zhang, Linjing Han.Project administration: Keliang Wu, Yingzhao, Xiaosheng Lin.Writing – original draft: Keliang Wu, Linjing Han.Writing – review & editing: Keliang Wu.


Glioblastoma is a type of brain cancer with aggressive and invasive nature. Such features result from increased proliferation and migration and also poor apoptosis of glioma cells leading to resistance to current treatments such as chemotherapy and radiotherapy. In recent studies, micro RNAs have been introduced as a novel target for treating glioblastoma via regulation of apoptotic signaling pathway, remarkably PI3K/AKT, which affect cellular functions and blockage or progression of the tumor. In this review, we focus on PI3K/AKT signaling pathway and other related apoptotic processes contributing to glioblastoma and investigate the role of micro RNAs interfering in apoptosis, invasion and proliferation of glioma through such apoptotic processes pathways. Databases NCBI, PubMed, and Web of Science were searched for published English articles using keywords such as 'miRNA OR microRNA', 'Glioblastoma', 'apoptotic pathways', 'PI3K and AKT', 'Caspase signaling Pathway' and 'Notch pathway'. Most articles were published from 7 May 2015 to 16 June 2020. This study focused on PI3K/AKT signaling pathway affecting glioma cells in separated subparts. Also, other related apoptotic pathways as the Caspase cycle and Notch have been also investigated. Nearly 40 miRNAs were found as tumor suppressors or onco-miRNA, and their targets, which regulated subcomponents participating in proliferation, invasion, and apoptosis of the tumoral cells. Our review reveals that miRNAs affect key molecules in signaling apoptotic pathways, partly PI3K/AKT, making them potential therapeutic targets to overcome the tumor. However, their utility as a novel treatment for glioblastoma requires further examination and investigation. The most invasive and aggressive subtype of brain cancer in humans is glioblastoma, globally showing the highest incidence in Western Europe, Northern Ame-rica, and Australia; its prevalence in the United States has been more than 9 per 100,000 people . AccordiBased on many studies on various cancers, including glioblastoma, changes in the expression level of some miRNAs could significantly alter tumor progression, and they contribute to biological processes of such cells as proliferation, differentiation, migration, and apoptosis , 12. TheDeregulation of the apoptotic pathway is the most crucial strategy of glioblastoma cells to fight current treatments . Hence, Search StrategyWe conducted an electronic search for published articles in PubMed, NCBI, Scopus, and Google Scholar with no restrictions on publication date. The search of the literature was done independently by two first authors who applied no limitation in the language of the literature. The initial search was conducted using the following terms: ("micro RNAs" OR "miRNAs" OR "MiRNA") AND ("glioblastoma"), ("PI3K" AND "AKT"), in the title and/or abstract. Inclusion and Exclusion CriteriaOur search initially yielded 191 records in used databases that were managed in the EndNote X7.2.1 software. A total of 16 duplicate references were removed. After screening the remained articles, 5 of them were not in English, and 11 articles were not found in full text, so those were excluded. The full texts of the remaining 159 articles were carefully read. Then, 18 studies were excluded because there were no data on pathways of miRNA regulation. Additionally, another 7 case studies were not related to glioblastoma and those were also excluded. 1. AKT Signaling Pathway The AKT pathway is known to play an effective role in the biological functions of cells . In partCorrelation of AKT Pathway with Oncogenic EGFR and c-METEGFR) gene is one of the most common changes that result in glioblas-toma ..EGFR) geEGFR and inhibit the AKT pathway leading to a reduction in viability of gliomas . In. In39). 1.3. Oncogenic PI3K/AKT/mTOR Signaling PathwayAs the results of previous studies, PI3K/AKT pathway is altered in 80% of glioma cells , 50. PI3The increase in expression of some miRNAs can modulate PI3K/AKT pathway in gliomas; for instance, miR-579, miR-548, and miR-4698 inhibit proliferation and invasion of glioblastoma through affecting this pathway , 61. miRAs a study, miR-153 is purposed as a tumor suppresser that affects RICTOR as the primary target. It has been suggested that the upregulation of miR-153 is negatively correlated with the downregulation of RICTOR and reduction of AKT activity . The resFollowing mTOR signaling and P70S6K1 elevation, HIF-1 is activated, affecting Sox2; one of the two gene targets resulting in proliferation, migration, and tumorigenesis . It has Additionally, miR-21 exerts its oncogenic effects targeting other downstream of mTORC1 as S6K and 4E-BP1, 71. Act1.4. AKT Inhibits the Pro-apoptosis DownstreamIn glioblastoma, the AKT pathway is identified to promote tumor development due to the inhibition of apoptotic effectors and activation of anti-apoptotic components . Upon AKFurthermore, AKT activates anti-apoptotic components that play key roles in cellular functions  77). As. As77). 1.5. AKT/NFkB/Bcl2 Signaling PathwayOne of the mechanisms of NFκB (Nuclear factor-kappa B) activation is the AKT phosphorylation of IκB mediated by IKKβ, a key regulator of NFκB  88). Ac. Ac88). The activated pathway of NFkB in glioblastoma can exert cell proliferation via inducing anti-apoptotic factors, including the Bcl2 family, Cyclin D1, and surviving  95). Bc. Bc95). 1.6. AKT/MDM2 Signaling and p53 Regulation0/G1 cell cycle arrest . S. S0/G1 ce arrest   and tumor necrosis factor receptor (TNFR) . The TRAFurthermore, the TNF receptor triggers NFκB signaling cascade, which involves biological processes like cell survival and inflammation . PreviouLRRFIP1 gene  gene via TP53 signaling pathway that its mechanism is not clear enough . FollowiSeveral miRNAs have an effective role in up- or down-regulation of proliferation and apoptosis through notch signaling pathways in glioblastoma . The Notin vivo findings, increased expression of miR-34a can inten-sely inhibit glioma growth via targeting the c-MET and Notch signaling pathway  to improve our understanding of their regulatory functions and their corresponding targets in other apoptotic path-ways, 2) to find some ways to control therapeutic responses and better management of glioblastoma .The authors declared no conflict of interest.None.


Background: Aging is characterized by the gradual loss of physiological integrity, resulting in impaired function and easier death. This deterioration is a major risk factor for major human pathological diseases, including cancer, diabetes, cardiovascular disease and neurodegenerative diseases. It is very important to find biomarkers that can prevent aging.Methods: Q-Exactive-MS was used for proteomic detection of young and senescence fibroblast. The key senescence-related molecules (SRMs) were identified by integrating transcriptome and proteomics from aging tissue/cells, and the correlation between these differentially expressed genes and well-known aging-related pathways. Next, we validated the expression of these molecules using qPCR, and explored the correlation between them and immune infiltrating cells. Finally, the enriched pathways of the genes significantly related to the four differential genes were identified using the single cell transcriptome.Results: we first combined proteomics and transcriptome to identified four SRMs. Data sets including GSE63577, GSE64553, GSE18876, GSE85358, and qPCR confirmed that ETF1, PLBD2, ASAH1, and MOXD1 were identified as SRMs. Then the correlation between SRMs and aging-related pathways was excavated and verified. Next, we verified the expression of SRMs at the tissue level and qPCR, and explored the correlation between them and immune infiltrating cells. Finally, at the single-cell transcriptome level, we verified their expression and explored the possible pathway by which they lead to aging. Briefly, ETF1 may affect the changes of inflammatory factors such as IL-17, IL-6, and NFKB1 by indirectly regulating the enrichment and differentiation of immune cells. MOXD1 may regulate senescence by affecting the WNT pathway and changing the cell cycle. ASAH1 may affect development and regulate the phenotype of aging by affecting cell cycle-related genes.Conclusion: In general, based on the analysis of proteomics and transcriptome, we identified four SRMs that may affect aging and speculated their possible mechanisms, which provides a new target for preventing aging, especially skin aging. In vivo, senescence is an important mechanism to prevent damaged cells from transforming into tumor cells  and senescent (34∼42 passage) fibroblasts were verified using SA-b-Gal staining and then used for further analysis. Informed consent to participate in the study have been obtained from participants . Three human foreskin specimens involved in this study were collected in June 2021.Primary normal human foreskin fibroblasts were isolated 3 donors  and cultured in DMEM  with 10% FBS  as previously described . The yousenescent fibroblasts was collected using TRIzol (Invitrogen). 2 μg RNA was used for reverse transcription , and gene expression was analyzed using an Applied Biosystems ® 7,500 machine . The primers were shown in The RNA of young and p < 0.05), and were considered significant. But for those protein matching unique peptides, individual ions scores >38 indicate identity or extensive homology (p < 0.01) were considered significant (The whole-cell extracts of young and senescent fibroblasts were prepared in NP40 buffer (Beyotime). After separation on SDS-PAGE, the protein from different bands were excised and used for trypsin digestion. The peptides were separated using an Ultimate 3,000 RSLCnano system and then analyzed on the Q Exactive . Proteome discoverer 1.4  with Masccot was used for protein identification, as previously described . The tannificant . The reshttps://www.ncbi.nlm.nih.gov/geo/). The GSE85358 and GSE18876 matrices are merged into large matrices using R language, and the batch effect is removed by using the “combat” function of the R software “sva” package (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp).Gene Expression Omnibus (GEO) database stores curated gene expression DataSets, as well as original Series and Platform records in the GEO repository. Data sets such as GSE63577, GSE64553, GSE151177, GSE85358, and GSE18876 are downloaded on the GEO database  is a reduced-dimensional data processing method. We use the “prcomp” function in the “stats” package (version 3.6.1) of R language for PCA analysis, and then use the “scale” function to normalize z-score. Finally, the visualization is carried out by using the “scatterplot3d” package (version 0.3.41) in R software.We conducted Venn analysis by using the “venn.diagram” function of the “VennDiagram” package  version  . In the We did a PPI analysis using the STRING (V11.5) online website, setting minimum required interaction score as 0.4. Enrichment analysis and network visualization are carried out by using “string” and “string enrichment” plug-ins in Cytoscape (V3.8.2)  softwarehttps://cibersortx.stanford.edu/index.php) (https://xcell.ucsf.edu/)  , MCP Coudex.php) , and Xcesf.edu/)  softwareAll experiments were performed in accordance with relevant guidelines and regulations. All the experiments in this study were approved by the Clinical Medical Ethics Committee of Xiangya Hospital of Central South University (202103574).All the analyses in this study are carried out using R software (version 4.1.2), and all the codes used are stored in the proteomics from 3 senescence and 3 young fibroblasts) were normal and there were no outlier samples  and analyzed the functional enrichment of these genes. The results of KEGG and GO-BP enrichment analysis of 151 genes significantly related to ETF suggested that the enrichment pathway was mainly concentrated in IL-17 signaling pathway  in GSE151177 data set for analysis. After three steps of strict initial quality control screening , the res pathway  and apop pathway . This su pathway . PPI ana pathway .36 genes significantly related to MOXD1 were analyzed by GO-BP enrichment analysis. the results showed that regulation of Wnt signaling pathway, collagen fibril organization, and supramolecular fiber organization pathways were enriched . In addiWe validated the expression of SRMs by qRT-PCR experiment using the senescence model of passage fibroblasts. The results showed that the expression of MOXD1, PLBD2, and ASAH1 was up-regulated in the aging group, while the expression of ETF1 was down-regulated in the aging group, which was consistent with the difference between the protein group and the transcriptional group .key SRMs. Then the correlation between SRMs and aging-related pathways was excavated and validated. Next, we validated the expression of SRMs at the tissue level and qPCR, and explored the correlation between them and immune infiltrating cells. Finally, at the single-cell transcriptome level, we validated their expression and explored the possible pathway by which they lead to aging.In this study, we first combined proteomics and transcriptome to identified four Eukaryotic translation termination factor 1 (ETF1) encodes a class-1 polypeptide chain release factor. The encoded protein plays an essential role in directing termination of mRNA translation from the termination codons UAA, UAG and UGA. In our study, there was a significant negative correlation between ETF1 and the famous aging promoting gene CDKN1A (p21) . What’s MOX (monooxygenase X), is a member of the copper monooxygenase family that includes dopamine beta-monooxygenase (DBM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) . As a meN-acylsphingosine amidohydrolase 1 (ASAH1) encodes a member of the acid ceramidase family of proteins. It is essential for the formation of mature lysosomal enzymes, which are overexpressed in many human cancers and may play a role in cancer progression . Rachel Little research has been done on phospholipase B domain containing 2  in human tissues. In the present research, PLBD2 was significantly up-regulated in aging tissues and fibroblasts and was significantly positively correlated with p21 and WNT16. In addition, the molecules significantly related to PLBD2 are mainly enriched in HIF-1 signaling pathway and ficolin-1-rich granule lumen pathway. Although there is no previous research guidance, our work suggests that PLBD2 may play a key role in the aging process.In general, based on the analysis of proteomics and transcriptome, we identified SRMs that may affect aging and predicted their possible mechanisms, which provides a new target for preventing aging, especially skin aging.


Paris polyphylla Sm. (C-P) drug pair in the treatment of adenomyosis (AM) based on network pharmacology and animal experiments.  To explore the molecular mechanism of the Cinnamomi ramulus and  Via a network pharmacology strategy, a drug-component-target-disease network (D-C-T-D) and protein–protein interaction (PPI) network were constructed to explore the core components and key targets of C-P drug pair therapy for AM, and the core components and key targets were verified by molecular docking. Based on the results of network pharmacology, animal experiments were performed for further verification. The therapeutic effect of the C-P drug pair on uterine ectopic lesions was evaluated in a constructed AM rat model.  A total of 30 components and 45 corresponding targets of C-P in the treatment of AM were obtained through network pharmacology. In the D-C-T-D network and PPI network, 5 core components and 10 key targets were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the PI3K signaling pathway was the most significantly enriched nontumor pathway. Molecular docking showed that most of the core components and key targets docked completely. Animal experiments showed that the C-P drug pair significantly ameliorated the pathological changes of endometriotic lesions in AM model rats and inhibited PI3K and Akt gene expression, and PI3K and Akt protein phosphorylation. In addition, treatment with the C-P drug pair promoted AM cell apoptosis; upregulated the protein expression of Bax, Caspase-3, and cleaved Caspase-9; and restrained Bcl-2 expression.  We propose that the pharmacological mechanism of the C-P drug pair in the treatment of AM is related to inhibition of the PI3K/Akt pathway and promotion of apoptosis in AM ectopic lesions. Adenomyosis (AM) is a gynecological disorder characterized by the abnormal appearance of endometrial glands and stroma in the myometrium. Increased dysmenorrhea, abnormal uterine bleeding (AUB), and infertility are the most typical symptoms of AM and severely affect the quality of life of patients . HormoneParis polyphylla Sm. (Chonglou) is recorded in Shennong's Herbal Classic (Shen Nong Ben Cao Jing) and is believed to be able to relieve heat, remove toxic material, detumescence, and analgesic effects. Gongxuening capsules , which are commonly used in the clinical treatment of AM, are developed from Paris polyphylla Sm. and have been used to treat uterine diseases and blood stasis syndrome for decades [Among alternative medicines, traditional Chinese medicine (TCM) has accumulated considerable clinical experience in AM treatment –7. Cinna decades –12.Paris polyphylla Sm. (C-P), from Guizhi-Fuling Wan and Gongxuening capsules as a drug pair and explored their synergistic regulatory effects on an AM rat model in this study [Paris polyphylla Sm. and Panax notoginseng (Sanqi) reduced the levels of matrix metalloproteinases (MMP-2 and MMP-9), increased the expression of Bax and Caspase-3, and significantly reduced the proliferation and invasion ability of endometrial cells in AM model rats [In TCM treatment theory, prescriptions based on the concept of “drug pairs” can often achieve better results than individual drug prescriptions, as two drugs can often increase the effectiveness of each other. We extracted the monarch drugs (the drugs that play major roles in a formula), namely, Cinnamomi ramulus and is study . A previdel rats . Using CTCMs are characterized by multiple components and multiple targets, which makes it difficult to further study the mechanism of the C-P drug pair in the treatment of AM. In recent years, the emergence of network pharmacology has provided an effective means to study the pharmacological mechanisms of TCMs. Construction and analysis of drug–component–target–disease (D-C-T-D) networks can enable the core components of TCMs and potential targets for disease treatment to be effectively discovered . In thishttps://old.tcmsp-e.com/index.php). The active ingredients obtained from the TCMSP database were screened under the conditions of oral bioavailability (OB) ≥30% and druglikeness (DL) ≥0.18, and the corresponding targets of the active ingredients were further obtained. The ingredients and corresponding targets of Paris polyphylla Sm. were obtained from the HERB database (https://herb.ac.cn/) and a literature search [https://www.swisstargetprediction.ch/) was used to predict the targets for the components obtained from the literature search [The ingredients and targets of Cinnamomi ramulus were obtained from the TCMSP database , Coremine , and PharmGKB (https://www.pharmgkb.org/) databases using “Adenomyosis” as the retrieval keyword. The correlation score was used as a reference for each included target. The common targets between C-P and AM were identified using a Venn diagram.AM-related targets were searched in the GeneCards , and “Homo sapiens” was used as the screening criterion to obtain the PPI relationship [The overlapping targets between the C-P active ingredient targets and the AM-related targets were imported into the STRING database (tionship . The reshttps://david.ncifcrf.gov/) was utilized to perform GO biological process enrichment and KEGG pathway analysis on the overlapping targets between C-P and AM [DAVID 6.8 (P and AM . The tophttps://pubchem.ncbi.nlm.nih.gov/) database. The 3D structures of key target proteins were downloaded from the RCSB database (https://www.rcsb.org/search). Chem3D software was used to preprocess the components, and AutoDock Vina software was used for molecular docking analysis of the core components and key targets. The binding energy between the component and the protein was used to assess the degree of binding. PyMOL software was used to visually display part of the structure.The five components with the highest degrees were identified as core components in the D-C-T-D network, and the 2D structures of the core components were downloaded from PubChem (Paris polyphylla Sm. were mixed at a standard ratio of 5 : 4 and were subjected to reflux extraction twice with 10 times the volume of distilled water for 1 h each. The extracts were then mixed thoroughly and concentrated to a relative density of 1.20–1.25 (70–80°C). The dosage of the C-P drug pair liquid used in subsequent animal studies was 2.7 g/kg/day. Mifepristone was purchased from Beijing Zizhu Pharmaceutical Co., Ltd. . The final dosage was 1.25 mg/kg/day [The herbal medicines were supplied by the Affiliated Hospital of Shandong University of Traditional Chinese Medicine  and verified by Prof. Feng Li. Cinnamomi ramulus and g/kg/day .Rabbit anti-Caspase-3 (bs-0081R) and Caspase-9 (bs-0049R) polyclonal antibodies were obtained from Beijing Bioss Biotechnology Co., Ltd. . A Bax monoclonal antibody (60267-1-Ig) and a Bcl-2 polyclonal antibody (12789-1-AP) were obtained from Wuhan Proteintech Biotechnology Co., Ltd. . PI3 kinase p85 alpha  (ab182651), GAPDH  (ab181602) and Akt  (ab38449) antibodies were obtained from Abcam, Inc. .Forty female Wistar rats and 30 male Wistar rats purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., were housed in the SPF Animal Experimental Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. All rats had free access to a commercial diet and tap water. All the experimental procedures conformed to the regulations described in the Guide to the Care and Use of Laboratory Animals of the USA National Institutes of Health. The ethical review was approved by the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (AWE-2019-009). According to the random number table method, 40 female Wistar rats were divided as follows: 10 mice served as the blank group, and the remaining 30 were prepared for modeling. Animal models were established according to previous studies . The pitμm-thick sections were prepared for hematoxylin and eosin (H&E) staining. All images were acquired using a NanoZoomer S60 Digital slide scanner .The uterine tissues were fixed in 4% paraformaldehyde solution and embedded in paraffin, and 5 Bax, Bcl-2, Caspase-3, and Caspase-9 protein expressions in uterine tissues were detected using the immunohistochemistry streptavidin-peroxidase (SP) method. The regions were randomly selected under the microscope, and the cumulative optical density, area, and average density of the regions were measured using ImageJ (version 1.8.0).The expression of genes was quantified using the real-time PCR ΔΔCT method with a LightCycler 480II platform . Total RNA was purified using a Simply P Total RNA Extraction Kit according to the manufacturer's instructions. cDNA was synthesized with a ReverTra Ace qPCR RT Kit. Quantitative PCR was performed using a LightCycler 480II Real-Time PCR Detection System. All primers were synthesized by Integrated DNA Technology . The primer sequences for mRNA analysis were as follows: phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) forward 5′-CTGCAGTTCAACAGCCACAC-3′ and reverse 5′-CCAGCTGCCATCTCAGTTCA-3′; Akt1 forward 5′-AAGGTTTGCTGGGTGAGTGA-3′ and reverse 5′-CTCCTCAGGCGTTTCCACAT-3′. GAPDH expression was used to normalize gene expression.Uterine tissues (50 mg) collected from each group were homogenized and lysed in RIPA lysis buffer and then analyzed to detect the protein concentration using a BCA kit. Tissue total proteins were loaded onto 10% SDS–PAGE gels and transferred onto a PVDF membrane. The membrane was blocked using 5% skim milk and then incubated overnight with primary antibodies  at 4°C. The membrane was then incubated with a secondary antibody. Western blot analysis was performed using a ChemiDoc Imaging System . The density of the signal was quantified by ImageJ.t-tests, whereas the significance of differences among three or more experimental groups was determined by one-way analysis of variance. A value of P < 0.05 was considered to indicate statistical significance. Diagrams were drawn using GraphPad Prism 8.All data were analyzed using GraphPad Prism 8 (version 8.0.2) and are expressed as the mean ± standard deviation (SD). Comparisons between two groups were performed using Student's Paris polyphylla Sm. were obtained from the HERB database and a literature search, and 360 targets corresponding to the active components of Paris polyphylla Sm. were obtained. A total of 384 potential targets of the C-P drug pair were identified after removing the repetition. A total of 504 targets related to AM were obtained through database retrieval. A Venn diagram revealed 45 intersecting targets between the C-P drug pair and AM  and 118 signaling pathways with a GG terms . The resThe 2D structures of the five core components in the D-C-T-D network were obtained from the PubChem database. The 3D structures of 10 key targets in the PPI network were obtained from the RCSB database. Five core components and 10 targets were docked. Binding energy was used to evaluate the degree of binding of ingredients to proteins, and a binding energy <−5 kcal/mol was considered to indicate that component and target could bind. The results of our study showed that the core components had good overall docking with the key targets , indicatIn the control group, the uterine morphology was intact and the muscular layer was continuous. The boundary between the muscular layer and the endometrium was clear, and no gland or endometrium tissue invasion was observed. However, the muscle layer in the model group was thickened with gland invasion and the boundary was blurred. Compared with that in the model group, the muscle layers in the C-P and mifepristone groups were almost intact. The boundary between the muscle layer and the intima layer was fairly clear, and fewer glands invaded the muscle layer (a).The results of real-time PCR analysis indicated that PIK3CA and Akt1 mRNA expression was significantly higher in the AM model group than in the control group. The C-P drug pair and mifepristone significantly suppressed PIK3CA and Akt1 mRNA expression in AM rats compared with that in model rats . The expWe observed changes in the apoptosis-related indicators Bax, Bcl-2, Caspase-3, and Caspase-9 in AM rats after C-P treatment based on tissue- and protein-level detection. The immunohistochemistry results showed that Bax , CaspaseParis polyphylla Sm. are commonly used in TCM [Paris polyphylla Sm. can remove blood stasis. Cinnamomi ramulus and Paris polyphylla Sm. are commonly used to warm meridians, remove blood stasis and relieve pain.Although AM is a condition of benign gynecological lesions, the lesions show abnormal tumor characteristics including endometrial cell proliferation, apoptosis, invasion, and migration. Compared with radical hysterectomy, TCM treatment has positive effects on women's fertility. Cinnamomi ramulus and d in TCM –31. The κB signaling pathway [Paris polyphylla Sm. and its derivatives have been widely used to treat abnormal endometrial bleeding [Paris polyphylla Sm. is also rich in a variety of saponins that have been widely used to combat abnormal cell proliferation and migration and to promote apoptosis of abnormal cells [Modern pharmacological studies have shown that the active components of Cinnamomi ramulus can inhibit the activity, migration, and glycolysis of endometrial stromal cells by inhibiting pyruvate kinase PKM (PKM2) transcription induced by the NF- pathway . Paris pbleeding . Paris pal cells , 34.TCM is characterized by the use of multiple drugs with multiple components and targets for the treatment of diseases. This complexity can hinder the exploration of the molecular mechanisms of TCM drugs in the treatment of AM from the perspective of precision medicine. Network pharmacology is useful for constructing D-C-T-D networks and PPI networks of drug–disease intersection targets based on the abovementioned characteristics of TCM. Through network analysis, the core components and key targets of TCM in the treatment of diseases can be obtained. This research method is beneficial for precision research on TCM and provides an effective means for identifying new targets for disease treatment.In our study, a total of 30 components and 45 corresponding targets of the C-P drug pair related to AM were obtained through network pharmacology. In the D-C-T-C network, ecdysone, pinnatasterone, 20-hydroxyecdysone, flavone and diosgenin exhibited high degree numbers. These results suggest that these compounds may play a major role in the pharmacological mechanism of C-P in the treatment of AM. 20-Hydroxyecdysone has been reported to be a natural active component with the potential to promote apoptosis and autophagy in cancer cells . In addiThrough analysis of the PPI network, we obtained 10 potential core targets of C-P therapy for AM: PIK3CA, IL10, IL6, ESR1, CCL2, TP53, VEGFA, FGF2, PTGS2, and CXCL8. PIK3CA is the catalytic subunit of the dimeric protein PI3K. As the core mediator of the PI3K signaling pathway, PI3K is a critical mediator of cell survival, and its downstream MTOR protein is an important protein involved in autophagy to avoid apoptosis . IL6 andThrough KEGG enrichment analysis of overlapping C-P and AM targets, we found that the PI3K signaling pathway was the most significant nontumor pathway, and GO enrichment analysis showed that phosphorylation (activation) of the PI3K signaling pathway was a significantly enriched biological process. In the molecular docking results, PIK3CA (PI3K) was able to dock with multiple core components. Therefore, PIK3CA may represent an important target of C-P in the treatment of AM. Previous studies have demonstrated that PI3K-Akt activation in AMs promotes endometrial cell proliferation . In addiThe PI3K/Akt signaling pathway is closely related to Bcl-2 family proteins. When stimulated, active Akt leads to the phosphorylation of the proapoptotic protein Bad and inhibits the translocation of Bad from the cytoplasm to the mitochondria, thereby inhibiting mitochondrial dysfunction. Mitochondrial dysfunction stimulates downstream Caspase-3 and Caspase-9. The cleaved forms of Caspase-3 and Caspase-9 regulate other protein substrates, ultimately triggering apoptosis . MoreoveNetwork pharmacology and animal experiments showed that the C-P drug pair can significantly delay disease progression in AM rats. We propose that the pharmacological mechanism of C-P in the treatment of AM is related to inhibition of the PI3K pathway and promotion of apoptosis in AM ectopic lesions. However, unfortunately, the pharmacological components of C-P are not well characterized, and whether C-P regulates apoptosis through the PI3K/Akt pathway remains unclear. We will further explore the effective components and pharmacological mechanism of C-P in the treatment of AM through subsequent experiments.


The fluorescence intensity ratio of the two emissions was found to be ca. 370 nm/590 nm for RhB@Zr-MOF. The fluorescence intensity values of the two emissions of RhB@Zr-MOF can also be affected by the structures of analytes containing different organic groups. Due to the existence of the dual-emission properties in RhB@Zr-MOF, the relative fluorescence intensity of the emission peaks was introduced as a detection index instead of absolute fluorescence intensity. RhB@Zr-MOF, which possesses the characteristics of a built-in self-calibrating fluorescence sensor, was investigated for detecting cations, nitroaromatics and pesticides. Aside from high sensitivity and selectivity, recyclability is the most important property for sensing pesticides. This work shows that RhB@Zr-MOF can maintain its stability after 5 cycles of detecting nitenpyram, with LOD of 0.2 μM. These results demonstrate that dye@MOFs with dual-emission properties can be employed as multifunctional fluorescence sensors for different types of analytes, and that RhB@Zr-MOF provides a new paradigm for analyte sensing.A RhB@Zr-MOF composite with dual-emission properties was successfully constructed, which comprises a zirconium-based metal–organic framework and the luminescent dye molecule, Rhodamine B (RhB), embedded  A RhB@Zr-MOF composite with dual-emission properties was demonstrated as a self-calibrating sensor for cations, nitro explosives and nitenpyram. However, pesticide residues can threaten human health, food safety and ecosystems.4–6 On the basis of the problems mentioned above, technologies that can detect pesticides effectively and sensitively are particularly desirable compared with classical methods, such as gas chromatography (GC),7 high-performance liquid chromatography (HPLC)8 and mass spectrometry (MS),9 which are costly, involve tedious sample preparation procedures and complex instrumental methods, and require highly skilled labor power.10–12Pesticides,13,14 When storing or transporting explosives, flames or high temperatures are absolutely not allowed. Meanwhile, it is necessary to avoid friction between explosives. Because of the hazards associated with explosives and explosives-like toxic molecules, developing sensitive, accurate and cost-effective methods of detecting them is essential. Many conventional materials have been selected to detect them, such as nanoscale materials and some polymers.15,16 So far, fluorescence sensors have become the most effective technology for detection, as they possess the merits of sensitivity and rapidness.Explosives or explosives-like toxic molecules, which exist everywhere in our daily life, are associated with issues of human health, national defense, industrial production and environmental protection.3+, Cr3+, Al3+ and Ga3+) is extremely vital because of their deposition in various biological systems.17,18 In comparison with conventional techniques for sensing trivalent metal ions, such as chromatography, accelerator mass spectroscopy (AMS), flame atomic absorption spectroscopy (FAAS), and inductively coupled plasma mass spectrometry (ICP-MS), chemical sensors, especially fluorescence sensors, have been proposed as a promising candidate.19–21 Fluorescence sensors for detecting trivalent metal ions have the merits of simplicity, rapidness, sensitivity and low limits of detection (LODs), allowing them to overcome the drawbacks of the conventional methods.Detecting trivalent metal ions , also known as porous coordination polymers, are hybrid molecular networks comprising functional organic ligands and metal ions or metal clusters formed 4 to prepare a luminescent Zr-MOF (CCDC: 1000802)43 containing nodes of Zr6 clusters. Furthermore, RhB@Zr-MOF was fabricated by encapsulating the luminescent dye molecule, Rhodamine B (RhB), into Zr-MOFs. RhB@Zr-MOF shows a dual-emission luminescence property, and acts as a multifunctional, self-calibrating fluorescence sensor for detecting cations, nitro explosives and pesticides. Due to the energy-transfer interactions between the host Zr-MOF and the guest RhB, the RhB@Zr-MOF composite can selectively detect Fe3+, 4-NP and nitenpyram by measuring the relative fluorescence intensities of two emissions instead of their absolute fluorescence intensities. The fluorescence intensity changes mainly come from Zr-MOF, with the calibration of the relatively weak intensity of encapsulated RhB molecules. A range of methods, including thermogravimetric analyses (TGA), powder X-ray diffraction (PXRD), and infrared radiation (IR), have been employed to characterize RhB@Zr-MOF. RhB@Zr-MOF displays high thermal stability, recyclability and low LODs for sensing various analytes, especially nitenpyram. The mechanism for detecting nitenpyram was also investigated thoroughly towards expanding the scope of RhB@Zr-MOF.In this work, we have selected 1,3,5-benzenetribenzoic acid (BTB), and ZrCliv) chloride  and 1,3,5-benzenetribenzoic acid (BTB) were purchased from Alfa. Rhodamine B (AR) was purchased from Sigma-Aldrich. KCl, NaCl, AgNO3, CuCl, PbCl2, MgCl2, CoCl2, CdCl2, BaCl2, HgCl2, MnCl2, CuCl2, CaCl2, NiCl2, FeCl2, AlCl3, FeCl3, CrCl3, GaCl3 and InCl3 were purchased from Aladdin. 1,2-dinitrobenzene , 1,3-dinitrobenzene , 1,4-dinitrobenzene , 2-nitrophenol (2-NP), 3-nitrophenol (3-NP), 4-nitrophenol (4-NP), 2-nitrotoluene (2-NT), 3-nitrotoluene (3-NT), 4-nitrotoluene (4-NT) and 2-nitro-m-xylene (NX) were purchased from Sigma-Aldrich. Isoxaflutole, fluroxypyr, teflubenzuron, rotenone, acetamiprid, etoxazole, carbendazim, carbaryl, thiamethoxam and nitenpyram were also purchased from Sigma-Aldrich. Ethanol, methanol, N,N′-dimethylacetamide (DMA) and N,N′-dimethylformamide (DMF) were purchased from Aladdin. All reagents in this work were commercially available and utilized without further purification.Zirconium was carried out using a PerkinElmer TG-7 analyzer by heating the samples from 25 °C to 800 °C with a ramping rate of 10 °C min−1 under a steady flow of N2. N2 adsorption measurements were collected using a Quantachrome Autosorb-iQ. The samples were immersed in CH2Cl2 for further guest-exchange and degassed at 80 °C for 24 h for experimentation. Fluorescence spectroscopy data were gathered using an LS-55 with a xenon lamp and quartz carrier at ambient temperature. UV-vis spectra were obtained on a Shimadzu UV-2700 spectrophotometer in the wavelength range from 200 nm to 800 nm.The PXRD data were collected on a Siemens D5005 diffractometer with Cu-Kα radiation, with angles ranging from 3° to 40° at ambient temperature. IR spectra were measured on an Alpha Centaurt FT/IR spectrophotometer using KBr pellets, over a wavenumber range of 4000–400 cm43 A mixture of BTB  and ZrCl4  was placed into a mixed solvent of DMF (2 mL)/acetic acid (6 mL) and was smoothly stirred for 30 min at ambient temperature. The mixture was then poured into a 25 mL Teflon-lined autoclave, which was heated from 25 °C to 120 °C and maintained at this temperature for 72 hours. Colorless blocks of crystals were obtained after cooling the mixture to ambient temperature at a rate of 5 °C h−1, which were washed with H2O several times and dried at ambient temperature. Yield: ca. 80% (108 mg) yield based on Zr(iv).Zr-MOF was synthesized by following the literature report with a few modifications.4 , RhB , DMF (2 mL) and acetic acid (6 mL) was put together and was smoothly stirred for 30 min at ambient temperature. The mixture was then poured into a 25 mL Teflon-lined autoclave, which was heated from 25 °C to 120 °C and maintained at this temperature for 96 hours. Pink powder was obtained after cooling to ambient temperature at a rate of 5 °C h−1, which was washed with DMF and H2O several times and dried at ambient temperature.The mixture of BTB , ZrCl2O, and each mixture was ultrasonicated to form a stable suspension.Fluorescence spectra of RhB@Zr-MOF in various solvents were studied. To prepare each sample, 3 mg of finely-ground RhB@Zr-MOF was soaked in 3 mL of DMF, DMA, methanol, ethanol or H2O (3 mL) and the mixture was ultrasonicated to form a stable suspension. An aqueous solution of each of the cations, K+, Na+, Ag+, Cu+, Pb2+, Mg2+, Co2+, Cd2+, Ba2+, Hg2+, Mn2+, Cu2+, Ca2+, Ni2+, Fe2+, Al3+, Fe3+, Cr3+, Ga3+ and In3+ (20 μL), was added to each suspension sample. The final concentration of metal salts in each sample was 0.060 mM.For each sample, finely-ground RhB@Zr-MOF (3 mg) was soaked in HThe procedure of sensing nitro explosives and pesticides was almost the same as that for sensing cations, except that the aqueous solutions were replaced with ethanol solutions. The final concentrations of the samples of nitro explosives and pesticides were both 0.060 mM.44 The geometries were optimized by the B3LYP45,46 functional. The 6-31G* basis set was used for all atoms. All geometry optimizations were performed without symmetry constraints in conjunction with the PCM continuum solvation model,47 using the most common solvent, water.All calculations were performed using the Gaussian 09W software package.ca. 15.9 Å × 11.8 Å × 5.6 Å), a cationic dye molecule with luminescence properties, has been extensively utilized in numerous fields, including printing, fiber products, cosmetics, pharmaceuticals, etc.48–50 Zr-MOFs, as robust structures, possess high chemical and thermal stabilities, making them suitable for encapsulating luminescent dye molecules. In this work, a Zr-MOF with a kdg topology, Zr6(μ3-O)4(μ3-OH)4(OH)6(H2O)6(BTB)2·6DMF·H2O, was synthesized, where the Zr6 cluster can be considered as a 6-connected node, and the BTB ligand can be simplified to a 3-connected linker. This Zr-MOF has 1D channels and the dimensions of the channels are 14.37 Å × 14.37 Å. Using a “one-pot” method, Rhodamine B molecules were loaded into the channels of Zr-MOF to form RhB@Zr-MOF, where the size of RhB was apt to fit in the channels of Zr-MOF. The color of RhB@Zr-MOF soaked in H2O was pink compared with the white color of Zr-MOF, which demonstrated that RhB molecules were not released, meaning that the adsorption of RhB was not on the surface of Zr-MOF and RhB@Zr-MOF had been successfully synthesized  surface area of 117 m2 g−1. This value is smaller than that of Zr-MOF, which has a BET surface area of 613 m2 g−1  is at 372 nm (λex = 300 nm), displaying a single emission. The main emission of RhB in ethanol solution (1 g L−1) is at 610 nm (λex = 300 nm), also displaying a single emission, while the solid state of RhB exhibits no fluorescence, which may be due to aggregation quenching effects.51 After RhB had been encapsulated in the channels of Zr-MOF, the main emissions of RhB@Zr-MOF in ethanol solution (1 g L−1) were at 368 nm, originating from Zr-MOF, and 590 nm, originating from RhB molecules (λex = 300 nm). These two emissions are both slightly blue-shifted compared with those of Zr-MOF and RhB.The luminescence emission spectra of Zr-MOF and RhB@Zr-MOF are shown in 2O. The different degrees of quenching effects may be attributed to the diverse interaction forces between RhB@Zr-MOF and the solvents used, further influencing the electron transfer process.52In order to detect the capability of RhB@Zr-MOF for small-molecule sensing, the luminescence properties of RhB@Zr-MOF in various solvent solutions were investigated. As shown in As a potential sensor, RhB@Zr-MOF with dual-emission properties was applied to detect cations, nitro explosives and pesticides as analytes. Different types and concentrations of analytes can cause different degrees of fluorescence enhancement or quenching. RhB@Zr-MOF possesses a strong emission peak at 368 nm and a weak emission peak at 590 nm, allowing it to serve as a self-calibrating sensor. Here, the relative peak-height, which means the difference of intensities between the strong and the weak peak values, was used to represent the detection signals Fig. S3.2O are relatively far apart and do not overlap, making this material suitable for sensing cations and allowing it to avoid mutual interferences. The relative peak-height of RhB@Zr-MOF in solvent with no analytes is defined as I0R, and with analytes it is defined as IR. IR/I0R, which represents the relative luminescence intensity (RLI). Aqueous solutions of K+, Na+, Ag+, Cu+, Pb2+, Mg2+, Co2+, Cd2+, Ba2+, Hg2+, Mn2+, Cu2+, Ca2+, Ni2+, Fe2+, Al3+, Fe3+, Cr3+, Ga3+ and In3+ were studied, and different cations show different quenching efficiencies compared with a blank sample  of RhB@Zr-MOF, fluorescence intensity will arise.55 The RLI values of Mn2+, In3+, Fe2+, Ni2+, Ga3+, Cr3+ and Fe3+ range from 0.77 to 0.98, displaying fluorescence quenching , especially Fe3+. Next, the titration method was adopted for RhB@Zr-MOF sensing Fe3+ at different concentrations ranging from 0.003 mM to 0.300 mM  equation, (I0R/IR) = Ksv[A] + 1, was adopted, where [A] is the molar concentration, and Ksv is the quenching constant (M−1).56 The Ksv of RhB@Zr-MOF for sensing Fe3+ is 1.29 × 104 M−1, and the plot displays a broad concentration range, implying that RhB@Zr-MOF is a good candidate for detecting Fe3+  were then studied, and these different analytes show different quenching efficiencies compared with a blank sample . The RLIhan Fe3+ . The LODThe same samples of RhB@Zr-MOF were reused for 5 cycles for the detection of 4-NP and the original intensities remained nearly unchanged Fig. S6. MeanwhiKsv of RhB@Zr-MOF is 9.01 × 104 M−1 for detecting nitenpyram, and the plot displays a broad concentration range, implying that RhB@Zr-MOF can detect nitenpyram more rapidly and sensitively than Fe3+, but slightly more slowly than 4-NP , at the B3LYP/6-31G* level, to further study the mechanism (2 groups has a relatively higher electron affinity than the other pesticides, resulting in the electrons transferring from the HOMO of RhB@Zr-MOF to the LUMO of the excited state of the pesticide.60The mechanism of analyte detection was investigated thoroughly. From the perspective of resonance energy transfer, the spectral overlap between the emission of this Zr-MOF and the absorption of RhB can be observed, as shown in Fig. S12,d 590 nm . From thechanism . Nitenpy3+ is 1.6 μM, that for 4-NP is 0.9 μM and that for nitenpyram is 0.2 μM. Furthermore, RhB@Zr-MOF sensors were utilized for detecting pesticides in real samples. Finally, the mechanism for the detection was analyzed to extend the range of use of RhB@Zr-MOF as a multifunctional sensor.In summary, RhB@Zr-MOF, a self-calibrating fluorescence sensor, has been successfully constructed and has been demonstrated as a multifunctional, efficient sensor for cations, nitro explosives and pesticides. RhB@Zr-MOF reveals superior recyclability and LODs as a dual-emission chemical sensor. The relative peak-height, defined as the difference of intensities between the strong and the weak peak values, has been employed to represent the detection signals. The LOD of RhB@Zr-MOF for detecting FeThere are no conflicts to declare.RA-010-D0RA02843F-s001


Danggui-Shaoyao-San (DSS) is a traditional Chinese medicine formula that has been widely used to treat a variety of disorders, including renal diseases. Despite being well-established in clinical practice, the mechanisms behind the therapeutic effects of DSS on diabetic nephropathy (DN) remain elusive.  To explore the therapeutic mechanism, we explored the action mechanism of DSS on DN using network pharmacology strategies. All ingredients were selected from the relevant databases, and active ingredients were chosen on the basis of their oral bioavailability prediction and drug-likeness evaluation. The putative proteins of DSS were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, whereas the potential genes of DN were obtained from the GeneCards and OMIM databases. Enrichment analysis using gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) was performed to discover possible hub targets and gene-related pathways. Afterwards, the underlying molecular mechanisms of DSS against DN were validated experimentally in vivo against db/db mice. α, IL-6, and ICAM-1, as well as extracellular matrix deposition, by blocking the JNK pathway activation, which protects against kidney injury.  We identified 91 phytochemicals using the comprehensive network pharmacology technique, 51 of which were chosen as bioactive components. There were 40 proteins and 20 pathways in the target-pathway network. The experimental validation results demonstrated that DSS may reduce the expression of TNF- This study discovered the putative molecular mechanisms of action of DSS against diabetic kidney damage through a network pharmacology approach and experimental validation. The increasing prevalence of diabetic nephropathy (DN) parallels the dramatic rise in the prevalence of diabetes globally. Nearly half of all type 2 diabetic (T2D) patients and one-third of type 1 diabetic (T1D) patients eventually suffer from CKD . CurrentTCM places emphasis on the notion of the integrity of the entire human body. Different from the single-target curative effect of chemical drugs, TCM compound ingredients have an overall curative effect, which is usually regulated by “multicomponent” and “multitarget” . NetworkIn this current study, we adopted a network pharmacology approach to analyze the underlying mechanisms of DSS in DN. Furthermore, the potential targets of DSS against DN hypothesized by the network pharmacological approach were confirmed through in vivo experiments. The detailed graphical abstract of this work is illustrated in http://tcmspw.com), the encyclopedia of TCM (ETCM), and the TCM integrative pharmacology-based research platform (TCMIP), which lists the pharmacokinetic properties of active phytochemicals such as oral bioavailability (OB) and drug-likeness (DL), among others. The OB index is an important pharmacokinetic parameter that represents the proportion of the medication in the blood circulation. The similarity between a novel compound and a pre-existing drug can be expressed in terms of the DL index; a high DL indicates that although a compound is not yet used as a drug, it may become one in the future. In this study, the DSS bioactive components meriting further analysis were those with an OB ≥ 30% and DL ≥ 0.18.Active DSS compounds were retrieved from the TCM systems pharmacology database and analysis platform . A false discovery rate cut-off <0.05 was applied for clustering during the functional annotation.GO and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed using the DAVID 6.8 functional annotation tool (w/v) at room temperature for 30 minutes with a reflux device before boiling for 1 hour and filtering the extract. The boiling and extraction steps were performed two times. The extracted filtrate was combined and then concentrated to a final extract concentration of 0.64 g/ml, referred to as DSS extract. The extract was dried to a powder form by freeze-drying and stored at −20°C, and the powder was redissolved with Milli-Q water to obtain DSS extract before the treatment.DSS comprises ASR, PRA, CR, PC, AMR, and AR (a ratio of 3 : 16 : 8 : 8 : 4 : 4). It was provided by the pharmacy department of the Shenzhen Traditional Chinese Medicine Hospital and prepared in the following manner: the mixture is submerged in distilled water . Eight db/m mice were used as a control group (n = 8). The db/db group and DSS group received gavages of 0.9% saline (10 ml/kg/day) and the DSS extract (6.4 g/kg/day), respectively. The study was approved by the Animal Ethics Committee of Shenzhen Top Biotech Co., Ltd. and conducted in full compliance with the directives of the National Institutes of Health guidelines. All mice were killed through cervical dislocation without consciousness at the end of the study. Blood samples were collected by eye enucleation, and the kidneys were excised and promptly snap-frozen to detect various markers.Male diabetic mice (db/db) and mice (db/m) of 10 weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. . The mice were maintained in the specific pathogen-free animal room of Shenzhen Top Biotech Co., Ltd.  on a 12-h light/dark cycle with a relative humidity of 40–60% and temperature of 20–25°C. Animals were free to access food and water. Fasting blood glucose greater than 11.1 mmol/L after 30 days was diagnosed as diabetes. The diabetic mice were randomly assigned to the db/db group and the DSS group  kit was used to assess the Scr levels according to the manufacturer's instructions.μm sections were prepared from the kidneys after dehydration, embedment in wax, and slicing for the periodic-acid-schiff (PAS), Masson's trichrome staining (Masson), and hematoxylin and eosin staining (H&E). Images were visualized with a Zeiss optical microscope. Ninety glomeruli from three mice in each group were assessed for the degree of glomerular mesangial expansion and glomerular area employing Image-J  in a blinded fashion, under ×400 magnification by PAS staining. Quantification of fibrosis was further performed under ×200 magnification by Masson trichrome staining [3-staining .α , IL-6 , Phospho-SAPK/JNK , and GAPDH . And then the blots were incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h at room temperature. The specific reaction was imaged with an advanced ECL kit  and the ChemiDoc MP Imaging System . ImageJ software was employed for the densitometric study.In brief, proteins extracted from the kidneys were lysed with RIPA buffer . The protein concentration was detected by the BCA Protein Assay Kit . Prepared protein lysates were loaded onto 8–12% SDS-PAGE gels and electrophoretically transferred to a polyvinylidene fluoride membrane. After being blocked with 5% nonfat milk for 1 h at room temperature, the membranes were probed with primary antibodies, including anti-JNK1 + JNK2 + JNK3 , anti-c-Jun , Phosphoa-c-Jun , anti-ICAM-1 , TNF-P value <0.05 was considered significant.The data were expressed as means ± SEM. SPSS  was employed for analyses. One-way analysis of variance was used to detect differences between groups, followed by Tukey's multiple comparisons test. A A total of 752 ingredients were identified through the TCMSP database, ETCM, and TCMIP v2.0 (up to July 2022). Based on the cut-offs of OB ≥ 30% and DL ≥ 0.18 detailed above, 51 candidate bioactive components were subjected to further analyses after eliminating overlapping genes. Detailed information is shown in Ninety-one potential DSS targets were predicted by the TCMSP database. A total of 1,153 targets related to diabetic nephropathy were collected from the GeneCards and OMIM databases. After eliminating the overlaps, we identified 126 common  ), suggesting that these genes potentially play a significant role in DSS treatment of DN. To better understand the potential mechanism of DSS in DN, we constructed a disease-targets-compound network using Cytoscape 3.8.0 software ). Nodes with different colors and shapes in the figure represent different types of information. Green ovals represent common targets, blue rectangular nodes represent active ingredients of TCM, purple hexagons represent DN, and orange prisms represent DSS.To determine how the overlapping genes interact, the 126 common targets were imported into the STRING database, and a PPI network diagram was drawn ). As stated above, more adjacent genes in the PPI map played more important roles. By calculating the number of nodes connected to each gene, the top 30 genes in terms of centrality were identified as the most important genes of DSS with respect to the treatment of CKD ); these included IL-6, AKT1, TNF, CAT, JUN, and PTGS2.We carried out a GO enrichment analysis using the DAVID database to identify the associations of the 126 common genes with DN; 69 GO terms were obtained, of which the top 20 were significantly enriched in terms of MFs . These tTo further explore how DSS affected DN through the 126 common genes, they were upregulated in the DAVID database and a KEGG enrichment analysis was performed. We identified 140 signaling pathways; the top 20 items are listed in P < 0.01). Administrating DSS significantly reduced the Scr (P < 0.01) level in db/db mice ). These data indicate that DSS prevented renal functional decline in db/db mice.Renal function was assessed by the Scr level. The Scr levels in the db/db group increased significantly compared to those in the control group , mesangial matrix enlargement, nodular glomerulosclerosis, and arteriolar hyalinosis . On PAS α were increased in the db/db mice but decreased after the DSS treatment (The western blot analysis has demonstrated that JNK and c-Jun were upregulated in the db/db mice expression compared with the db/m mice while these proteins were significantly decreased in the DSS group. We also found that the treatment with DSS significantly reduced the phosphorylation levels of JNK and c-Jun in db/db mice Figures . Meanwhireatment . These dDiabetic nephropathy is a multifactorial, complex disease process caused mainly by hyperglycemia, oxidative stress, AGEs, and angiotensin II. All of these factors are linked to various proteins or pathways throughout the development and progression . In partIn this study, the compounds in DSS with an OB of 30% and DL > 0.18 were considered pharmacokinetically active and may be largely responsible for the therapeutic effects of DSS in DN. Paeoniflorgenone was the most abundant compound, followed by paeoniflorin, lactiflorin, paeoniflorin qt, albiflorin qt, benzoyl, paeoniflorin, beta-sitosterol, and stigmasterol, which is consistent with the results of Luo et al. [in vivo to further support this hypothesis.We demonstrated that DSS acted on several targets and signaling pathways. Hub targets of the pharmacokinetically active compounds included IL-6, AKT1, TNF, CAT, JUN, and PTGS2. Inflammation, cell proliferation, apoptosis, and fibrosis were all linked to these genes. Thus, DSS may exert its antirenal damaging effects in DN by preventing fibrosis, lowering inflammation, and modulating mitochondrial homeostasis, cell proliferation, and apoptosis, which are key mechanisms in the development of DN . Among tin vivo data showed that DSS decreased Scr levels and considerably reduced pathological damage in db/db mice. DSS substantially decreased JNK, P-JNK, c-Jun, and P-c-Jun expression in vivo, according to the western blotting. We also found that DSS reduced the expression of ICAM-1, IL-6, and TNF-α; this finding was validated by H&E staining, which revealed inflammatory infiltration. Inflammation is the initial reaction of the body to kidney damage. JNK is a mitogen-activated protein kinase subfamily protein that regulates critical biological functions, such as cell proliferation, differentiation, and apoptosis [α and IL-6, is linked to the continued activation of JNK1 [α, IL-6, IL-1, ICAM-1 to exert its biological impact [α mediates inflammatory processes and is also implicated in the progression of diabetic complications [α [α concentrations enhance sodium absorption, resulting in renal hypertrophy and salt retention, which are characteristic abnormalities during the early stages of diabetic neuropathy [We explored the curative effects of DSS in db/db mice with the goal of providing further support for this hypothesis. The poptosis . The JNKpoptosis . Seen inpoptosis . JNKs no of JNK1 , 36. Actl impact –39. Intel impact . Recentll impact , 42. As l impact . Numeroul impact . TNF-α mications . Almost tions [α . There iuropathy . We demoThe active pharmacological components and mechanisms by which DSS suppresses inflammation in DN were demonstrated using an integrated approach, including network pharmacology and experimental validation. We found that DSS suppresses inflammation primarily by regulating the JNK signaling pathway. In conclusion, the combination of network pharmacology and experimental validation was useful in defining the mechanism of the action of DSS.