Ulcerative colitis (UC) is a chronic inflammatory bowel disease of the colonic mucosa. Esculetin is a type of natural coumarin that has many pharmacological activities such as antioxidant, anticancer, anti-inflammatory, etc. A previous study showed that esculetin improved intestinal inflammation and reduced serum proinflammatory cytokines in UC. The present study aimed to utilize network pharmacology and molecular docking to explore the potential mechanism of esculetin against UC. The potential gene targets of esculetin were predicted through SwissTargetPrediction and Super-PRED web servers. UC-related genes were obtained from DisGeNet, OMIM, and GeneCards databases. The overlap between gene targets of esculetin and UC-related genes were identified as the potential targets of esculetin against UC. The interaction between these overlapping genes was analyzed by the STRING database and the core genes were identified by Cytoscape platform. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the core genes were then performed. And the results of these analyses were further confirmed through molecular docking. A total of 50 overlapping genes were identified as the potential action targets of esculetin against UC. Among them, 10 genes  were finally identified as the core genes. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis results showed that the top signaling pathway associated with the core genes of esculetin against UC was the prolactin (PRL) signaling pathway. Molecular docking results showed that esculetin has a strong binding affinity to the core genes, as well as PRL and prolactin receptor. This study suggests that esculetin may have a crucial impact on UC through the PRL signaling pathway and provides insights into the potential mechanism of esculetin in the treatment of UC, which may shed light on the mechanism and treatment of UC. North America and Europe have long been recognized as regions with a high prevalence of UC, with the highest reported prevalence of 505 per 100,000 in Europe (Norway) and 286 per 100,000 in North America (United States). Asia, a region with historically low prevalence of UC, has experienced a striking increase in the 21st century. The exact etiology of UC is not yet fully understood and may involve interactions between the environment, immune system, gut microbiota, and genetic susceptibility.Ulcerative colitis (UC) is a chronic inflammatory bowel disease of the colonic mucosa characterized by alternating periods of exacerbation and remission.Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb., Fraxinus szaboana Lingelsh., and Fraxinus stylosa Lingelsh. It is one of the simplest coumarin compounds with 2 hydroxyl groups at carbons 6 and 7. Esculetin has been shown to have many pharmacological activities in vitro and in vivo, including antioxidant, anticancer, anti-inflammatory, antidiabetic, anti-atherosclerotic, and so on.–8 A recent study demonstrated that administration of esculetin ameliorated intestinal inflammation and reduced serum levels of the pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in a mouse model of dextrose sodium sulfate (DSS)-induced UC. However, the mechanism by which it protects against UC remains to be discovered.Esculetin  is a type of natural coumarin extracted mainly from the dried bark of  Molecular docking is a method used to model the interaction between small molecule ligands and proteins at the atomic level, which helps to characterize potential molecular interactions that may occur at the binding site. Based on the research strategy of network pharmacology, our study attempted to predict the core genes and potential signaling pathways of esculetin for the treatment of UC. The affinity between the screened receptors and esculetin was further verified using molecular docking, thus exploring the potential mechanism of esculetin against UC.In recent years, network pharmacology has become a widely used tool in drug research to reveal the interrelationships between compounds, targets, pathways, and associated diseases.The flow chart of the study design was shown in Figure https://www.ebi.ac.uk/chembl/), and then we utilized its simplified molecular-input line-entry system string (O = c1ccc2cc(O)c(O)cc2o1) to search the predicted target genes of esculetin in 2 small molecule target prediction databases: SwissTargetPrediction web server , and Super-PRED web server . All the target genes were standardized by the UniProt database (https://www.uniprot.org/), and the species was restricted to “Homo sapiens” in the search. Final target gene predictions of esculetin were obtained after duplicate removal.To determine the predicted target genes of esculetin, we first identified the simplified molecular-input line-entry system string of esculetin through the ChEMBL database , OMIM , and GeneCards . The search was performed using the term “ulcerative colitis” as the keyword. Genes that appeared in at least 2 of the above 3 databases were identified as final UC-related genes.The identification of UC-related genes was obtained from 3 well-established databases: DisGeNet (https://bioinfogp.cnb.csic.es/tools/venny/). These overlapping genes were identified as the potential action targets of esculetin against UC and used for further analysis.A Venn diagram was plotted between the predicted target genes of esculetin and the UC-related genes using the online tool Venny 2.1.0  was used to analyze the potential action targets of esculetin against UC. The organism was selected as “Homo sapiens.” After downloading the data from the STRING database, the PPI network was constructed and visualized using the Cytoscape v3.10.0 platform, and the CentiScaPe 2.2 plug-in was employed for the identification of the core genes. In CentiScaPe 2.2, betweenness, closeness and degree were selected for analysis, and the higher the value, the more important the gene.A PPI network analysis through the STRING database  pathway enrichment analysis of the core genes were performed using the clusterProfiler package in R v4.3.0 and RStudio 2023.06. A https://pubchem.ncbi.nlm.nih.gov/). The PDB formats of the core genes, including AKT1 (PDB ID: 3O96), STAT1 (PDB ID: 7NUF), CCND1 (PDB ID: 5VZU), SRC (PDB ID: 1HCS), PTGS2 (PDB ID: 5F19), EGFR (PDB ID: 5D41), NFKB1 (PDB ID: 1SVC), ESR1 (PDB ID: 6V8T), MMP9 (PDB ID: 6ESM), and SERPINE1 (PDB ID: 4G8R) were downloaded from the PDB database (https://www.rcsb.org/). In addition, the KEGG pathway enrichment analysis concluded that the prolactin (PRL) signaling pathway might play a key role in the treatment of UC with esculetin, which is normally activated by PRL binding to the prolactin receptor (PRLR). Therefore, the PDB formats of PRL (PDB ID: 1RW5) and PRLR (PDB ID: 3D48) were also downloaded. Molecular docking was performed using AutoDockTools v4.2 . Visualization of AutoDockTools results was performed using PyMOL v2.5 (https://pymol.org/2/), Protein–Ligand Interaction Profiler web server (https://plip-tool.biotec.tu-dresden.de/), and ProteinsPlus webserver (https://proteins.plus/).Molecular docking was performed to predict the binding affinity between esculetin and its potential therapeutic targets for the treatment of UC, which could provide a basis for further experimental verification. The SDF format of esculetin (PubChem CID5281416) was obtained from the PubChem database , and NFKB1 are common inflammation-associated genes that are closely involved in the release of inflammatory mediators and cytokines such as IL-1β, IL-6, interferon gamma, TNF-α, inducible nitric oxide synthase (prostaglandin E2).,24 The polarization imbalance of M1/M2 macrophages in UC has been the focus of recent attention. AKT1 is strongly associated with the M1 phenotype of macrophages, which plays a key role in colitis. EGFR is an upstream molecule of the mitogen-activated protein kinase (MAPK) signaling pathway. SRC is one of the major protein tyrosine kinases that regulate the MAPK signaling pathway as well as the nuclear factor-kappa B signaling pathway. Both the MAPK and nuclear factor-kappa B signaling pathways are critically involved in the pathogenesis of UC due to the abundant release of cytokines and inflammatory mediators upon their activation.A recent study has confirmed the potential of esculetin to ameliorate DSS-induced UC by reducing inflammation and regulating cytokine release. It has been reported that the mucosal expression and serum levels of MMP9 were significantly higher in UC patients compared to the control group, and the increased MMP9 expression contributed to the severity of mucosal damage in active UC. In addition, serum levels of MMP9 were significantly associated with C-reactive protein, white blood count, platelets, and Mayo endoscopic score in UC patients. The role of MMP9 in UC is not well understood and may involve the release of cytokines and chemokines, angiogenesis, regulation of tight junction proteins, goblet cell differentiation, and so on. Our molecular docking results showed that esculetin had a strong binding affinity to MMP9 with a value of −7.45 kcal/mol. It is suggested that esculetin may be a potential therapeutic agent for UC through its interaction with MMP9.Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic endopeptidases with extracellular matrix remodeling and degradation properties. Most MMPs are transcriptionally upregulated in response to proinflammatory cytokines, cell-cell, or cell-extracellular matrix interactions. As both a circulating hormone and a cytokine, the primary function of PRL is to promote mammary gland development, lactation, and pregnancy. Aside from its actions on reproductive processes, PRL plays a role in maintaining the constancy of the internal environment by regulation of the immune system, osmoregulation, angiogenesis, and so on. The secretion of PRL is mainly under the inhibitory control of hypothalamic dopamine (DA), and it is also subject to a negative feedback loop of its own. The critical role of DA in suppressing endogenous PRL secretion has been well established. Tuberoinfundibular dopamine neurons located in the dorsomedial arcuate nucleus secrete DA into the pituitary portal blood vessels. The D2 DA receptor is abundantly expressed in the pituitary gland, which mediates the inhibition of PRL secretion. In addition to the pituitary gland, PRL can also be produced in extrapituitary locations, such as decidua, ovary, prostate, mammary gland, adipose tissue, brain, and immune cells.The KEGG pathway enrichment analysis results indicated that the PRL signaling pathway may play an important role in the treatment of UC with esculetin. PRL is a 23 kDa peptide hormone composed of 199 amino acids that is principally secreted by the lactotrophs in the anterior pituitary gland. In addition to the 23 kDa monomeric PRL, 2 other major forms are present in the circulation: 40 to 60 kDa big PRL (dimer of the monomeric form) and >150 kDa big-big PRL (complexes of the monomeric form and IgG autoantibodies). However, the monomeric form of PRL is the most active and the higher molecular weight forms have minimal biological activity. PRLR consists of an extracellular domain, a single transmembrane domain, and an intracellular domain. When PRL binds to PRLR, it causes receptor dimerization, resulting in the initiation of several intracellular signal transduction, including canonical Janus kinase 2/signal transducer and activator of transcription (JAK2/STAT) pathway, MAPK pathway, and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. JAK2 is a non-receptor tyrosine kinase that is rapidly activated upon stimulation with PRL, promoting the phosphorylation and nuclear localization of STATs, including STAT3 and STAT5. The JAK2/STAT3 pathway is an extremely important intracellular signal transduction pathway that has been widely recognized as a critical modulator of a variety of biological processes, including innate and adaptive immunity and inflammation. Blockade of the JAK2/STAT3 pathway has been demonstrated to modulate innate and acquired immune responses and to attenuate chronic intestinal inflammation. Comparatively, activation of JAK2/STAT5 signaling may exert a protective effect against UC. Phosphorylation of STAT5 promotes the synthesis of Foxp3, which promotes the differentiation of CD4+ T cells into regulatory T cells. Regulatory T cells always play a role in immune homeostasis and suppressing inflammatory responses. Furthermore, activated PI3K/Akt pathway is also implicated in the regulation and release of proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α, which in turn participates in the development of UC.PRL binds to its receptor PRLR, which belongs to the class I cytokine receptor family that has no intrinsic kinase activity. Similarly, decreased levels of DA were detected in the colonic mucosa of TNBS-induced rats, in the colonic mucosa of DSS- induced mice, and in the feces of TRUC (−/− Rag2−/−T-bet ulcerative colitis) mice. In UC, a decrease in DA levels may lead to a weakening of its inhibitory effect on PRL secretion, ultimately resulting in an increase in PRL levels. A clinical study from China with 72 UC patients and 72 healthy controls showed that UC patients had significantly higher PRL levels than healthy controls . In the present study, by inputting the core genes of esculetin against UC for KEGG pathway enrichment analysis, the main significantly enriched pathway was the PRL signaling pathway. Subsequently, we used molecular docking to predict the binding affinity of the esculetin ligand to PRL and PRLR. The results showed that the binding energy values of esculetin to PRL and PRLR were both <−5 kcal/mol, indicating that esculetin could form stable binding conformations with PRL and PRLR. Since the biological activity of PRL is highest when in a monomeric form, binding of esculetin to PRL may reduce PRL activity. On the other hand, if esculetin competes with PRL for its receptor PRLR, it may block activation of the PRL-induced pathway. This ultimately leads to inhibition of the downstream JAK2/STAT, MAPK, and PI3K/Akt pathways associated with UC. These above results offered an initial theoretical basis for further experimental investigation (Fig. It was reported that DA levels in the inflamed mucosa of UC patients were markedly lower than in controls. so the role of MMP9 in UC remains to be clarified. Thirdly, STAT3 and STAT5, as downstream of the PRL signaling pathway, seem to play opposite roles in UC. This will cause some disagreement on the role of the PRL pathway in UC. Fourthly, the changing profile of PRL expression levels in UC patients need to be supported by more clinical data.However, the present study has some limitations. Firstly, the pharmacological mechanism of esculetin in the treatment of UC in our study is based on computational technologies and still requires further validation through pharmacological and clinical studies. Secondly, some reports suggest that MMP9 upregulation is a consequence rather than a cause of intestinal inflammation,In summary, our study utilized a network pharmacology approach to investigate the potential mechanism of esculetin in the treatment of UC. Our study ultimately identified 10 core genes, including AKT1, STAT1, CCND1, SRC, PTGS2, EGFR, NFKB1, ESR1, MMP9, and SERPINE1, which may be potential targets of esculetin against UC. The KEGG pathway enrichment analysis indicated that esculetin may have a crucial impact on UC through the PRL signaling pathway. Our molecular docking analysis also showed that esculetin effectively interacted with PRL and PRLR in addition to the 10 core genes. Overall, our study provides a reference for further investigating the mechanism of esculetin against UC. Furthermore, we also want to draw the attention of researchers to the role of PRL signaling pathway on UC, which may shed light on the mechanism and treatment of UC.Conceptualization: Bin Cai.Funding acquisition: Ting Cai, Bin Cai.Methodology: Ting CaiProject administration: Bin Cai.Visualization: Bin Cai.Writing – original draft: Ting Cai, Bin Cai.Writing – review & editing: Bin Cai


Qihuang Granule (QHG) is a traditional prescription  that has exhibited potential in safeguarding against age-related maculopathy (AMD). Salvia miltiorrhiza (SM) and Fructus lycii (FL) are the main components of QHG. Ferroptosis, a newly discovered, iron-dependent, regulated cell death pathway, have been implicated in the pathogenesis of AMD. This study delves into the intricate mechanism by which SM/FL and QHG confer protection against AMD by modulating the ferroptosis pathway, employing a combination of network pharmacology and experimental validation.Bioactive compounds and potential targets of SM and FL were gathered from databases such as TCMSP, GeneCard, OMIM, and FerrDb, along with AMD-related genes and key genes responsible for ferroptosis regulation. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein–protein interaction (PPI) network were performed to discover the potential mechanism. The construction of an interaction network involving AMD, ferroptosis, SM/FL potential target genes was facilitated by the STRING database and realized using Cytoscape software. Subsequent validation was accomplished through molecular docking and in vitro cell experiments.2O2.Noteworthy active compounds including quercetin, tanshinone IIA, luteolin, cryptotanshinone, and hub targets such as HIF-1α, EGFR, IL6, and VEGFA were identified. KEGG enrichment unveiled the HIF-1 signalling pathway as profoundly enriched, and IL6 and VEGF were involved. The molecular docking revealed the significant active compounds with hub genes and quercetin showed good binding to HIF-1α, which is involved in inflammation and angiogenesis. Experimental results verified that both herbs and QHG could regulate key ferroptosis-related targets in the retinal pigment epithelium and inhibit the expression of HIF-1α, VEGFA, and IL-6, subsequently increase cell viability and decrease the ROS content induced by HThis study demonstrates the molecular mechanism through which SM/FL and QHG protect against AMD and emerges as a plausible mechanism underlying this protection.The online version contains supplementary material available at 10.1186/s12906-023-04205-3. Age-related macular degeneration (AMD) is a prelavent neurodegenerative disease characterized by the progressive deterioration of the macula, which is responsible for sharp and detailed vision. Nowadays, AMD affects more than 50% of people over 80 worldwide. AMD is the primary cause of visual impairment and blindness on a global scale, particularly among the elderly population. Recent statistics reveal a significant prevalence of AMD globally, with an estimated 196 million individuals affected by 2020. The impact of AMD on visual impairment is substantial, accounting for approximately 8.7% of all blindness worldwide. In developed countries, AMD is the leading cause of blindness, particularly among older individuals [The mechanisms involved in the development of AMD are intricate and multifactorial. These factors include genetic susceptibility, age-related dysfunction of normal retinal homeostasis, impaired lipid metabolism, immune activation leading to chronic inflammation, oxidative stress, and extracellular matrix (ECM) dysfunction . DespiteWhile several tissues are affected in AMD, including photoreceptors, retinal pigment epithelium (RPE), Bruch’s membrane and choriocapillaris, the dysfunction of RPE is an early and crucial event in the molecular pathways that results in clinically relevant changes in AMD patients . In the AMD is clinically categorized into early and late stages, with the majority of cases falling into the early stage. This classification includes medium-sized drusen and retinal pigmentary changes. Early-stage AMD is prevalent and imposes a significant economic burden on society . Current2O2-induced inflammatory injury in human RPE cells and improve visual acuity and fundus conditions in dry AMD patients [In recent years, the use of traditional Chinese medicine (TCM) for treating AMD has gained attention as a potential solution. “Qihuang” granule (QHG) is a traditional prescription of TCM and had been studied extensively. It contains compounds such as Salvia miltiorrhiza (SM) and Fructus lycii (FL), which have been found to have protective effects against Hpatients . In addiFerroptosis is a recently discovered form of regulated cell death that is dependent on iron and characterized by excessive lipid peroxidation and iron overload. It has been implicated in various conditions such as neurodegeneration, ischaemia–reperfusion injury, and cancer –12. StudNetwork pharmacology is a meaningful approach for drug discovery , 16.Our https://tcmspw.com/tcmsp.php). TCMSP GeneCards is a comprehensive database integrating information from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and GeneCards databases. It provides information on the interactions between Chinese herbal compounds and targets, disease-associated genes, and drug targets, helping researchers understand the mechanisms of action and potential pharmacological effects of Chinese herbal compounds. To obtain relevant targets of the main compounds of SM and FL, oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were selected as screening conditions in this study [https://www.uniprot.org/) was chosen to determine the gene name and identifiers of their targets.The ingredients of SM and FL were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  and OMIM  [http://www.zhounan.org/ferrdb/) focuses on genes and proteins related to iron metabolism. It collects information on genes, proteins, and metabolic pathways associated with iron metabolism and related diseases. FerrDb provides detailed data on the functions of iron metabolism genes, expression patterns, and mutations associated with diseases [https://bioinfogp.cnb.csic.es/tools/venny/) intersection.Potential AMD-related targets were identified through GeneCards  (https:/im.org/) , OMIM (Odiseases . Overlaphttps://string-db.org/) with the species set to “Homo sapiens” and the minimum required interaction score was set to 0.40 [https://cytoscape.org/, Version 3.7.2), CytoNCA plug-in was used to calculate the parameters, and the hub targets that were related to SM/FL-induced ferroptosis in AMD were identified according to BC, CC, and degree. Finally, the “AMD-Ferroptosis-SM/FL Potential Targets Genes” visual network was constructed with Cytoscape software.Taking the intersection of all the decoction targets, AMD targets, and ferroptosis targets, we identified the targets that are related to SM/FL-induced ferroptosis in AMD. To further identify the core regulatory targets, PPI analysis was performed by submitting the overlapping targets of active compounds of SM/FL, ferroptosis, and AMD to the STRING biological database  enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out to further study the functions of the identified potential anti-AMD target genes based on R 4.0.2 and related R packages . Only functional terms and pathways with p values < 0.05 were considered statistically significant and retained.Molecular docking is a widely employed method in drug discovery due to its capacity to accurately predict the conformation of small molecule ligands within their appropriate target binding sites and to assess the binding affinity –24. In t2. The cells showed obvious extension and the density was between 70 and 90%, indicating that the cells were in good condition. This research has been reviewed by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine, and it is believed that the project meets the requirements for exemption from ethical review and agrees to exempt the project from review.Human RPE cells  were cultured in DMEM , supplemented with 10% foetal bovine serum , 100 U/mL penicillin and 50 U/mL streptomycin  at 37 ℃ in 5% CO2O2 . To examine the effect of SM/FL or QHG on RPE cells undergoing oxidative stress, cells were plated in 96-well plates at a density of 5 × 103 cells per well and preincubated with 2 g/l SM/FL or 2 g/l QHG for 24 h. Then the cells were further incubated with H2O2 (200 μm) to induce oxidative stress. The cells were divided into four groups: control group (without drug), H2O2 group, H2O2 + SM/FL group and H2O2 + QHG group.To resemble AMD pathophysiology in ARPE-19 cells, we induced oxidative stress with H2O2 (200 μm) for 12, 24, and 48 h. Cell viability was analyzed with CCK-8 method  in accordance with the manufacturer’s protocol. After treatment, cells were harvested and incubated with 10 μl of CCK-8 solution for 2 h at 37℃. The absorbance was measured at 450 nm with a microplate reader , and the optical density represents the proliferation of RPE cells.RPE cells were preincubated with SM/FL or QHG for 24 h, and then the cells were further incubated with H2O2 (200 μm) for 6 h. Protein expression of HIA-1α, VEGFA, IL6, SLC7A11, and GPX4 in each group was measured by Western blotting. Cells were washed with cold PBS, harvested in RIPA buffer , incubated on ice for 20 min and centrifuged for 5 min at 500 rpm. Then, protein concentration was quantified using a BCA protein assay kit . Proteins were separated by SDS-PAGE  and transferred to PVDF membranes by a Trans-Blot Turbo Transfer System. Membranes were blocked with 5% skim milk in for 1 h and incubated with the following primary antibodies overnight at 4 °C: polyclonal rabbit antihuman HIA-1α , polyclonal rabbit antihuman VEGFA , polyclonal rabbit antihuman IL-6, polyclonal rabbit antihuman GPX4 , polyclonal rabbit antihuman SLC7A11 and anti-β-actin. Protein bands were visualized by incubation with an anti-rabbit secondary antibody  or anti-mouse secondary antibody  and chemiluminescence substrates . Finally, the bands were quantified by Image Lab software .RPE cells were preincubated with SM/FL or QHG, and then incubated with H2O2 (200 μm) for 6 h and cellular ROS levels were measured by dihydroethidium (DHE) staining. The cells were washed with PBS twice and diluted, and 10 mM of DHE  was incubated with the cells for 30 min at 37 °C, after which cells were washed with PBS again. ROS production was observed with a fluorescence microscope . ImageJ software was applied to quantify and calculate the fluorescence intensity.ARPE-19 cells with different treatment were incubated with Hp-value less than 0.05 were considered as statistically significant.All the experiments were performed at least three times. The experimental data were statistically analyzed using GraphPad Prism software and SPSS20.0 Software. Single-factor ANOVA was applied to assess differences in experimental data between groups. SM and FL are the primary components of QHG, and information about these components is presented in Table HIF-1α, EGFR, IL6, VEGFA, and NFE2L2, with degree values of 20, 18, 18, 16, and 14, respectively . A total of 12 core GO terms were enriched in the biological processes. These included RNA poly II-specific DNA-binding transcription factor binding, DNA-binding transcription factor binding, ubiquitin protein ligase binding, ubiquitin-like protein ligase binding, among others .To determine the effect of SM/FL or QHG on RPE cells undergoing oxidative stress, cell viability was measured. As shown in Fig. ons Fig. a, b   Fig. a, b, c.F2O2, SM/FL and QHG on the expression of the intracellular ferroptosis markers GPX4 and SLC7A11. Compared with RPE cells cultured in DMEM, cells exposed to H2O2. showed significant downregulation of GPX4 and SLC7A11. Notably, the effect of H2O2 was almost reversed in RPE cells that were pretreated with SM/FL or QHG   has established its significance in disease prevention and treatment, offering a holistic approach through multiple ingredients that often influence diverse targets and pathways. In our investigation, we harnessed the power of network pharmacology to unravel the potential mechanisms by which Salvia miltiorrhiza (SM) and Fructus lycii (FL), the primary compounds of Qihuang Granule (QHG), exert their protective effects against age-related macular degeneration (AMD). Specifically, we delved into the ferroptosis-related pathway, a pathway that mounting evidence has linked to AMD pathogenesis through its role in inducing oxidative stress-mediated damage and inflammation , 26, 27.HIF-1α, EGFR, IL6, VEGFA, and NFE2L2 emerged as the top five hub targets based on degree values within the protein–protein interaction (PPI) network. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses illuminated these core targets' involvement in biological processes, including RNA poly II-specific DNA-binding transcription factor binding, DNA-binding transcription factor binding, ubiquitin protein ligase binding, and ubiquitin-like protein ligase binding, mediated by the HIF-1, MAPK, P13K-Akt, Relaxin, and Ras signaling pathways.Through network pharmacology, we successfully identified 56 shared target genes among bioactive compounds in SM/FL and target genes implicated in AMD. Notably, quercetin, a key component of SM/FL, stood out as the compound with the highest number of associations with AMD-related genes and ferroptosis. This suggests quercetin's paramount role in treating AMD by targeting ferroptosis. Subsequently, we pinpointed 12 core targets through which SM/FL may protect against AMD via ferroptosis regulation. Among these, 2O2-induced declines in cell viability and the increase of HIF-1α, VEGFA, and IL6. Additionally, both SM/FL and QHG elevated the expression of key ferroptosis-related proteins SLC7A11 and GPX4 while mitigating ferroptosis reactions and oxidative stress responses in RPE cells. These outcomes serve as a valuable corroboration of our network pharmacology analyses.Molecular docking analysis provided further substantiation by confirming the interaction between active compounds in SM/FL and hub genes. These findings underscore the effectiveness of SM/FL in treating AMD from a bioinformatics perspective, elucidating the underlying mechanisms of QHG's actions. In line with the predictions from network pharmacology, our in vitro experiments revealed that pre-treatment with SM/FL or QHG effectively countered HQi Huang Granule (QHG), a traditional prescription comprising SM and FL as its primary components, has previously demonstrated its efficacy in improving visual acuity and fundus conditions in dry AMD patients. SM, renowned for its antioxidative, neuroprotective, anti-inflammatory, and antineoplastic properties, has been shown to mitigate RPE damage and hypoxia by suppressing inflammation in the retina –32. FL, RELA and TP63 [Our study pinpointed four active ingredients within SM/FL that play pivotal roles in AMD treatment by regulating ferroptosis, underscoring their potential for further research. Quercetin, the most abundant compound in SM/FL, boasts diverse biological functions such as antioxidant, anti-inflammatory, and anti-viral activities –38. Our and TP63 , 45 In Rand TP63 , 47.Luteolin, known primarily for its anti-inflammatory activity, protects RPE cells from oxidative stress-induced cell death, mitigates epithelial-mesenchymal transformation, and suppresses IL-1β-induced adhesion in AMD –50. CrypAs seen in the AMD-Ferroptosis-SM/FL Potential Targets Genes network, many target genes can be regulated by different active compounds and one active compound can target several genes. These results suggest that this therapy has multicomponent, multitarget biological attributes. Additionally, the PPI results suggest that the 12 target proteins are not independent of each other but are linked and interact. These results also indicate that SM/FL can be involved in the alleviation and treatment of AMD through the regulation of multiple proteins.HIF-1α can upregulate the expression of VEGF in general and in RPE cells. It is thus a central molecule for triggering CNV formation [HIF-1α emerged as a pivotal hub target, with the HIF-1α pathway ranking first in PPI and KEGG analyses. HIF-1α, a subunit of HIF-1, acts as a master regulator of hypoxia-inducible genes associated with inflammation, angiogenesis, cell proliferation/survival, and glucose/iron metabolism . Notablyormation  These reormation , 62.Our in vitro experiments further solidified the connection between SM/FL, QHG, ferroptosis, and AMD. Western blotting demonstrated decreased expression of HIF-1α, VEGFA, and IL6, alongside increased SLC7A11 and GPX4 expression in the SM/FL and QHG groups compared to the H2O2 group. This translates to not only potential therapeutic benefits but also a reduction in proinflammatory factors that exacerbate AMD. The elevated cell viability and stability observed in SM/FL and QHG-treated cells corroborate the network pharmacology findings.One limitation of this study pertains to the absence of consideration for interactions between active ingredients, as well as the caveat that compound absorption in humans is not solely determined by the oral bioavailability (OB). Further exploration is warranted to elucidate the intricate relationship between HIF-1α, VEGFA, and IL6 in the context of AMD.In conclusion, our comprehensive approach, integrating network pharmacological analysis and in vitro experiments, sheds light on the mechanisms that underlie the protective effects of Salvia miltiorrhiza (SM) and Fructus lycii (FL), as well as their composite, Qihuang Granule (QHG), against age-related macular degeneration (AMD). These findings not only advance our understanding of the therapeutic mechanisms of traditional Chinese medicine but also present a promising strategy for deciphering the scientific rationale and therapeutic modalities of traditional Chinese medicine formulas in addressing complex diseases.Additional file 1.Additional file 2.


Quercetin (QR) is a natural flavonol compound widely distributed in the plant kingdom with extensive pharmacological effects. To find the potential clinical indications of QR, 156 differentially expressed genes (DEGs) regulated by QR were obtained from the Gene Expression Omnibus database, and new potential pharmacological effects and clinical indications of QR were repurposed by integrating compounds with similar gene perturbation signatures and associated-disease signatures to QR based on the Connectivity Map and Coexpedia platforms. The results suggested QR has mainly potential therapeutic effects on multiple sclerosis (MS), osteoarthritis, type 2 diabetes mellitus, and acute leukemia. Then, MS was selected for subsequent animal experiments as a representative potential indication, and it found that QR significantly delays the onset time of classical MS model animal mice and ameliorates the inflammatory infiltration and demyelination in the central nervous system. Combined with network pharmacology technology, the therapeutic mechanism of QR on MS was further demonstrated to be related to the inhibition of the expression of inflammatory cytokines  related to TNF-α/TNFR1 signaling pathway. In conclusion, this study expanded the clinical indications of QR and preliminarily confirmed the therapeutic effect and potential mechanism of QR on MS. Repurposed drugs can reveal new targets and pathways that can be further exploited . The advls.gov/) . At presls.gov/) . ThalidoMoreover, with the continuous reduction of the cost of high-throughput sequencing technology and the iterative accumulation of data related to drug research and development, massive data obtained by bioinformatics technology to find new pharmacological effects and indications of the marketed drugs and reveal their potential therapeutic mechanism, which has become a hot spot in current drug repurposing research . Computain vitro and animal studies (Quercetin (QR) is a natural flavonoid that widely exists in nature, such as in vegetables, fruits, and medicinal herbs. Its antioxidant, anti-inflammatory, and antiallergic properties have been observed in numerous  studies . Present studies , hyperur studies , COVID-1In this study, differentially expressed genes (DEGs) significantly regulated by QR were obtained from Gene Expression Omnibus (GEO) database. The new potential pharmacological effects and clinical indications of QR were repurposed by integrating clinically approved drugs with similar gene expression signatures to QR and candidate diseases associated with QR-regulated DEGs based on CMap and Coexpedia platform. The classic animal model was used to verify the drug repurposing result and explore the mechanism of action .https://www.ncbi.nlm.nih.gov/geo/), the data information on gene expression related to QR regulation was retrieved under the restriction of “homo sapiens,” and the GEO online analysis tool GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r) was used for data preprocessing and the DEGs in the dataset were screened by the statistical cut-offs of p-value <0.05 and |log2-fold change| (|logFC|) > 1. Based on CMap (https://clue.io/query) and Drugbank 83 (https://go.drugbank.com/), the approved drugs with the same transcriptional regulation direction as 84 the QR-regulated DEGs were screened out [Normalized Connection Score (Norm CS) ≥ 1.3], and the mechanisms of action between different drugs were compared. Disease Ontology (DO) analysis of QR-regulated DEGs was performed using the Coexpedia platform (https://www.coexpedia.org/).Based on the GEO database , TTD, PharmgKB (https://www.pharmgkb.org/), OMIM and DisGeNET (https://www.disgenet.org/). In addition, to collect the QR targets, CTD (https://ctdbase.com), DrugBank (https://go.drugbank.com/), PubChem (https://pubchem.ncbi.nlm.nih.gov/), HIT (http://www.badd-cao.net:2345/), PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), and SwissTargetPrediction (http://www.swisstargetprediction.ch/) databases were used. The QR targets were matched with the targets of the relevant indication to obtain the common targets of QR treatment indication.The potential indications enriched in DO combined with the pharmacological effects of approved drugs screened by CMap were used for drug repurposing of QR, and the most representative indication was selected for subsequent research. The representative indication targets were retrieved based on MalaCards (https://cn.string-db.org/), where the organism was set as “homo sapiens” and the data file was imported into Cytoscape for interaction network visualization. Then, the CytoHubba plugin (http://apps.cytoscape.org/apps/cytohubba) was 103 used to analyze the PPI network and calculate the core targes.The common targets were put into the STRING database (https://metascape.org/) for functional enrichment analysis (p < 0.05) were visualized using the OmicShare tools (https://www.omicshare.com/tools).All the common targets were imported into Metascape  was added to Freund’s incomplete adjuvant  to prepare the 10 mg ml-1 solution, and the same volume of myelin oligodendrocyte glycoprotein peptide  solution (2 mg ml-1) was dissolved in sterile PBS to prepare oil antigen emulsion.C57BL/6 female mice, 8–10 weeks of age (18–20 g), were obtained from SPF Biotechnology Co., Ltd , and housed in specific pathogen-free conditions at the First Affiliated Hospital of Henan University of Chinese Medicine , Henan, China. All efforts were made to reduce animal suffering, and the Institutional Committee on Care and Use of Research animals approved the procedures used in this study. The inactivated −1 QR  group, the EAE group and EAE + QR group (n = 6 in each group). EAE was induced by MOG 35-55, as previously described M. L. . Briefly−1 QR   inflammation and demyelination. The mice were anesthetized with 1% pentobarbital sodium and perfused with 0.9% sodium chloride solution on day 21 p.i. The left brain was quickly removed and fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE inflammatory infiltration staining and LFB myelin staining. The scoring criteria for inflammatory infiltration  were as https://www.rcsb.org/). For docking analysis, all protein and molecular files were converted into PDBQT format with all water molecules excluded, and polar hydrogen atoms were added. The molecular docking results were evaluated through the criteria of binding structure, binding energy, and possible interactions between the ligand and the critical residues of the protein.To analyze the binding affinities and modes of interaction between QR and the core target proteins, AutoDock Vina, a silico protein-ligand docking software, was employed . The twohttp://atb.uq.edu.au/) database. Initially, the entire system used the steepest descent minimization. Then, the balance of acceptor, ligand, and solvent was achieved by running 1 ns NVT. Finally, molecular dynamics sampling with 100 ns and a time step of 2 fs was performed for analysis.After molecular docking, molecular dynamics simulation was used to simulate the complex of target proteins and QR to detect the stability and flexibility of the complex. This study used the GROMACS 2020.5 software package, GROMOS54a7_atbff force field, and SPC216 water model for molecular dynamics simulation. In order to ensure the total charge neutrality of the simulation system, a corresponding number of sodium ions are added to the system to replace water molecules to form a solvent box of appropriate size. Then the periodic boundary condition is applied to the three directions of the system. Use the gromos54a7_atb force field to obtain the QR’s force field parameters from ATB  and reversely transcribed into cDNA using Fast All-in-One RT Kit . Then, RT-PCR was performed using cDNA as the template with Super SYBR Green qPCR Master Mix  and QuantStudio 6 Flex PCR System . GAPDH was used as the reference control, and the results were analyzed using the 2SD). p < 0.05 was considered statistically significant.All statistical analyses were performed using GraphPad Software . The Student’s t-test was used to analyze the weight changes, the difference in the mean clinical severity of mice, and HE staining and LFB staining scores. All values are presented as mean ± standard deviation (+ monocytes before and after 2 weeks of QR (150 mg d-1) supplementation in three healthy volunteers. The results show that a total of 156 DEGs of QR have been screened, including 135 upregulated DEGs and 21 downregulated DEGs with p-value <0.05 and |log FC| > 1 as the threshold: , and the top 10 disease entries were screened for display according to the p-value. As shown in Based on Coexpedia platform, the QR-regulated DEGs are mainly enriched in 84 significant disease entries  enrichment analysis are shown in p < 0.05). The top 15 main pathways are TNF signaling pathway, IL-17 signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and NF-κB signaling pathway (The KEGG enrichment indicated that 44 common targets were enriched in 87 pathways ( pathway . KEGG fu pathway  indicatep < 0.01), and the mean clinical scores and cumulative clinical scores were lower (p < 0.01) than the EAE group mice  to other PPI network protein nodes. Studies have shown that TNF-α binding to Tumor Necrosis Factor Receptor Type 1 (TNFR1) can trigger a series of proinflammatory cytokines and chemokines, thereby increasing the immune cascade of CNS . In addi-1 indicates that there is a certain binding activity between the ligand small molecule and the receptor protein; the binding energy < −5.0 kcal mol-1 indicates that there is a good binding activity between the two; the binding energy < −7.0 kcal mol-1 indicates that the ligand and receptor have strong binding activity  of TNFα-QR and TNFR1-QR in protein were obtained. Compared with the first frame, the TNFα-QR complex reached stability after 10 ns. The RMSD oscillation amplitude of QR is about 0.14 nm TNFR1-QR complex was stable after 28 ns(RMSD 0.46–0.56 nm). The RMSD oscillation amplitude of QR is about 0.10 nm. It is suggested that during the 100 ns simulation process, the systems of TNFα-QR and TNFR1-QR quickly reached stability without excessive fluctuations .Another stability factor is the fluctuations of protein during simulation that were evaluated by analyzing RMSF values . On RMSFp < 0.01) observably compared with that in NC mice, while their expressions were significantly reduced  after QR treatment  were identified by PPI network analysis. To verify the above findings, we determined the mRNA levels of these six target genes in the spinal cord tissues of NC, EAE, and EAE + QR groups by reverse transcription-polymerase chain reaction (RT-PCR). The expressions of TNF, IL6, IL1B, IFNG, IL17A, and IL2 in the CNS of EAE mice were elevated  mainly treat allergic and autoimmune inflammatory diseases (including MS) through anti-inflammatory and immunosuppressive effects . Muscle MS is an autoimmune disease of the CNS characterized by immune cell infiltration and demyelination. It has the characteristics of easy recurrence and a high disability rate. This study used an internationally recognized animal model of MS (EAE) to verify whether QR has a therapeutic effect on MS . The resIt is currently recognized that the pathogenesis of MS is caused by the cellular immune response mediated by the body’s own T lymphocytes . NotablyIn conclusion, based on the drug repurposing strategy, we have successfully discovered new clinical indications of QR and verified QR’s therapeutic potential and potential mechanism on MS through experiments. This study laid an experimental foundation for the preclinical study of QR in treating MS and provided a reference for expanding the clinical indications of other approved or investigational drugs. Of course, as an early exploratory study, a series of more in-depth studies on the mechanism of action are still needed. Thus, this study must be viewed in light of its strengths and limitations, many of which represent opportunities for future research.


Purpose: The aim of present study was to explore the pharmacological mechanisms of Niaoduqing granules on the treatment of podocyte injury in diabetic nephropathy (DN) via network pharmacology and experimental validation.Methods: Active ingredients and related targets of Niaoduqing, as well as related genes of podocyte injury, proteinuria and DN, were obtained from public databases. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analysis were performed to investigate the potential mechanisms. High glucose (HG) -induced MPC5 cell injury model was treated with the major core active ingredients of Niaoduqing and used to validate the predicted targets and signaling pathways.Results: Totally, 16 potential therapeutic targets were identified by intersecting the targets of Niaoduqing and disease, in which 7 of them were considered as the core targets via PPI network analysis. KEGG enrichment analysis showed that AGE-RAGE signaling pathway was identified as the most crucial signaling pathway. The results of in vitro experiments revealed that the treatment of Niaoduqing active ingredients significantly protected MPC5 cells from HG-induced apoptosis. Moreover, Niaoduqing could significantly attenuate the HG-induced activation of AGE-RAGE signaling pathway, whereas inhibited the over-expression of VEGF-A, ICAM-1, PTGS-2 and ACE in HG-induced MPC5 cells.Conclusion: Niaoduqing might protect against podocyte injury in DN through regulating the activity of AGE/RAGE pathway and expression of multiple genes. Further clinical and animal experimental studies are necessary to confirm present findings. Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus, which involves the entire kidneys . In Chinvia inhibiting tubular epithelial-to-mesenchymal transition (EMT) and regulating TGF-beta1/Smad pathway (p < 0.05), and reduce the level of blood urea nitrogen , blood creatinine  and urine protein . Compared with using ACEI/ARB alone, the better renal function is observed in patients received combination therapy with Niaoduqing  according to the condition of oral bioavailability (OB) ≥ 30% and drug-like properties (DL) ≥ 0.18 (http://www.swisstargetprediction.ch/) (https://pubchem.ncbi.nlm.nih.gov/). The conversions from protein names to the unique entry gene IDs were performed through the uniprot database (https://www.uniprot.org/).Niaoduqing granules consist of nine components, including Bai Shao, Bai Shu, Che Qian Cao, Da Huang, Dan Shen, Fu Ling, Huang Qi, Ku Shen and Sang Bai Ye. The active ingredients of the above night components were screened through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  ≥ 0.18 . Relatedion.ch/) . In Swisp-value < 0.05. Volcano plots and heat maps were used to represent the DEGs.DN related databases  were obtained from Gene Expression Omnibus (GEO) database. After removing duplicate and missing data, we screened the differentially expressed genes (DEGs) according to the following criteria: │log FC│≥1 and https://www.genecards.org/) (https://omim.org/) (http://db.idrblab.net/ttd/) (https://www.disgenet.org/home/) (https://www.ncbi.nlm.nih.gov/) and PharmgKB database (https://www.pharmgkb.org/)  , the Onlim.org/) , Therapeet/ttd/) , DisGeNEg/home/) , NCBI (hkb.org/)  with “diAll disease targets obtained from the above databases and the DEGs of DN obtained from GSE1009, GSE96804 and GSE111154 datasets were pooled together, and those targets appearing in at least two databases and datasets were defined as DN-related targets in present study. Similar protocol was applied to screen the proteinuria-related genes: only the overlapping targets appearing in at least two databases were identified as proteinuria-related genes.Niaoduqing-related targets were intersected with the DN-related targets, the proteinuria-related targets and the podocyte injury-related targets to identify the potential therapeutic targets of Niaoduqing on the treatment of podocyte injury and proteinuria in DN. “venn” and “VennDiagram” packages from R language were used to create the Venn diagram to depict the intersections between different databases and datasets. Cytoscape 3.6.1 software was used to construct the relationship network among night components, active ingredients and the potential therapeutic targets.https://cn.string-db.org/, Version: 11.5), with the minimum required interaction score set at “median confidence (0.400)” level. Cytoscape 3.6.1 and its CytoNCA plugin were used to further analyze the original PPI network. Three topological parameters, including betweenness centrality, closeness centrality and degree value, were calculated and considered as the evidence for the screen of core targets. The higher the values were, the more important the targets were (The protein-protein interactions (PPI) results among potential therapeutic targets were obtained from the SRTING database (ets were . Nodes wGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using “Cluster profiler” package from R language to investigate the probable molecular mechanisms of Niaoduqing on the treatment of podocyte injury and proteinuria in DN. The top 10 enriched enters of molecular function (MF), biological process (BP) and cellular components (CC) in GO analysis were presented in bar chart. The top 20 enriched pathways were shown in bubble chart.https://www.rcsb.org/). The water molecules, metal ions and small molecule ligands were removed, and the active pockets were identified by PyMOL software. Thirdly, AutoDock Vina 1.1.2  was obtained from Bioss Biotech . Anti-AKT antibody , anti-phospho-AKT antibody , anti-caspase-3 antibody  and anti-Bax antibody  was acquired from Cell Signaling Technology . Anti-NF-κB antibody  and anti-phosphor-NF-κB p65/RelA antibody  was purchased from ABclonal Biotech . Goat anti-mouse IgG second antibody (C1308), goat anti-rabbit IgG second antibody (C1309) and anti-actin antibody  was acquired from Pulilai Biotech . Annexin V—Alexa Flour 488/PI Apoptosis Kit (FXP022) was purchased from 4A Biotech . CCK-8 (CK04) was obtained from Dojindo Laboratorise . Fetal bovine serum (FBS) was purchased from Zeta Life .Niaoduqing Granules were obtained from KangCheng Pharmaceutical Industry, China (No. Z20073256). The lyophilized powder of quercetin  was acquired from Sigma . The lyophilized powders of kaempferol  and luteolin  were obtained from Selleck . Anti-PI3K antibody  was purchased from Affinity Biosciences . Anti-phospho-PI3KCA antibody  model, extra glucose  was added to growth medium and differentiated MPC5 cells were cultured under high glucose condition (44 mM). Since the solution of Niaoduqing granules showed toxic effect on the survival and proliferation of MPC5 cells, the mixture of three major active ingredients of Niaoduqing (Que + Lut + Kae) were used as an alternative for the in vitro experiments. MPC5 cells were randomly divided into four groups: the control group, Que + Lut + Kae (1 μg/mL) group, HG group and HG + Que + Lut + Kae group.The conditionally immortalized mouse podocyte cell line Mouse Podocyte Clone 5 (MPC5) cells were purchased from Jennio Biotech . MPC5 cells were maintained in RPMI 1640 medium supplemented with 15% FBS, 2 mM L-Glutamin, 100 IU/mL penicillin-streptomycin, and 5 U/mL recombinant mouse interferon-γ  at 33°C in a humidified atmosphere with 5% COvalue of experimental group) − ]/[-] × 100%.MPC5 cells were re-plated in 96-well plates  and cultured at 37°C overnight. Then, growth medium was removed and 100 μL of culture medium with different concentrations of Niaoduqing granules  and Que + Lut + Kae mixture  was added. Twenty four and 48 h after treatment, 100 μL of basic medium and 10 μL CCK-8 solution were added to each well and incubated for another 2 h. The optical density (OD) value was measured at 490 nm. The cell viability was calculated using the following formula: Cell viability% = [. The primer sequences for RT-qPCR are shown in Total RNA was extracted from each group of MPC5 cells 48 h after treatment using Trizol method . The cDNA was synthesized using Evo M-MLV RT kit . The mRNA was quantified using the 2× SYBR Green Pro Taq HS Premix II , with Protein was extracted from different groups of MPC5 cells using RIPA lysis buffer  containing 1:100 protease inhibitors and 1:100 phosphatase inhibitors. The protein was quantified using bicinchoninic acid kit  according to the manufacturer’s instruction. After adding 5 × protein loading buffer, all samples were denatured by boiling at 100°C for 10 min and separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis was performed at a constant voltage of 60 V for 60 min initially and then switched to 120 V. The gel was further blotted to PVDF membrane for 120 min at a constant current of 200 mA. After blocking with 5% skimmed milk, membranes were incubated with primary antibody at a concentration of 1:1000 at 4°C overnight and further incubated with secondary antibody at a concentration of 1:5000 for 1 h at room temperature. The protein blots were visualized using enhanced chemiluminescence reagent . Quantitative analysis was completed using ImageJ.Apoptosis was determined using an Annexin V/Alexa Fluor 488/propidium iodide Apoptosis Detection Kit  according to the manufacturers’ instructions. Briefly, MPCs cells were washed twice with cold phosphate-buffered saline (PBS) and then re-suspended in 100 µL of 1 × binding buffer. The cell suspension was incubated with AnnexinV-Alexa Flour 488 (5 µL) for 5 min in dark at room temperature, then 10 µL of PI solution and 400 µL of PBS was added. Samples were measured on Accuri C6 flow cytometer (BD Biosciences) and data were analyzed by FlowJo 8.0 software .t-test and Bonferroni test in ANOVA were used to make comparisons between two and multiple groups. p < 0.05 was considered as a significant difference . GraphPad Prism 8  was used to visualize the results.Data shown in present study repeated at least three times. All data were showed as mean and standard deviation of the mean (SD) and analyzed by SPSS23.0 . Student’s A total of 206 active ingredients were identified, including 32 from Sang Bai Ye, 13 from Bai Shao, 8 from Bai Shu, 10 from Che Qian Cao, 16 from Da Huang, 67 from Dan Shen, 16 from Fu Ling, 20 from Huang Qi and 45 from Ku Shen . There whomo sapiens” as organism. After taking the intersection of targets appearing in at least two databases and datasets using Venn diagram, 986 overlapping targets was identified as DN-related targets  did not show significant cytotoxicity up to 4 μg/mL (p > 0.05). Therefore, 1 μg/mL of Que + Lut + Kae was considered as a safe concentration and further used in our study.To determine the cytotoxicity and appropriate concentration of the crude Niaoduqing granules solution and its purified core active ingredients, differentiated MPC5 cells were exposed to drugs at different concentrations and CCK-8 assays were performed to detect the cell viability. As shown in Vegf-α (***p < 0.001), Ptgs-2 (***p < 0.001), Icam-1 (*p = 0.026) and Ace (***p < 0.001) expression, but had no effect on Ren (p = 1.000), Spp-1 (p = 0.052) and Nos-3 (p = 0.524) expression. Compared with HG group, the upregulation of mRNA expression levels of Vegf-α (***p < 0.001), Ptgs-2 (***p < 0.001), Icam-1 (*p = 0.034) and Ace (***p < 0.001) were significantly attenuated in HG + Que + Lut + Kae group. There was no statistical difference of expression levels for all target genes between control and Que + Lut + Kae group .To validate the potential therapeutic targets of Niaoduqing in HG-induced podocytes injury model, RT-qPCR was performed to detect the relative expression levels of the above predicted targets. As shown in p = 0.028) and NF-κB , as well as the mRNA levels of target genes including Rage (***p < 0.001), Tnf-α (**p = 0.002), Tgf-β1 (**p = 0.004), Col1a1 (***p < 0.001) significantly increased in HG group. Strikingly, Niaoduqing ingredients treatment significantly reduced HG-induced the increase of phosphorylation of NF-κB  and upregulation of the expression of Rage (***p < 0.001), Tnf-α (**p = 0.001), Tgf-β1 (**p = 0.004), and Col1a1 (***p < 0.001). Although Que + Lut + Kae treatment also inhibited HG-induced phosphorylation of PI3KCA  and AKT , but with no statistical significance. The details of AGE/RAGE signaling pathway were shown in To further investigate the potential mechanisms and evaluate the results of our network pharmacology analysis, the activity of AGE/RAGE signaling pathway in diabetic complications (hsa04933) was detected, which was the most enriched signaling pathway in KEGG analysis. As shown in p = 0.001) and cleaved-Caspase-3 (**p = 0.012) protein levels significantly increased in HG-induced group, while Que + Lut + Kae treatment reduced HG-induced increase of protein levels of Bax (**p = 0.006) and cleaved-Caspase-3 (**p = 0.006). Our data suggested that Niaoduqing might reduce proteinuria through the protection of podocyte from high glucose induced apoptosis.Previous study showed that Niaoduqing had better effect on controlling proteinuria in patients with early stage DN . Since pDiabetic nephropathy poses a significant threat to the global public health and places enormous economic burden due to high morbidity, high mortality but poor control rate worldwide. To date, we still do not have effective treatment approach to stop or delay the progression of DN . Podocytin vitro experiment. Although only three core ingredients could not fully represent Niaoduqing compound, they might be considered as one of the best alternative methods for clarifying the therapeutic effect of Niaoduqing in vitro experiment. Quercetin is an effectively ingredient in alleviating diabetes and related complications . Two of the four hub targets (VEGF-A and ICAM1) were involved in this pathway. AGE/RAGE pathway has been demonstrated to be involved in the development of DN . In the  with DN . Targeti with DN . In presvia activating Wnt signaling pathway and interfering with metabolism of fatty acids  expansion and collagens accumulation are identified as the key links of fibrosis development, in which EMT and endothelial to mesenchymal transition (EndMT) are the main sources of matrix-producing myofibroblasts . Inflammty acids . Fibroblty acids . The defty acids , Compare pathway . The los pathway . In presRecently, there are many potential drugs that have been proven to be effective against DN. As the commonly used anti-hypertension drugs, both ACEI and ARB show good effect on inhibiting kidney fibrosis, but their therapeutic effects are not completely consistent . The autvia serologic pharmacology method as previously described is an optional method to solve this problem  might be one of the core signaling pathways. Further evidence of


Rostellularia procumbens (L.) Nees (R. procumbens) extract and to make a preliminary investigation of its anti-nephritis mechanism.The purpose of this study was to demonstrate the in vitro anti-nephritis activity of R. procumbens and CGN. Then, the potential targets for R. procumbens against CGN were imported into the DAVID database for Gene Ontology (GO) biological annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A lipopolysaccharide (LPS)-stimulated rat mesangial cell HBZY-1 model in vitro was used to examine the anti-inflammatory activity of R. procumbens extract. RNA-seq was utilized to investigate differentially expressed genes (DEGs) and enriched signaling pathways between groups. Finally, qPCR was used for the validation analysis of the experimental results.A prediction network was built that describes the relationship between R. procumbens exerts its therapeutic effect on CGN through the AGE-RAGE signaling pathway in diabetic complications, PI3K-Akt, IL-17 signaling pathway, and so on. R. procumbens n-butanol extract (J-NE) can effectively relieve inflammation in HBZY-1. The results of KEGG pathway enrichment suggest that J-NE attenuated CGN was associated with the IL-17 signaling pathway, and the results of RNA-seq were consistent with network pharmacology. Targets enriched in the IL-17 signaling pathway, including Chemokine (C-C motif) ligand 7 (CCL7), Lipocalin 2 (LCN2), Chemokine (C-C motif) ligand 2 (CCL2), and Chemokine (C-X-C motif) ligand 1 (CXCL1), have been identified as crucial targets attenuating CGN by J-NE.The results of network pharmacology showed that R. procumbens is a promising pharmacological candidate for the treatment of CGN in the present era.The online version contains supplementary material available at 10.1186/s12906-023-04079-5. The global prevalence is estimated at 8–16% . A patieay 2022) , which cay 2022) . To addrTherefore, we should actively seek treatment for CGN to prevent the disease from continuing to a stage that requires dialysis or kidney transplantation.R. procumbens, a medicinal plant of the Acanthaceae family, was first published in Sheng Nong’s Herbal Classic. The plant name has been checked with The Plant List . It is mainly composed of lignans, flavones, and triterpenes [R. procumbens [Information about terpenes , which aterpenes –8, anti-terpenes , antiplaterpenes , 10, 11,terpenes , 13 actiIn this study, network pharmacology was used to predict the potential pharmacodynamic components and effective mechanism of J-NE in the treatment of CGN. RNA-seq was used to comprehensively analyze the mechanism of action of J-NE in the treatment of CGN, and qPCR was used for final verification.R. procumbens were retrieved from relevant China and foreign literature, and a database of R. procumbens compounds was established [The main compounds contained in ablished . Pharmacablished .https://pubchem.ncbi.nlm.nih.gov/) and imported into the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) to predict potential target information. The predicted targets were ranked according to their scores, and a threshold of 10 was chosen to screen approximately 18% of the targets as potential targets for candidate compounds.The SMILES molecular formulas of candidate active compounds were obtained from the PubChem database (https://www.genecards.org/) was searched using the keyword “chronic glomerulonephritis” to obtain targets associated with CGN.The GeneCards database . The networks were then loaded into the Cytoscape 3.7.2 software, which was used to examine the close relationships between the targets.The predicted targets of candidate compounds were compared and crossed with the collected “chronic glomerulonephritis” targets to obtain the potential targets of https://david.ncifcrf.gov/summary.jsp) for GO functional annotations and KEGG pathways enrichment analysis. Biological process, molecular function, and cellular component were selected for GO functional annotations. p ≤ 0.05 was set as the threshold to screen for significantly different terms or pathways.The targets were entered into the DAVID database  Nees. was harvested in October 2019 from the Wuchang district, Hubei province of China, and was authenticated by Prof. Hezhen Wu, Hubei University of Chinese Medicine (HBUCM). Collection of plant materials complied with local or national guidelines. Voucher specimens (JC-2019-01) were stored in the pharmaceutical college.Petroleum ether, ethyl acetate, n-butanol, and DMSO  were purchased from Sinopharm Chemical Reagent Co., Ltd . The ethanol used for extraction was food grade. LPS (L4391) was purchased from Sigma (USA). The Rat TNF-α Elisa Kit (E-EL-R2856c) was purchased from Elabscience Biotechnology Co., Ltd . MTT (1334) was purchased from Bioforxx (Germany).R. procumbens  was weighed, crushed, sieved (24 mesh), and percolated with ten volumes of 80% ethanol to obtain a percolation liquid. The resulting diafiltrate was concentrated to remove ethanol under reduced pressure. Through systematic solvent extraction, the R. procumbens is finally divided into petroleum ether extraction material (J-PE), ethyl acetate extraction material (J-EE), and water-saturated n-butanol extraction material (J-NE).Fifteen kg of R. procumbens was dissolved in DMSO  to prepare a 200 mg/mL stock solution. It was diluted to the target concentration with DMEM when used (The concentration of DMSO in each group is less than 0.5%).Rat kidney mesangial cells (HBZY-1) were supplied by China Center for Type Culture Collection , and were cultured in a humidified incubator at 37℃ containing 5% carbon dioxide using DMEM  supplemented containing 100 U/mL penicillin/streptomycin mixture  and 10% fetal bovine serum . When growth to 80–90% confluence, cells were washed once with phosphate-buffered saline (PBS) and digested by 0.25% trypsin , spare. Every fraction from MTT) assay. HBZY-1 cells were seeded in 100 µl media at 4000 cells/well in 96-well plates with three parallel wells in each group. After the cells adhered, fractions were added to each well at a final concentration of 50, 100, 200, and 400 µg/mL and then treated for 24 h. After cells were incubated with MTT solution (0.5 mg/mL) for 4 h at 37 °C, discarding the cell supernatant containing MTT, each well was added 150 µl DMSO. The 96-well plate was shaken with a plate shaker to dissolve the formazan, and the optical density (OD) was measured at the absorption wavelength of 490 nm.Cell proliferation was detected by 3--2,5-diphenyltetrazolium bromide  were set up. After the cells adhered, the model and the experimental group were induced with 1 µg/mL LPS for 24 h. Then the experimental group was treated with each fraction  for 24 h. The supernatant was taken for TNF-α concentration measurement by Elisa kit.HBZY-1 cells were seeded into 6-well plates at 3 × 105 cells/well. The control, model, and experimental groups  were set up. After modeling and administration in the same way as in 2.11, the supernatant was discarded, 1 mL of TRIzol reagent  was added to each well to digest and lyse the cells, and the cells were collected with an enzyme-free EP tube.HBZY-1 cells were seeded into 6-well plates at 3*10Total RNA was extracted according to the requirements of the instructions, and the qualified total RNA test by Agilent Bioanalyzer 2100  was the starting sample for mRNA sequencing. The starting total RNA was accurately quantified with QUBIT RNA ASSAY KIT . The purified and fragmented total RNA was used to build a cDNA library, and the quality of the cDNA library was checked using an Agilent Bioanalyzer 2100. Q-PCR method was used to accurately quantify the effective library concentration (library effective concentration > 2 nM). Qualified samples were sent to the Illumina Hiseq2500 platform for sequencing to obtain sequencing data.2 2.7.0d  was used to map clean reads to the reference genome. According to the bam files obtained by the alignment, QoRTs3 1.3.0 (http://hartleys.github.io/QoRTs/) was used to perform statistics and graphs. The quality of the sequencing data was evaluated from the aspects of alignment rate, alignment region, and gene region coverage.The reference genome version of this project was Rnor_6.0. After filtering the original sequencing data, the alignment software Star5 was used to analyze and screen out DEGs . We use bioinformatics (http://www.bioinformatics.com.cn/) to draw volcano plots and Venn diagrams of DEGs. The Venn diagram was used to count the number of DEGs in common between each group. DEGs in common were uploaded to the DAVID database to analyze GO terms and KEGG pathways. Then identify the signaling pathways that DEGs are mainly involved in.DESeqTotal RNA was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit . qPCR was performed using TB Green® Premix Ex Taq™ II . Samples were analyzed in triplicate and normalized by subtracting the average Ct value of the sample from the average Ct value of the housekeeping gene GAPDH. Primer information is shown in the supplementary file Table S.p < 0.05 was considered statistically significant.GraphPad Prism 8.0  was used for statistical analysis. Statistical differences between groups were evaluated by one-way analysis of variance (ANOVA). In this research, twenty-eight compounds were screened out and used as candidate active compounds. Twenty-eight candidate active compounds were shown in the supplementary file Table S.R. procumbens against CGN were obtained  , while the TNF-α concentration after 100 µg/mL J-NE treatment was significantly decreased (p < 0.01). The J-EE also had the effect of reducing the secretion of TNF-α, but its effect is not as good as that of J-NE. Therefore, we considered that the active ingredients for the treatment of CGN are concentrated in the n-butanol fraction (J-NE).Figure As shown in the volcano plot Fig. A, comparR. procumbens exerted its effects in the relief of CGN and was closely related to the six pathways, including the IL-17 signaling pathway, Cytokine-cytokine receptor interaction, and NF-kappa B signaling pathway. The IL-17 signaling pathway was the one we were interested in and ranked first according to the p-value, which was also consistent with the results of network pharmacology. Therefore, we considered that the targets enriched in the IL-17 signaling pathway including CXCL10, CSF3, MMP13, CCL7, MMP3, LCN2, TNFAIP3, CCL2, CXCL1, and PTGS2 were targets for J-NE against CGN. We selected some genes and verified their expression at the transcriptional level using qPCR  Nees.Traditional Chinese medicine and pharmacology are a great treasure-house of the Chinese nation and a glorious crystallization of its great culture. Just as ancient TCM books offer clues for Ms. Tu Youyou to discover artemisinin, we have discovered a TCM that can relieve nephritis, which name is R. procumbens attenuating CGN. Through KEGG enrichment analysis, it was concluded that R. procumbens might act through the PI3K-Akt, NF-kappa B, IL-17 signaling pathway, and so on. However, it is necessary to further clarify in experiment ways whether R. procumbens affects as ancient TCM books say and whether it works through pathways analyzed in network pharmacology. In this study, based on network pharmacology, further analysis was performed using RNA-seq, and we found J-NE may have a therapeutic effect on CGN via influencing the expression of CCL7, LCN2, CCL2, and CXCL1, consequently changing the inflammatory response and controlling the IL-17 signaling pathway. qPCR was used for the validation analysis of the experimental results.By employing the network pharmacology method, we preliminarily predicted the mechanism of CGN is a kind of disease that includes many pathological types, is caused by a variety of factors, and originates in the glomerulus. A part of chronic nephritis occurs because of the continuation of acute nephritis, but most of them are caused by immune complexes deposition in the glomerulus. These immune complexes directly promote glomerular inflammation and mesangial hyperplasia. Activation of the local and systemic renin-angiotensin system and complement also leads to glomerulosclerosis and tubulointerstitial fibrosis, which leads to loss of renal function . MesangiIn addition to measuring the concentration of TNF-α in the cell supernatant, we also examined the expression of IL-6 at the transcriptional level. As seen in Fig. R. procumbens extract were used to treat LPS-stimulated HBZY-1 cells. We found that J-NE was significantly effective in the in vitro treatment of CGN. Three cell groups including control group, model group, and J-NE (100 µg/mL) group were set up, and the possible mechanism of J-NE against CGN using RNA-seq technology was explored. The IL-17 signaling pathway was the one we were interested in and ranked first according to the p-value, which was also consistent with the results of network pharmacology. The targets enriched in the IL-17 signaling pathway, including CCL7, LCN2, CCL2, and CXCL1, were targets for J-NE against CGN.Three different polarities of R. procumbens, could reduce the mRNA expression level of MCP-1/CCL2, MCP-3/CCL7, and CXCL1 induced by LPS, which inhibits inflammation.Mesangial cells were involved in glomerular inflammation because they are equipped with a set of innate pathogen-recognition receptors that enable them to recognize a large number of pathogen-associated molecular patterns and damage-associated molecular patterns and translate the recognition of these danger signals into the pro-inflammatory secretion of mediators, namely cytokines and chemokines , such asLCN2, also known as neutrophil gelatinase-associated lipocalin (NGAL), was mainly expressed in neutrophils and played an important role in innate immunity . It was R. procumbens extracts significantly ameliorated ADR-induced renal pathological changes in mice and that the therapeutic mechanism of J-NE was associated with the inhibition of podocyte apoptosis [Here, we demonstrated that J-NE inhibits the production of TNF-α and the expression level of MCP-1/CCL2, MCP-3/CCL7, and CXCL1, thus providing a potential mechanism for its anti-inflammatory activity. A previous study in our laboratory found that treatment with poptosis . This stRegrettably, we detected changes in chemokine expression levels in a single cell model in vitro that elicit responses from other populations of cells if in vivo. Although our findings may not be the direct cause of the reduction in inflammatory factors in vitro, they were undoubtedly surprising findings pointing to future research directions.J-NE can have a therapeutic effect on CGN by affecting the expression of CCL7, LCN2, CCL2, and CXCL1, thereby altering the inflammatory response and controlling the IL-17 signaling pathway, and is a promising drug candidate for the treatment of CGN.Additional file 1: Table S1. 28 candidate active compounds for Network pharmacology.  Nees against chronic glomerulonephriti. Table S3. Primer information.e et al. ). Table


Ultra-high-performance concrete (UHPC) is a cement-based material with excellent impact resistance. Compared with traditional concrete, it possesses ultra-high strength, ultra-high toughness, and ultra-high durability, making it an ideal material for designing structures with impact resistance. The research on the impact resistance performance of UHPC and its composite structures is of great significance for the structural design of protective engineering projects. However, currently, there is still insufficient research on the impact resistance performance of UHPC composite structures. To study the impact resistance performance, experiments were conducted on UHPC targets using high-speed projectiles. The results were compared with impact tests on granite targets. The results indicated that when subjected to projectile impact, the UHPC targets exhibited smaller surface craters compared with the granite targets, while the penetration depth was lower in the granite targets. Afterwards, the process of a projectile impacting the UHPC composite structure was numerically simulated using ANSYS 16.0/LS-DYNA finite element software. The numerical simulation results of penetration depth and crater diameter were in good agreement with the experimental results, which indicates the rationality of the numerical model. Based on this, further analysis was carried out on the influence of impact velocity, impact angle, and reinforcement ratio on the penetration depth of the composite structure. The results show that the larger the incident angle or the smaller the velocity of the projectile is, the easier it is to deflect the projectile. There is a linear relationship between penetration depth and reinforcement ratio; as the reinforcement ratio increases, the penetration depth decreases significantly. This research is of great significance in improving the safety and reliability of key projects and also contributes to the application and development of ultra-high-performance materials in the engineering field. In recent years, military actions and terrorism have become increasingly frequent worldwide, posing serious threats to buildings and facilities in the field of protection engineering . TherefoExtensive experiments and numerical simulation studies have been conducted on the impact resistance properties of ordinary concrete. However, relatively less research has been conducted on the penetration resistance of UHPC and rock ,11,12,13Although rocks have better penetration resistance, they are more difficult to process than concrete. Rocks are naturally occurring materials and, unlike concrete, they are difficult to shape or mold. Concrete can be poured into molds, making it easy to achieve the desired shape. In comparison, working with rocks typically requires specialized tools and techniques such as drilling, cutting, or blasting to achieve the desired form or size. In this regard, Yang et al.  conducteThe aforementioned research mainly focuses on the anti-penetration performance of granite, UHPC, and ceramic/armored steel/UHPC composite structures. However, there is relatively less research on the anti-penetration performance of UHPC/granite composite structures. Furthermore, there are limitations to the application of ceramics and armored steel in protective engineering due to their high cost. Based on previous research on the anti-penetration performance of UHPC and granite, this paper compares the anti-penetration performance of UHPC and granite under the same penetration conditions. A UHPC/granite anti-penetration composite structure is designed, and the anti-penetration performance of the composite structure is studied using ANSYS 16.0/LS-DYNA finite element software. The effects of penetration speed, projectile hit angle, and target reinforcement ratio on the penetration depth are systematically investigated. A flow chart for the methodology of this paper in provided in The mix proportions of the UHPC material in this article is shown in Pour the prepared UHPC slurry into the mold; first, pour in half of the height of the slurry, place the mold on a vibrating table, and vibrate for 1 min. Then, pour in the remaining half of the slurry and vibrate for another minute. Finally, smooth the surface of the specimen. Place the specimens in a curing room with a temperature of 20 °C and a humidity of 95% for 28 days of curing. After curing, remove the specimens from the molds and grind the two ends of the loading surface of the specimens to ensure that the flatness of the two end faces of the specimens is less than 5‰.According to the Chinese Standard GB/T 31387-2015 [In the present projectile impact test on UHPC targets, three cylindrical targets of the same size were made, with a diameter of 1300 mm and a thickness of 600 mm, as shown in The projectile material used in the impact test is 35CrMnSiA, with a yield strength of 1300 MPa. The length of the projectile is approximately 307 mm, and its mass is approximately 1000 g. The diameter of the projectile is 30 mm, as shown in The Φ35 mm caliber cannon is used as the experimental launching equipment, and the projectile velocity is controlled between 200 and 350 m/s by adjusting the amount of propellant charge. The test results were compared with the anti-penetration test results of granite mentioned in reference , as showh/d) with projectile velocity for the experiments in this study and the reference [eference . It can Based on the above analysis, it can be concluded that granite performs better than UHPC in reducing projectile penetration depth, while UHPC demonstrates better performance in suppressing impact crater diameter. When UHPC and granite are combined to form a composite structure, it can achieve superior penetration resistance capabilities.Based on the analysis in the previous section, in order to further maximize the penetration resistance performance of UHPC and granite, we designed a UHPC/granite composite structure and conducted numerical simulation research. The UHPC/granite composite structure is shown in C, P are the strain rate parameters.In numerical simulations, the MAT_PLASTIC_KINEMATIC model in LS-DYNA is chosen for the projectile and rebar material models. MAT_PLASTIC_KINEMATIC is commonly used to describe the plastic behavior of metals, and the model can be represented as follows:The rebar parameter values for the MAT_PLASTIC_KINEMATIC model are given in In LS-DYNA, the Riedal–Hiermaier–Thomas  (Mat_RHTAfter conducting the convergence test, two element sizes for targets and the projectile are selected in the present study. The finite element model of the UHPC/granite composite structure is shown in To verify the accuracy of the numerical model, we manufactured a UHPC/granite composite structural target plate, according to the configuration shown in D is commonly used in numerical simulations under the RHT model to reflect the damage caused by projectiles when penetrating granite targets; D = 0, the material is considered intact; when D = 1, the material is considered completely damaged. The damage factor The angle of penetration has a significant impact on the depth of penetration. In general, the same projectile will produce different penetration depths at different angles of penetration. When the projectile penetrates at a perpendicular angle to the target surface, the penetration depth is maximum. As the angle of penetration becomes more oblique, the penetration depth decreases. This is due to the resistance of the target material that the projectile encounters during penetration. A larger angle of penetration requires more energy to overcome the resistance of the target material, resulting in less energy available for penetration, thus reducing the penetration depth.In order to analyze the effects of projectile impact angle on penetration depth and trajectory deviation, the penetration process of a projectile impacting a composite structure at angles of 0°, 10°, 20°, and 30° are simulated, as shown in the ae of the impact angle; the curve of ae changing with the impact angle is shown in ae with respect to the impact angle is shown in According to the numerical simulation results in hx represents the horizontal displacement of the projectile after penetrating into the composite structure, while hy represents the vertical displacement of the projectile within the composite structure. From the figure, it can be seen that the penetration trajectories deviate to varying degrees at different penetration velocities. In the initial phase of penetration, the projectile’s motion paths almost overlap, and the deflection becomes greater as the velocity decreases.Adding steel reinforcement to the UHPC/granite composite structure can further enhance its resistance to penetration. On one hand, the steel reinforcement provides effective confinement to the UHPC/granite composite target, including toughening and crack arrest capabilities, strengthening the target’s resistance to penetration and reducing the formation of penetrating cracks caused by projectile impact. On the other hand, steel reinforcement itself possesses high strength. During the process of penetrating the UHPC/granite composite target, the deformation and failure of the steel reinforcement consume a significant amount of energy from the projectile when the projectile collides with the steel reinforcement, thereby reducing its destructive effect on the target.In order to analyze the effect of reinforcement ratio on the penetration resistance of composite structures, a set of calculation models for UHPC/granite composite targets with reinforcement ratios of 4.6%, 3.45%, 2.3%, 1.15%, and 0% were established. The effects of different target reinforcement ratios on the penetration depth were studied at four impact velocities of 200 m/s, 300 m/s, 400 m/s, and 500 m/s. The simulation results are shown in re with reinforcement ratio under different penetration velocities. It can be observed that the value of the equivalence coefficient re decreases as the reinforcement ratio increases at a given penetration velocity, indicating better penetration resistance performance of the structure. The reinforcement ratio and the equivalence coefficient re exhibit a linear relationship.The relationship between penetration depth and impact velocity for UHPC/granite composite structures with different reinforcement ratios is shown in re with penetration velocity is shown in The variation curve of the equivalence coefficient re and penetration velocity, the following equivalence coefficient calculation formulas for four types of reinforced composite structures and the plain concrete composite structure can be obtained:By performing linear fitting on the equivalence coefficient The impact tests were conducted on UHPC targets with velocities ranging from 216 to 340 m/s. Compared with granite targets, UHPC targets perform better in reducing crater diameter, while granite targets have a greater advantage in reducing penetration depth.The numerical models can effectively predict the penetration depth of projectiles into UHPC/granite composite structures. Meanwhile, compared with experimental results, the damage on the target surface obtained by the numerical model is consistent with the experimental observations.For the UHPC/granite composite structure, the penetration depth significantly decreases with the increase in the projectile incidence angle, and there is a linear relationship between penetration depth and impact angle.The greater the impact angle of the projectile, the more likely the trajectory of the projectile is to deflect during the penetration process. At the same time, the higher the velocity of the projectile, the less likely the trajectory is to deflect.re of the reinforcement ratio is not only correlated with the reinforcement ratio but also related to the penetration velocity.For the UHPC/granite composite structure, there is a linear relationship between penetration depth and reinforcement ratio. Furthermore, the equivalent coefficient This study conducted impact resistance tests on UHPC targets and compared the differences in penetration depth and crater diameter between UHPC targets and granite targets. In order to further enhance the impact resistance performance, a UHPC/granite composite structure was designed. We carried out numerical simulations using ANSYS 16.0/LS-DYNA finite element software to analyze the impact resistance performance of the UHPC/granite composite structure and investigate the influence of impact speed, impact angle, and reinforcement ratio on its impact resistance performance. The main conclusions are as follows:


If that's possible, then it could give us a reason to think that when we experience the present as being special, that's not because it's objectively so. Instead, our experience of the present being special is a feature of having a psychology like ours. While we are sympathetic to the thought that there is no objective present, we do not think that these experiments give us a reason to think this. That said, VR experiments, such as Gruber and Smith's, hold much promise for being able to illuminate various aspects of our temporal psychology.Gruber and Smith  have conC, which cause behavior, and unconscious processes, U, which construct a schematic representation of the environment. Hartle proposed that we experience the present as being special because of the sensory information at each time entering into C. For Gruber et al. . We have a genuine experience of time passing but time itself does not physically pass.According to Hartle's  IGUS modr et al. , the sucAlternatively, it could be that we do not experience time passing at all, rather people who claim to have an experience of time passing have false beliefs about their experience  responded affirmatively to both probe questions and Gruber et al. took this to mean that participants experienced the past snapshot as being present again. That is, Gruber et al. think that participants experienced both sensory feeds, the present feed and the past feed, as having equal status as being the present. If that's right, it gives us a reason to think that our experience of the present being special is C, and recognize the past feed shows events that they have already experienced before. We think that they conceptualize the past feed as a recording (which it in fact is), so what they experience as the present is them viewing a recording. This is a bit like when a person recalls a past event—rather than thinking that they are experiencing the past event as being present again, what they experience as the present is the recalling of a memory of a past event. We think the participants' responses to the probe questions are consistent with this explanation.However, there is an alternative and more straightforward explanation of participant's responses to these probe questions. Participants in this experiment chose which feed is fed into Participants agreed with the statement that events in the past feed appeared as real as the first time, and that it felt like they were there. This is meant to be evidence that people don't just experience events as being present when they are viewed in the present feed but also when they are viewed in the past feed. But when participants responded that things in the past feed seemed just as real as when they were in the present feed, this doesn't require that they experienced the event of seeing the second row of dominoes as being present twice. We can imagine someone who recalls a vivid memory of an event responding that the event in their mind seems just as real as when it occurred. All this tells us is that the VR environment is rich and immersive, not that people are experiencing events in the present and past feed as having equal status as an objective present.Gruber et al. say that they didn't ask people if they felt they were “in the past” because no participant has ever been in the past; but this seems to be what we want to know! Knowing whether participants experience the same event as being the present twice requires knowing whether participants experience past events being present again. Of course, we are sympathetic to the thought that probing this directly would be problematic. Participant responses will no doubt be influenced by their knowledge that things are not that way, making people reluctant to report that they seem that way too.An additional problem is raised by some of our own work on judgments about time—people have difficulty grasping different models of time and their implications. Sometimes over half a sample has to be dropped due to comprehension failures . As a general principle, caution is warranted when interpreting participant reports.Perhaps future studies could coach people. Once it is explained that we can have genuine experiences of things being a certain way that are not veridical with how things are (such as experiencing a past event again as present), then affirmative responses might be more interpretable. The unsolicited responses by participants reported by Gruber and Smith  suggest To bring out this line of thought further consider a different set of critical probe questions. We wonder what people believe their action affordances are while viewing the present feed and past feed. Imagine that you are in the VR experiment and while in the present feed you watch the experimenter as they lay out a third row of dominoes. Now imagine switching to the past feed, does it seem open to you that you can stop the experimenter laying out the third row of dominoes? The motivating thought here is that if people are experiencing the past feed as the present, then affordances for action that were apparent when the event was in the present feed should be similarly apparent again . But as we have already noted, that seems unlikely. When you experience the experimenter laying out the third row of dominoes in the present feed, you update your schematic representation of the world such that there is now a third row of dominoes. Switching to the past feed does not undo this.C it has already been incorporated into the schematic representation of the world. Thus, our present experience (or at least how it seems to us), while being similar to when the event was experienced for the first time, is nevertheless quite different from it. The consequences of the event in memory are already part of your schematic representation of the world and so the affordances that were present when the event was first experienced are also not the same. For example, if the experimenter knocks over the third row of dominoes in the present feed and then I switch to the past feed, I don't then think that I have an affordance to knock over the dominoes. My schematic representation of the world already represents things such that the affordance to knock over the dominoes is not available.This thought is entirely consistent with Hartle's IGUS model. Our experience of the present is not just a function of the current sensory information being taken from the world but also our schematic representation of the world constructed from past sensory information. When the past feed is being fed into In summary, we suspect that when participants switch to the past feed, they experience it as the recording that it is, rather than as an objective present. We do not think that VR experiments are well placed to answer questions about the nature of time itself . However, we do think that they provide a powerful means of probing our temporal psychology. Immersive VR experiments could be used to adjudicate between different accounts of the mechanisms and processes responsible for our temporal beliefs and experiences, including what could cause people to make erroneous statements about their own temporal phenomenology.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.